WO2024151990A1

WO2024151990A1 - Effective oncolytic herpes simplex virus vector retargeting

Info

Publication number: WO2024151990A1
Application number: PCT/US2024/011461
Authority: WO
Inventors: Joseph C. Glorioso Iii; Bonnie L. HALL; Justus B. Cohen; Selene INGUSCI; William F. Goins; Marco MARZULLI
Original assignee: University Of Pittsburgh - Of The Commonwealth System Of Higher Education
Priority date: 2023-01-13
Filing date: 2024-01-12
Publication date: 2024-07-18

Abstract

Disclosed are recombinant oncolytic Herpes Simplex Virus (oHSV) vectors comprising a non-HSV ligand that specifically binds to a protein present on the surface of a cancer cell. Related nucleic acids, viral stocks, compositions, and methods of killing a cancerous cell are also disclosed.

Description

EFFECTIVE ONCOLYTIC HERPES SIMPLEX VIRUS VECTOR RETARGETING

CROSS REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63/438,900, filed January 13, 2023, the disclosure of which is incorporated by reference in its entirity herein.

STATEMENT REGARDING

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Grant Numbers P01CA163205 and R01CA222804 awarded by the National Cancer Institute. National Institutes of Health. The Government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 22,617 Byte XML file named ^L‘769768_ST26.XML,” created January’ 4, 2024.

BACKGROUND OF THE INVENTION

[0004] Replication competent viruses, including oncolytic Herpes Simplex Virus (oHSV) vectors, may present a promising therapy for the treatment of cancer. However, treatment efficacy in patient trials has been limited. Although progress has been made in the development of safe and effective oHSVs, there is a need for oHSVs that demonstrate improved tumor cell-specific oncolysis.

BRIEF SUMMARY OF THE INVENTION

[0005] An aspect of the invention provides a recombinant oncolytic Herpes Simplex Virus (oHSV) comprising glycoprotein gE, glycoprotein gl, glycoprotein gH, and UL24, wherein the oHSV comprises one or more of the following amino acid substitutions (i)-(iv): (i) a substitution of valine at position 154 of glycoprotein gE; (ii) a substitution of isoleucine at position 286 of glycoprotein gl; (iii) a substitution of alanine at position 732 of glycoprotein gH; and (iv) a substitution of cysteine at position 103 of UL24.

[0006] An aspect of the invention provides a recombinant oncolytic Herpes Simplex Virus (oHSV) comprising a non-HSV ligand that specifically binds to a protein present on the surface of a cancer cell, wherein the ligand is inserted between residues 5 and 25 of the oHSV glycoprotein gD.

[0007] Another aspect of the invention provides a recombinant oHSV comprising a non- HSV ligand which is a single domain antibody that specifically binds to Epidermal Grow th Factor Receptor (EGFR) or Epidermal Growth Factor Receptor Variant III (EGFRvIII) present on the surface of a cancer cell, wherein the single domain antibody comprises:

(A) the heavy chain complementary determining region (VH CDR) 1 amino acid sequence of SEQ ID NO: 1; the VH CDR2 amino acid sequence of SEQ ID NO: 2; and the VH CDR3 amino acid sequence of SEQ ID NO: 3;

(B) the VH CDR1 amino acid sequence of SEQ ID NO: 4; the VH CDR2 amino acid sequence of SEQ ID NO: 5; and the VH CDR3 amino acid sequence of SEQ ID NO: 6; or

(C) the VH CDR1 amino acid sequence of SEQ ID NO: 7; the VH CDR2 amino acid sequence of SEQ ID NO: 8; and the VH CDR3 amino acid sequence of SEQ ID NO: 9.

[0008] Further aspects of the invention provide nucleic acids, viral stocks, and compositions related to the inventive oHSVs.

[0009] Another aspect of the invention provides a method of killing a cancerous cell, comprising exposing the cell to any of the inventive oHSVs, viral stocks, or compositions under conditions sufficient for the oHSV to infect the cancerous cell, whereby replication of the oHSV within the cancerous cell results in cell death.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0010] Figure 1 is a schematic illustrating the general structure of the retargeted glycoprotein D (gD) according to an aspect of the invention. The numbers shown in Fig. 1 correspond to the amino acid residue position numbers of gD, wherein the signal peptide is not included in the numbering, and gD amino acid residue position number 1 begins immediately after the C-terminal amino acid residue of the signal peptide. The abbreviations shown are as follows: “SP^?’ signal peptide; “PFD” prefusion domain: “TM^?’ transmembrane domain: ’EGFR” epidermal growth factor receptor; single domain (SD) 1 , SD2, and SD3 are defined in Table 3.

[0011] Figure 2 is a schematic showing an alignment of the VHH amino acid sequences for SD1, SD2, and SD3. The CDRs are boxed. The SD1, SD2. and SD3 full length VHH amino acid sequences shown in Fig. 2 are defined in Table 2 and correspond to SEQ ID NOs: 22-24, respectively. The VH CDR1, VH CDR2, and VH CDR3 amino acid sequences of SD1, SD2, and SD3 are defined in Table 1.

[0012] Figure 3A is an image of a gel showing the results of a Western blot analysis of purified virus particles for glycoproteins gD and gB and the tegument protein VP 16 for each of the indicated retargeted gD constructs and wild-type (WT) gD; scEGFRA2-24 is an anti- EGFR single-chain antibody inserted between gD amino acid residues 1 and 25.

[0013] Figure 3B is a graph showing the glycoprotein levels (relative to VP 16) incorporated into the viral envelope for each of the indicated retargeted gD constructs and WT gD.

[0014] Figures 4A-4D are graphs showing the percentage of ICP4 positive cells following infection of Vero (A), U251 (B), SNB19 (C), or A549 (D) cells with purified viruses containing the indicated retargeted gD or WT gD. Statistical significance for each virus compared to scEGFR (A2-24) was determined by one-way ANOVA (*** P < 0.001). [0015] Figures 5A-5C are graphs showing the percentage of U251 (A), SNB19 (B), or A549 (C) cells surviving following infection with the indicated retargeted gD and WT gD viruses, relative to uninfected cells.

[0016] Figures 6A-6D are graphs showing plaque area (mm²) following infection of Vero (A), U251 (B), SNB19 (C), or A549 (D) cells with viruses containing the indicated retargeted gD or WT gD. Statistical significance for each virus compared to scEGFR (A2-24) was determined by one-way ANOVA (* P = 0.01; ** P = 0.001: *** P < 0.001).

[0017] Figure 7 is a graph showing the percentage of U251 cells surviving at the indicated number of hours after mixing with Vero cells infected with the indicated retargeted gD viruses (scE = scEGFR) compared to uninfected Vero cells.

[0018] Figure 8 is a graph showing systemic delivery of the virus to U251 tumor-bearing mice. A CMV promoter-driven firefly luciferase (fLuc) reporter gene was inserted into the viral backbone to follow virus infection by bioluminescent imaging (BLI). Virus was delivered to subcutaneous flank U251 tumors (1 x IO¹⁰ gc/mice) by IV vector delivery (dO) and followed by BLI. The graph depicts tumor values for total flux (photons/second).

[0019] Figure 9A is a graph showing tumor volume over time (mm3; mean ± SEM).

U251 cells were implanted in the right hind flank of BALB/c athymic nude mice and when tumors reached a volume of -100 mm³, the mice were treated with 1 x io¹⁰ gc of the indicated virus or vehicle control (PBS). Arrows indicate the days of treatment; four doses of SD2 were delivered IT or IV (n=3 mice per group). Statistical differences were determined by two-way ANOVA (* p < 0.05).

[0020] Figure 9B is a graph showing fLuc expression from the viral backbone as quantified by bioluminescent imaging in a time course beginning 1 day after vector delivery and expressed as photons per seconds (p/s; mean ± SEM).

[0021] Figure 10A is a schematic illustrating the backbone structure of KNGF.

[0022] Figure 10B shows images showing the results of an entry assay in which JI. 1-2,

J/C and J/TrkA cells were infected at 10 or 100 pfu/cell with KNGF virus and mCherry expression was visualized 24 and 72 hour post infection.

[0023] Figure 11 shows images showing the results of an entry and spreading assay performed on J/TrkA cells comparing KNGF and the selection cycle KNGF-J4 products at 24 and 96 hpi.

[0024] Figure 12 shows images showing the results of an infection assay on J/TrkA cells comparing KNGF, KNGF-J4, and three J4 isolates. Cells were infected at 1 pfu/cell and mCherry expression was evaluated at 5 days post infection.

[0025] Figure 13 shows images showing the results of infection assay carried out on JI. 1- 2, J/C, and J/TrkA cells comparing J4H (TrkA-targeted/nectin-1 competent virus) and J4HA38 (completely TrkA-retargeted/nectin-1 deficient virus).

[0026] Figure 14A shows a Western blot analysis comparing glycoproteins gB, gD, and gH in KNGF and a mutant containing the UL24 mutation (KNGF-24’). Equivalent genome copies (gc) of each virus were loaded per lane (n=4); 5xl0⁷gc/lane for gH and gB quantification or 7xl0⁷ gc/lanefor gD quantification.

[0027] Figure 14B shows a quantification of the Western blot in Fig. 14A. The relative amount of each glycoprotein was determined using ImageJ software, normalized to the viral capsid protein VP5, and presented relative to KNGF lane 1.

[0028] Figures 15A-15C are graphs showing plaque area (mm²) following infection of Vero (A), A549 (B), or U251 (C) cells with the indicated constructs. Statistical significance for each virus compared to K_wt was determined by one-way ANOVA (* P = 0.01; ** P = 0.001 ; *** P < 0.001).

DETAILED DESCRIPTION OF THE INVENTION

[0029] The inventive oHSVs may provide any one or more of a variety of advantages including, for example, tumor cell-specific oncolysis. One possible strategy for achieving tumor cell-specific oncolysis involves vector retargeting, whereby HSV infection is restricted to cells expressing tumor-associated cell surface receptors; effectively changing viral tropism. One strategy for vector retargeting involves modification of the viral envelope glycoproteins involved in the process of HSV receptor binding and cell entry. The attachment and fusion steps of HSV infection are mediated primarily by components of the viral envelope, a membranous structure containing at least 10 glycoproteins (gB. gC, gD, gE, gG, gH, gl, gj, gL. and gM) and four non-glycosylated integral membrane proteins (UL20, UL34. UL45, and UL49.5). Of the glycoproteins, gB, gD, gH, and gL are essential for wild type herpes viruses to infect their host cells, while the remainder are dispensable for viral attachment or internalization. Prior to HSV-1 entry, virions are adsorbed to the cell surface through binding of gC and gB to exposed glycosaminoglycans on the cell membrane. Towards virus entry into a host cell, viral envelope glycoprotein D (gD) binds to one of its cellular receptors, nectin-1 or herpesvirus entry mediator (HVEM). Receptor binding triggers conformational changes that activate gD, and ultimately enables virus entry via a cascade of events involving additional glycoproteins, namely the gH/gL heterodimer and the viral fusogen, gB. To retarget virus infection, the natural receptor-specific binding properties of gD may be eliminated by deletion or modification of specific amino acids in the N-terminus of gD, and alternative protein-binding moieties that recognize tumor-associated cell-surface proteins may be inserted at the site of deletion.

[0030] A challenge for retargeting is the selection of mutations to reduce or eliminate gD recognition of its cognate receptors, herpesvirus entry mediator (HVEM) and nectin-1. HVEM recognition can be abolished by various amino acid substitutions, deletions, or insertions, such as targeting ligands, in the N-terminal region of gD, roughly extending to residue 30. However, these modifications do not typically impair virus entry through nectin- 1 recognition. The gD residue 38 (tyrosine; Y38) is a component of the nectin-1 binding site. Earlier retargeted viruses contained a point mutation (Y38C) at this position and an scFv specific for human EGFR/EGFRvIII inserted between gD residues 1 and 25 (A2-24). EGFR- dependent infection was achieved by combination with a previously selected mutant glycoprotein B gene (gB:NT) that enhances the rate of HSV entry (Uchida et al., Virol., 84: 12200-12209 (2010); Uchida et al., Mol. Ther., 21 : 561-569 (2013)). However, it was observed that position 38 can revert to tyrosine in nectin-1 -expressing cells. Deletion of residue 38 (A38) may provide a stable nectin-1 -detargeted phenotype, but may reduce EGFR- dependent virus entry.

[0031] The inventive oHSVs may ameliorate any one or more of the disadvantages of the earlier retargeted viruses described above. For example, the inventive oHSVs may provide enhanced retargeted virus spread.

[0032] An aspect of the invention provides a recombinant oHSV comprising glycoprotein gE, glycoprotein gl, glycoprotein gH, and UL24, wherein the oHSV comprises one or more (e.g., two or more, three or more, or all four) of the following amino acid substitutions (i)- (iv): (i) a substitution of valine at position 154 of glycoprotein gE (gE:V154); (ii) a substitution of isoleucine at position 286 of glycoprotein gl (gl:1286); (iii) a substitution of alanine at position 732 of glycoprotein gH (gH:A732); and (iv) a substitution of cysteine at position 103 of UL24 (UL24:C103). In an aspect of the invention, the oHSV comprises all of the foregoing amino acid substitutions (i)-(iv).

[0033] The substitution of valine at position 154 of glycoprotein gE may be a substitution of the valine at position 154 of glycoprotein gE with any amino acid residue other than valine. The substitution in gE is relative to the sequence of GenBank Accession No. YP_009137143. In an aspect of the invention, the substitution of valine at position 154 of glycoprotein gE is a substitution of valine with methionine (gE:V154M).

[0034] The substitution of isoleucine at position 286 of glycoprotein gl may be a substitution of the isoleucine at position 286 of glycoprotein gl with any amino acid residue other than isoleucine. The substitution in gl is relative to the sequence of GenBank Accession No. AAW49017. In an aspect of the invention, the substitution of isoleucine at position 286 of glycoprotein gl is a substitution of isoleucine with phenylalanine (gl:I286F). [0035] The substitution of alanine at position 732 of glycoprotein gH may be a substitution of the alanine at position 732 of glycoprotein gH with any amino acid residue other than alanine. The substitution in gH is relative to the sequence of GenBank Accession No. YP 009137096. In an aspect of the invention, the substitution of alanine at position 732 of glycoprotein gH is a substitution of alanine wi th valine (gH:A732V). [0036] The substitution of cysteine at position 103 of UL24 may be a substitution of the cysteine at position 103 of UL24 with any amino acid residue other than cysteine. The substitution in UL24 is relative to the sequence of GenBank Accession No. QAU10282.1. In an aspect of the invention, the substitution of cysteine at position 103 of UL24 is a substitution of valine with tyrosine (UL24:C103Y).

[0037] The inventive oHSVs may, advantageously, provide EGFR-specific virus entry combined with a stable nectin-1 -detargeted phenotype. An aspect of the invention provides a recombinant oHSV comprising a non-HSV ligand that specifically binds to a protein present on the surface of a cancer cell, wherein the ligand is inserted between residues 5 and 25 of the oHSV glycoprotein gD. The ligand insertion site(s) in gD described herein are relative to the sequence of GenBank Accession No. CAA38245, wherein the first 25 amino acids of the sequence of GenBank Accession No. CAA38245 (counting from the N-terminus) are not included in the numbering and the Lysine at position 26 is the first amino acid of the ‘mature’ gD sequence, referred to herein as residue 1.

[0038] The non-HSV ligand may be specific for a molecule (protein, lipid, or carbohydrate determinant) present on the surface of a cell (such as a cancer cell). The non- HSV ligand of the inventive oHSV may be incorporated into a glycoprotein exposed on the oHSV surface, such as glycoprotein gD. to facilitate targeting the desired cell with the ligand. For example, the ligand can be inserted between residues 5 and 25 of the oHSV gD or between residues 6 and 25 of the oHSV gD. Preferred ligands for targeting glioblastoma multiforme (GBM) and other cancer cells include those targeting EGFR and EGFRvIII, CD133, CXCR4. carcinoembryonic antigen (CEA), ClC-3/annexin-2/MMP-2, human transferrin receptor and EpCAM, and the ligand can target such a receptor or cell-surface molecule, i.e., the ligand can be capable of specifically binding such receptor or cell-surface molecule. Preferably, the ligand specifically binds Epidermal Grow th Factor Receptor (EGFR) or Epidermal Growth Factor Receptor Variant III (EGFRvIII).

[0039] In an aspect of the invention, the ligand comprises the antigen binding domain of a single domain antibody. A single domain antibody (sdAb) (also referred to as a “nanobody”) includes only the variable domain of an antibody heavy chain and completely lacks both the antibody light chain and the constant region of the antibody heavy chain that are found in conventional antibodies. sdAbs include VHH sdAbs and VNAR sdAbs. VHH sdAbs are derived from heavy chain antibodies found in camelids (e.g.. dromedaries, camels, llamas, and alpacas). VNAR sdAbs are derived from heavy chain antibodies found in cartilaginous fishes (e.g., sharks). A single-domain antibody can be obtained by immunization of dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies in addition to screening techniques such as phage display. In an aspect of the invention, the ligand is a VHH sdAb. Examples of VHH sdAb are described in Schmitz et al.. Structure, 21(7): 1214-24 (2013). sdAbs can also be made from conventional antibodies (e.g., from mice, rabbits, or humans). Such sdAbs are described in the literature (e.g., Feng et al., Antibody Ther., 3(1): 10-17 (2020)). Alternatively, sdAbs can also be derived from sdAbs produced by humans. Humans occasionally produce single domain antibodies by the random creation of a stop codon in the light chain. Human single-domain antibodies targeting various tumor antigens including mesothelin, GPC2, and GPC3 are described in the literature (Tang et al., Mol. Cancer Ther., 12(4): 416-26 (2013); Li et al., PNAS, 114 (32): E6623-E6631 (2017); Feng et al., PNAS, 110 (12): E1083-91 (2013); and Gao et al., Nat. Comm., 6:6536 (2015)). Without being bound to a particular theory or mechanism, it is believed that ligands with a more compact size may be less disruptive of the structure of the recombinant viral glycoprotein.

[0040] A sdAb generally comprises four heavy chain framework regions (VH FR), whose sequences are relatively conserved. The four VH FRs are referred to as VH FR1, VH FR2, VH FR3, and VH FR4. The framework regions are connected by three heavy chain complementarity determining regions (VH CDRs). The three VH CDRs. referred to as VH CDR1, VH CDR2, and VH CDR3, form the “hypervariable region” of the sdAb, which is responsible for antigen binding.

[0041] The terms “antigen binding domain,” “functional fragment of a sdAb.” and “antigen binding portion” are used interchangeably herein to mean one or more fragments or portions of a sdAb that retain the ability to specifically bind to an antigen. The antigen binding portion may comprise, for example, one or more of the VH CDR1, VH CDR2, and VH CDR3 of the sdAb. The antigen binding portion may comprise all of the VH CDR1. VH CDR2, and VH CDR3 of the sdAb.

[0042] In an aspect of the invention, the antigen binding portion may comprise one or more of the VH FRs in addition to the VH CDRs described above. In this regard, the antigen binding portion may comprise, for example, one or more of the VH FR1, VH CDR1, VHFR2, VH CDR2. VH FR3. VH CDR3 and VH FR4 of the sdAb. The antigen binding portion may comprise all of the VH FR1. VH CDR1. VHFR2. VH CDR2. VH FR3. VH CDR3 and VH FR4 of the sdAb. [0043] In an aspect of the invention, the antigen binding portion may comprise, for example, the heavy chain variable region (VH) of the sdAb. The VH of the sdAb may comprise all of the VH FR1, VH CDR1, VHFR2, VH CDR2, VH FR3, VH CDR3 and VH FR4 of the sdAb.

[0044] In an aspect of the invention, the ligand specifically binds to EGFR or EGFRvIII and comprises one or more of the amino acid sequences of Table 1.

TABLE 1

[0045] In an aspect of the invention, the ligand specifically binds to EGFR or EGFRvIII and comprises one or more of the amino acid sequences of Table 2. In Table 2, the CDRs are underlined. TABLE 2

[0046] In an aspect of the invention, the recombinant oHSV comprises a non-HSV ligand which is a single domain antibody that specifically binds to EGFR or EGFRvIII present on the surface of a cancer cell, wherein the single domain antibody comprises:

[0047] In an aspect of the invention, the single domain antibody comprises:

(A) the VH FR1 amino acid sequence of SEQ ID NO: 10; the VH CDR1 amino acid sequence of SEQ ID NO: 1; the VH FR2 amino acid sequence of SEQ ID NO: 11; the VH CDR2 amino acid sequence of SEQ ID NO: 2; the VH FR3 amino acid sequence of SEQ ID NO: 12; the VH CDR3 amino acid sequence of SEQ ID NO: 3; and the VH FR4 amino acid sequence of SEQ ID NO: 13; (B) the VH FR1 amino acid sequence of SEQ ID NO: 14; the VH CDR1 amino acid sequence of SEQ ID NO: 4; the VH FR2 amino acid sequence of SEQ ID NO: 15; the VH CDR2 amino acid sequence of SEQ ID NO: 5; the VH FR3 amino acid sequence of SEQ ID NO: 16; the VH CDR3 amino acid sequence of SEQ ID NO: 6; and the VH FR4 amino acid sequence of SEQ ID NO: 17; or

(C) the VH FR1 amino acid sequence of SEQ ID NO: 18; the VH CDR1 amino acid sequence of SEQ ID NO: 7; the VH FR2 amino acid sequence of SEQ ID NO: 19; the VH CDR2 amino acid sequence of SEQ ID NO: 8; the VH FR3 amino acid sequence of SEQ ID NO: 20; the VH CDR3 amino acid sequence of SEQ ID NO: 9; and the VH FR4 amino acid sequence of SEQ ID NO: 21.

[0048] In an aspect of the invention, the single-domain antibody comprises:

(A) the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 22;

(B) the VH amino acid sequence of SEQ ID NO: 23; or

(C) the VH amino acid sequence of SEQ ID NO: 24.

[0049] In an aspect of the invention, the single domain antibody is a VHH single domain antibody.

[0050] In an aspect of the invention, the single-domain antibody is inserted between residues 5 and 25 of the oHSV glycoprotein gD.

[0051] In an aspect of the invention, any of the recombinant oHSVs described herein may further comprise glycoprotein gE, glycoprotein gl, glycoprotein gH, and UL24, wherein the oHSV comprises one or more of the following amino acid substitutions (i)-(iv): (i) a substitution of valine at position 154 of glycoprotein gE; (ii) a substitution of isoleucine at position 286 of glycoprotein gl; (iii) a substitution of alanine at position 732 of glycoprotein gH; and (iv) a substitution of cysteine at position 103 of UL24. The amino acid substitutions of (i)-(iv) may be as described herein with respect to other aspects of the invention.

[0052] In an aspect of the invention, the oHSV further comprises a mutation of glycoprotein gD that reduces or prevents binding of gD to nectin-1. The mutation of gD that reduces or prevents binding of gD to nectin-1 may be a deletion of the tyrosine residue at position 38 (Y38) of glycoprotein gD (A38). The deletion in gD is relative to the sequence of GenBank Accession No. CAA38245, wherein the first 25 amino acids of the sequence of GenBank Accession No. C AA38245 (counting from the N-terminus) are not included in the numbering and the lysine at position 26 is the first amino acid of the ‘mature’ gD sequence. [0053] Also, in certain aspects, the genome of the inventive vector contains a deletion of the internal repeat (joint) region comprising one copy each of the diploid genes ICPO, ICP34.5, LAT and ICP4 along with the promoter for the ICP47 gene. In other aspects, instead of deleting the joint, the expression of genes in the joint region, particularly ICPO and/or ICP47, can be silenced by deleting these genes or otherwise limited mutagenesis of them.

[0054] In an aspect of the invention, the genome of the inventive vector encodes glycoprotein B. In another aspect of the invention, the genome of the inventive vector encodes a mutant glycoprotein B. In a further aspect of the invention, the genome of the inventive vector contains mutant glycoprotein B gene (gB:NT) (Uchida et al., J. Virol., 84: 12200-12209 (2010); Uchida et al., Mol. Ther.. 21 : 561-569 (2013)).

[0055] The inventive vector can be produced by standard methods known to persons of ordinary skill in the field of HSV virology. However, to facilitate manipulation of the HSV genome and production of the inventive vector, an aspect of the invention also provides a nucleic acid encoding the inventive vector. A preferred nucleic acid is a bacterial artificial chromosome (BAC) encoding the inventive vector, which facilitates manipulation of the HSV in a bacterial system.

[0056] Generally, the inventive oHSV vector is most useful when enough of the virus can be delivered to a cell population to ensure that the cells are confronted with a suitable number of viruses. Thus, an aspect of the present invention provides a stock, preferably a homogeneous stock, comprising the inventive oHSV vector. The preparation and analysis of HSV stocks is well known in the art. For example, a viral stock can be manufactured in roller bottles containing cells transduced with the oHSV vector. The viral stock can then be purified on a continuous nycodenze gradient, and aliquotted and stored until needed. Viral stocks vary considerably in titer, depending largely on viral genotype and the protocol and cell lines used to prepare them. Preferably, such a stock has a viral titer of at least about 10⁵ plaque-forming units (pfu) per milliliter (ml), such as at least about 10⁶ pfu/ml or even more preferably at least about 10⁷ pfu/ml. In still more preferred aspects, the titer can be at least about 10⁸ pfu/ml, or at least about 10⁹ pfu/ml. and high titer stocks of at least about 10¹⁰ pfu/ml or at least about 10¹¹ pfu/ml are most preferred. Such titers can be established using cells that express a receptor to which the vector is targeted, for example.

[0057] An aspect of the invention additionally provides a composition comprising the inventive oHSV vector or viral stock and a carrier, preferably a physiologically-acceptable earner. The carrier of the composition can be any suitable carrier for the vector. The carrier tj pically will be liquid, but also can be solid, or a combination of liquid and solid components. The carrier desirably is a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent). Pharmaceutically acceptable carriers are well known and are readily available. The choice of carrier will be determined, at least in part, by the particular vector and the particular method used to administer the composition. The composition can further comprise any other suitable components, especially for enhancing the stability⁷ of the composition and/or its end-use. Accordingly, there is a wide variety of suitable formulations of the composition of the invention. The following formulations and methods are merely exemplary and are in no way limiting.

[0058] Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0059] In addition, the composition can comprise additional therapeutic or biologically - active agents. For example, therapeutic factors useful in the treatment of a particular indication can be present. Factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the vector and physiological distress. Immune system suppressors can be administered with the composition method to reduce any immune response to the vector itself or associated with a disorder. Alternatively, immune enhancers can be included in the composition to upregulate the body's natural defenses against disease, particularly against the cancer or tumor against which the inventive vector is to be used. Antibiotics, i.e., microbicides and fungicides, can be present to reduce the risk of infection associated with gene transfer procedures and other disorders.

[0060] The inventive oHSV may be used to target and kill cancerous cells, whether in vivo or in vitro. A preferred application is to employ the inventive vector therapeutically, particularly in human patients and/or against human tumors/cells (which can be xenografts in various mammalian species). However, the method can also be employed in animals, such as companion animals (e.g., cats and dogs), or animals of agricultural importance (e.g., cattle, sheep, horses, and the like), or of zoological importance. Exemplary tumors/cancerous cells, the treatment of which the inventive vectors can be employed, involve cancers of the central nervous system, and in particular glioblastoma multiforme.

[0061] In this regard, an aspect of the invention provides a method of killing a cancerous cell, comprising exposing the cell to the inventive oHSV, viral stock, or composition under conditions sufficient for the oHSV to infect the cancerous cell, whereby replication of the oHSV within the cancerous cell results in cell death. In an aspect of the invention, the cell is a tumor cell. The tumor cell may be a glioblastoma multiforme tumor cell. In a preferred aspect, the cell is human. The tumor may be within the brain of an animal. The animal may be a human. In an aspect of the invention, the oHSV is exposed to the cell by intracranially injecting the oHSV, stock, or composition to the animal.

[0062] In an aspect of the invention, the oHSV is exposed to the cell by intravenously (IV) injecting the oHSV, stock, or composition to the animal. In an aspect of the invention, the oHSV is exposed to the cell by intratumorally (IT) injecting the oHSV, stock, or composition to the animal.

[0063] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0064] This example demonstrates the preparation of retargeted gD according to an aspect of the invention.

[0065] To explore the possibility that more compact EGFR-targeting ligands may be less disruptive of the structure of the retargeted protein (as compared to an scFv), three single domain (VHH/SD) antibodies (Schmitz et al., Structure, 21(7): 1214-24 (2013)) (Table 3) were tested in place of a larger scFv. A schematic illustrating the general structure of the retargeted glycoprotein D (gD) is shown in Fig. 1 . The positioning of these ligands in the gDA38 vector varied (SD1, SD2, and SD3; Table 4). The full-length VHH amino acid sequences of SD1, SD2, and SD3 are shown in Fig. 2.

TABLE 3

TABLE 4

"Virus names describe the corresponding retargeted gD construct design. Deletion notations A2-24, A6-24, or A7-24 specify the insertion site of the noted targeting ligand: scEGFR (scFv) and SD (VHH). All contain the A38 nectin-1 detargeting mutation.

'Genome copy titers were determined by qPCR for UL5; DNA from purified virus particles.

"Plaque Forming Unit titers were determined by standard plaque assay on Vero cells. ^ADNot determined; high titer stocks could not be obtained.

EXAMPLE 2

[0066] This example demonstrates the incorporation of the gD into virions.

[0067] Western blot analysis of purified virus particles demonstrated that that when normalized to the tegument protein VP 16, the amount of retargeted gD incorporated into the envelope was variable, while gB protein levels remained consistent across all constructs (Figs. 3A-3B). For this analysis, 1 x 10⁸ genome copies (gc) of each purified virus were loaded per lane and probed with antibodies to the envelope glycoproteins gD and gB and the tegument protein VP16. Similar VP16 levels across the lanes demonstrated that the genome copy titers obtained provide a good estimate of viral particles for analysis. All modified gD viruses expressed protein of the predicted molecular weight. While gB levels were roughly consistent between the modified viruses, gD levels varied considerably; falling well below wt gD levels (Figs. 3A-3B).

EXAMPLE 3

[0068] This example demonstrates that retargeted gD mediates cell entry through both EGFR and EGFRvIII.

[0069] Receptor-dependent cell entry was assessed on both B78H1 and JI. 1-2 based cell lines that do not express canonical gD receptors, HVEM and necin-L and do not allow wt HSV entry. Stable cell lines were created that express either nectin-1 (B78-C and J-C), full- length EGFR (J-EGFR) or the deletion mutant EGFRvIII (B78-vIII). Cells were infected at a multiplicity of infection (MOI) of 1,000 gc per cell, infections were stopped at 6 h.p.i (hours post-infection) and stained with antibody to the IE protein ICP4 as a marker of entry and DAPI (4’,6-diamidino-2-phenylindole).

[0070] The entry-receptor specificities of the retargeted viruses were verified by infection of cells expressing either no gD receptor (B78H1 or JI. 1-2), human nectin-1 (B78-C or J-C), EGFRvIII (B78-vIII) or full length EGFR (J-EGFR). Wildtype (wt) gD mediated entry strictly through nectin-1 and the retargeted gD proteins mediated entry through both EGFR and EGFRvIII (Tables 5-6). In Tables 5-6, “+” indicates that stain was observed (cell entry), while indicates that no stain was observed (no cell entry). TABLE 5

TABLE 6

EXAMPLE 4

[0071] This example demonstrates the tumor cell entry efficiency of retargeted gD viruses.

[0072] Virus entry efficiency was tested on a panel of EGFR/vIII-expressing tumor cell lines and Vero cells. Cells were infected at an MOI of 1,000 gc/cell, infections were stopped at 6 h.p.i and stained with antibody to ICP4 as a marker of entry (virus⁴ cells) and DAPI (total cells). ICP4⁺/DAPI⁺ cells were quantified using Image J software (mean +/- SEM).

[0073] These data demonstrated that the constructs varied in entry efficiency in a celltype dependent manner (Figs. 4A-4D), with the SD2 (A6-24) virus displaying enhanced entry on all cell lines compared to the scEGFR (A2-24) virus. Vero cells that were used to propagate all virus stocks were used as a positive control. EXAMPLE 5

[0074] This example demonstrates the killing of tumor cell lines by retargeted gD. [0075] Cell killing was assessed by infecting cells at an MOI of 1,000 gc/cell and quantifying cell viability every 24 hours via the ALAMARBLUE™ cell viability' assay. Virus-mediated cell killing was tested on the same cell lines at the same gc-based infection input as described in Example 4 (Figs. 5A-5C). As seen with cell entry, these data demonstrated that the SD-based vectors provided enhanced cell killing when compared to the scEGFR virus at all time points tested. The extent of cell killing varied by cell line. These data likely reflect differences seen in both cell entry' and cell-to-cell spread.

EXAMPLE 6

[0076] This example demonstrates the cell-to-cell spread of retargeted gD in tumor cell lines.

[0077] To separate the individual components of virus entry and cell-to-cell spread, plaque formation was analyzed in a modified infectious center assay (Figs. 6A-6D), where infected Vero cells were overlay ed on cell monolayers and resulting plaque sizes were evaluated at 48h post infection. Cell-to-cell spread was assessed on a panel of EGFR/vIII expressing cell lines. Vero cells were infected at an MOI of 10,000 gc/cell, cells were glycine washed at 2 h.p.i to remove extracellular virus, single Vero cells were overlay ed on monolayers of cells, and images of plaques were taken at 48 hpi. Plaque area was quantified using Image J software.

[0078] These data demonstrate that plaque size varied considerably between the viruses, with scEGFR (A2-24) producing small plaques, indicative of minimal spread, and the SD- based viruses consistently produced substantially larger plaques. The SD2 (A6-24) virus showed a robust enhancement in plaque formation compared to the scEGFR (A2-24) vector on all cell lines tested.

EXAMPLE 7

[0079] This example demonstrates that the SD2 (A6-24) gD construct demonstrates a robust improvement in U251 cell killing compared to scEGFR (A2-24). [0080] When cell killing was assessed by the same technique described in Example 6, mixing infected Vero cells with uninfected U251 cells, SD2 (A6-24) demonstrated a robust improvement in cell killing compared to scEGFR (A2-24) (Fig. 7).

EXAMPLE 8

[0081] This example demonstrates the systemic vector delivery to U251 subcutaneous flank tumors in vivo.

[0082] A CMV promoter-driven firefly luciferase (fLuc) reporter gene was inserted into the viral backbone to follow virus infection by bioluminescent imaging (BLI). U251 flank tumors were established in athymic nude mice (2 x 10⁶ U251 cells per mouse); 1 x 10¹⁰ genome copies of SD2 (A6-24)- or scEGFR (A2-24)-retargeted virus were delivered i.v. when tumors reached -100 mm³ (dO). Virus infection was followed by BLI out to 17 dpi.

[0083] Initial data comparing sy stemic delivery of the luciferase-enhanced SD2 (A6-24) vector (SD2 in Fig. 8) and scEGFR (A2-24) vector (scE in Fig. 8) in a U251 flank tumor model (Fig. 8) suggested that virus was detectable in the tumor from 24 hours to 17 days post vims delivery, while detection of vims infection of other tissues was minimal. While the SD2 (A6-24) vector maintained a consistently high fLuc signal throughout the course of the experiment, the signal from the scEGFR (A2-24) vector began to taper 1 week following vims delivery.

[0084] In summary', all SD-based viruses provided evidence of substantial improvements in EGFR-dependent virus entry and lateral spread relative to the previous A38-containing vims, scEGFR (A2-24). In particular, the VHH-type ligand referred to as SD2 inserted at a modified position (A6-24), mediated increased entry' and spread compared to the previous vims.

[0085] The results of Examples 1-8 confirmed that infection by all of the retargeted viruses was strictly dependent on the presence of EGFR/EGFRvIII on the cell surface. While the efficiency of vims entry' and subsequent cell killing varied between different EGFR/EGFRvIII-positive cell lines, it was found that viruses containing the SD antibody EgAl generally outperformed comparable scFv-retargeted vimses in entry, spread and cellkilling assays, often by a substantial margin. Viruses containing the SD antibody 9G8 or 7D12 also generally performed better than scFv-retargeted viruses. It was also observed that the scFv was a more effective targeting ligand when inserted adjacent to the first residue of mature gD than after residue 5, while the 3 SD antibodies showed the opposite preference. Overall, these results indicate that HSV retargeting can best be accomplished using SD antibodies inserted into the N terminus of gD. Without being bound to a particular theory or mechanism, it is believed that these ligands are smaller and more compact and allow better incorporation into the virus envelope.

EXAMPLE 9

[0086] This example demonstrates that SD2 reduces tumor growth.

[0087] U251 tumor bearing mice were treated with the SD2 (A6-24) vector by intravenous (IV) or direct intratumoral (IT) delivery' every two days for a total of 4 doses. The vehicle (PBS)-treated tumors increased steadily in size over the 18-day time course to -1.000 mm³ (Fig. 9A). Multiple IT injections of ILuc-gD:SD2 resulted in tumor clearance by 18 days. For animals treated with multiple IV injections of the same vector, tumor size remained significantly smaller than for the PBS-treated animals for 9 days after treatment, followed by a progressive increase. Based on the area under the curve, multiple IV doses significantly reduced tumor growth when compared to the vehicle controls. The BLI signal in IT treated animals was higher than in IV treated animals at 1- and 3-days post-delivery, remained relatively high for 7 days, and then decreased in a manner consistent with the decrease in tumor size, reaching background levels when tumor regression was complete (Fig. 9B). In the IV treated animals, the BLI signal peaked at 3 days and remained largely constant within the tumor over the observation period, declining between 13 and 18 dpi.

[0088] These data show that by repeated IV delivery, the retargeted vector was able to transduce and replicate in the tumor cells, reducing tumor growth. Further, repeated IT delivery' was able to eradicate the tumor.

EXAMPLE 10

[0089] This example demonstrates the identification of a recombinant oHSV with enhanced fitness for TrkA-mediated infection.

[0090] Mutations that enhance retargeted virus spread using a KOS-based viral backbone (KNTc-AgD:GW; Hall et al., Int. J. Mol. Set., 21(22): 8815 (2020)) retargeted to the TrkA receptor were identified. To target HSV infection to TrkA-expressing cells, the signal peptide (SP) and HVEM-binding N-terminal region of gD were replaced with mouse pre-pro- NGF (ppNGF) to create gD:ppNGF(Y38). This design provided the ppNGF SP and processing sites and maintained gD residue Y38 to preserve the interaction with the viral entry receptor nectin-1. Recombinant gD:ppNGF(Y38) was introduced at the gD locus of KNTc-AgD:GW to create the KNGF virus (Fig. 10A).

[0091] An entry assay was performed in which JI. 1-2, J/C and J/TrkA cells were infected at MOI of 10 and 100 with KNGF virus and mCherry expression was visualized 24 and 72 hour post infection (Fig. 10B). The results showed that KNGF demonstrated MOI-dependent infection of J/TrkA cells (TrkA positive/nectin- 1 negative). However, TrkA-mediated infection yielded only individual infected cells and no apparent cell-to-cell spread.

EXAMPLE 11

[0092] This example demonstrates the identification of a J4H virus isolate that demonstrates enhanced infectivity on J/TrkA cells.

[0093] A genetic selection was performed by repeat passage on J/TrkA cells. For each cycle, J/TrkA cells were infected with KNGF virus (MOI 10 pfu/cell), viral supernatant was harvested at 3 days post infection (dpi) and amplified by nectin-1 mediated infection of U2OS cells to produce a virus stock (KNGF-JwJ. J/TrkA cells were infected with each KNGF-Jra stock and observed over time for evidence of cell-to-cell spread.

[0094] An infection assay was carried out on J/TrkA cells comparing the KNGF parental virus and the KNGF-J4 selection cycle. Cells were infected at MOI of 10 and mCherry expression was evaluated at 24 hpi and 96 hpi. The results are shown in Fig. 11. It was anticipated that the KNGF-J4 stock would represent a mixed population of viruses with potentially different genetic changes driving the ability⁷ to infect TrkA expressing cells.

[0095] Individual KNGF-J4 viral DNAs were rescued. These unique virus clones were characterized by infection of J/TrkA cells. Fig. 12 shows an infection assay comparing KNGF, the KNGF-J4 mixed pool, and three representatives of clonal isolates (J4C, J4D, and J4H) at 5 dpi.

[0096] Whole genome sequencing was performed for J4C, J4D, and J4H and Table 7 shows the detected mutations. TABLE 7

[0097] This selection technique identified the J4H virus isolate that demonstrated enhanced infectivity on J/TrkA cells. Whole genome sequencing of J4H identified substitution mutations in four coding genes: 1) a valine to methionine substitution at residue 154 of gE (gE:V154M), 2) an isoleucine to phenylalanine substitution at residue 286 of gl (gI:I286F), 3) an alanine to valine substitution at position 732 of gH (gH: A732V), and 4) a cysteine to tyrosine substitution in UL24 at position 103 (UL24:C103Y) (Table 7).

[0098] The enhanced infectivity provided by the J4H backbone allowed the production of nectin-1 -binding deficient and thereby completely detargeted, NGF retargeted virus (J4HA38) containing recombinant ppNGFgD(A38) at the gD locus. JI.1-2, J/C and J/TrkA cells were infected with 10 gc/cell of J4H and J4HA38 and mCherry expression was observed at 4 dpi. These data showed that while both viruses were able to infect and spread in J/TrkA cells, only J4H was able to infect J/C cells efficiently. The results are shown in Fig. 13.

EXAMPLE 12

[0099] This example demonstrates the effect of the UL24 mutation on gD, gB, and gH incorporation in virus particles.

[0100] Relative quantification of gB, gD, and gH glycoprotein incorporation into purified virus particles was compared for KNGF and KNGF-UL24’ (Table 9). The genome copy (gc) and plaque forming unit (pfu) titers for each virus are shown in Table 8. Equivalent genome copies of each virus were loaded per lane (Fig. 14A) and the relative amount of each glycoprotein was determined using ImageJ software, normalized to the viral capside protein VP5. and presented relative to KNGF lane 1 (Fig. 14B). KNGF-UL24’ is the original KNGF virus modified to contain the UL24:C103Y substitution.

TABLE 8

“Plaque Forming Unit titers were determined by standard plaque assay on U2OS cells.

' Genome copy titers were determined by qPCR for UL5; DNA from purified virus particles.

EXAMPLE 13

[0101] This example demonstrates that the J4H backbone of Example 11, with an EGFR- retargeted gD, augments lateral spread via EGFR-retargeted gD.

[0102] To determine if the mutations identified in the J4H backbone of Example 11 would also augment lateral spread via EGFR-retargeted rather than TrkA-retargeted gD, gD:SD2(A6-24) was introduced at the gD locus of J4H in place of gD:ppNGF(Y38). The resulting virus, J4H_SD2(A6-24), was compared to K_ SD2(A6-24), and K_wt for plaque formation in Vero, U251, and A549 cells by infectious center assay (Figs. 14A-14C). The structures of K_wt. J4H_SD2(A6-24), K_ SD2(A6-24) are described in Table 9.

TABLE 9

[0103] These data demonstrate a statistically significant increase in plaque size for the J4H SD2(A6-24) virus relative to both the K SD2(A6-24) and K wt viruses. These data suggest that the mutations identified in the J4H backbone can enhance cell-to-cell spread for other retargeted gD constructs as well.

[0104] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety⁷ herein.

[0105] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [0106] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted bv context.

Claims

CLAIM(S):

1. A recombinant oncolytic Herpes Simplex Virus (oHSV) comprising glycoprotein gE, glycoprotein gl, glycoprotein gH, and UL24, wherein the oHSV comprises one or more of the following amino acid substitutions (i)-(iv):

(i) a substitution of valine at position 154 of glycoprotein gE;

(ii) a substitution of isoleucine at position 286 of glycoprotein gl;

(iii) a substitution of alanine at position 732 of glycoprotein gH; and

(iv) a substitution of cysteine at position 103 of UL24.

2. The recombinant oHSV of claim 1, wherein the oHSV comprises all of the following amino acid substitutions (i)-(iv):

(i) a substitution of valine at position 154 of glycoprotein gE;

(ii) a substitution of isoleucine at position 286 of glycoprotein gl;

(iii) a substitution of alanine at position 732 of glycoprotein gH; and

(iv) a substitution of cysteine at position 103 of UL24.

3. The recombinant oHSV of claim 1 or 2, wherein:

(i) the substitution of valine at position 154 of glycoprotein gE is a substitution of valine with methionine;

(ii) the substitution of isoleucine at position 286 of glycoprotein gl is a substitution of isoleucine with phenylalanine;

(iii) the substitution of alanine at position 732 of glycoprotein gH is a substitution of alanine with valine; and

(iv) the substitution of cysteine at position 103 of UL24 is a substitution of cysteine with tyrosine.

4. A recombinant oncolytic Herpes Simplex Virus (oHSV) comprising a non- HSV ligand that specifically binds to a protein present on the surface of a cancer cell, wherein the ligand is inserted between residues 5 and 25 of the oHSV glycoprotein gD.

5. The oHSV of claim 4, wherein the ligand specifically binds EGFR or

EGFRvIII.

6. The oHSV of claim 4 or 5, wherein the ligand comprises the antigen binding portion of a single domain antibody.

7. The recombinant oHSV of claim 4 or 5, wherein the ligand is a VHH single domain antibody.

8. A recombinant oHSV comprising a non-HSV ligand which is a single domain antibody that specifically binds to Epidermal Growth Factor Receptor (EGFR) or Epidermal Growth Factor Receptor Variant III (EGFRvIII) present on the surface of a cancer cell, wherein the single domain antibody comprises:

9. The recombinant oHSV of claim 8. wherein the single-domain antibody comprises:

(A) the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 22;

(B) the VH amino acid sequence of SEQ ID NO: 23; or

(C) the VH amino acid sequence of SEQ ID NO: 24.

10. The recombinant oHSV of claim 8 or 9, wherein the single-domain antibody is inserted between residues 5 and 25 of the oHSV glycoprotein gD.

11. The recombinant oHSV of claim 8 or 9, wherein the single-domain antibody is inserted between residues 6 and 25 of the oHSV glycoprotein gD.

12. The recombinant oHSV of claim 8 or 9, wherein the single domain antibody is a VHH single domain antibody.

13. The recombinant oHSV of any one of claims 4, 5, 8. or 9, wherein the oHSV comprises glycoprotein gE, glycoprotein gl, glycoprotein gH, and UL24, and wherein the oHSV comprises one or more of the following amino acid substitutions (i)-(iv):

(i) a substitution of valine at position 154 of glycoprotein gE;

(ii) a substitution of isoleucine at position 286 of glycoprotein gl;

(iii) a substitution of alanine at position 732 of glycoprotein gH; and

(iv) a substitution of cysteine at position 103 of UL24.

14. The recombinant oHSV of any one of claims 4, 5, 8, or 9, wherein the oHSV comprises all of the following amino acid substitutions (i)-(iv):

(i) a substitution of valine at position 154 of glycoprotein gE;

(ii) a substitution of isoleucine at position 286 of glycoprotein gl;

(iii) a substitution of alanine at position 732 of glycoprotein gH; and

(iv) a substitution of cysteine at position 103 of UL24.

15. The recombinant oHSV of claim 13, wherein:

16. The recombinant oHSV of any one of claims 1, 2, 4, 5, 8, 9, or 15, comprising a deletion of the internal repeat (joint) region comprising one copy each of the diploid genes ICPO, ICP34.5. LAT and ICP4 along with the promoter for the ICP47 gene.

17. The oHSV of any of claims 1, 2, 4. 5, 8, 9. or 15, further comprising a mutation of glycoprotein gD that reduces or prevents binding of gD to nectin-1 .

18. The oHSV of claim 17, wherein the mutation of gD is deletion of residue 38 of glycoprotein gD.

19. A nucleic acid encoding the oHSV of any one of claims 1, 2, 4, 5, 8, 9, or 15.

20. A viral stock comprising the oHSV of any of claims 1, 2, 4, 5. 8, 9, or 15.

21. A composition comprising the oHSV of any of claims 1, 2, 4, 5, 8, 9, or 15 and a pharmaceutically-acceptable carrier.

22. A composition comprising the viral stock of claim 20 and a pharmaceutically- acceptable carrier.

23. A method of killing a cancerous cell, comprising exposing the cell to the oHSV of any of claims 1, 2, 4. 5, 8, 9, or 15, under conditions sufficient for the oHSV to infect the cancerous cell, whereby replication of the oHSV within the cancerous cell results in cell death.

24. The method of claim 23, wherein the cell is in vivo.

25. The method of claim 23, wherein the cell is a tumor cell.

26. The method of claim 25, wherein the tumor cell is a glioblastoma multiforme tumor cell.

27. The method of any of claim 23, wherein the cell is human.

28. The method of claim 25, wherein the tumor is within the brain of an animal.

29. The method of claim 28, wherein the oHSV is exposed to the cell by intracranially, intravenously, or intratumorally injecting the oHSV, stock, or composition into the animal.

30. The method of claim 29, wherein the animal is human.