Oncolytic viruses for systemic delivery and improved antitumor activity

Abstract

The present invention relates to oncolytic viruses that are more resistant to neutralization and phagocytosis by the immune system, as well as methods for their preparation and use to treat disorders and diseases, such as cancer. Described herein is an oncolytic herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2) treated in immune serum containing high levels of anti-HSV antibodies. In one preferred embodiment, the oncolytic virus comprises an extracellular CD47 domain inserted into the N-terminus of the glycoprotein to inhibit phagocytic activity. The oncolytic virus is suitable for systemic administration for cancer treatment.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 200,011, filed February 9, 2021, and U.S. Provisional Application No. 63 / 263,528, filed November 4, 2021, each of which is incorporated by reference herein.

[0002] Statement of Federally Sponsored Research or Development This invention was made with Government support under Grant G110207 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present invention relates to oncolytic viruses that are more resistant to clearance and neutralization by the immune system, as well as methods for their preparation and use to treat disorders and diseases, such as cancer, including metastatic cancer. [Background technology]

[0004] Cancer virotherapy is based on a pragmatic approach with a simple and practical therapeutic mechanism that exploits the inherent infection / cytolytic activity of viruses for the selective destruction of malignant cells. In recent years, the development of cancer virotherapy has made remarkable progress. Currently, one oncolytic virus derived from herpes simplex virus type I (HSV-1), Imlygic or T-VEC, has been approved for clinical application for the treatment of unresectable cutaneous, subcutaneous and nodal lesions in melanoma patients who have relapsed after initial surgery (Greig, 2016). In addition, there are multiple ongoing clinical trials investigating the efficacy of various oncolytic viruses constructed from different viruses (Macedo et al., 2020).

[0005] Currently, oncolytic virus therapy is administered primarily via intratumoral route. However, systemic delivery is desirable for the treatment of patients with many malignancies, especially those who develop metastatic disease. In fact, the vast majority of patients who undergo virus therapy may have metastatic disease. Despite the importance of this delivery route, an optimal systemic delivery strategy has yet to be developed. Studies have shown that although therapeutic effects were detected in some experiments after oncolytic HSV was delivered via systemic route, the overall therapeutic effect was significantly lower than that seen with intratumoral delivery (Fu et al., 2006; Nakamori et al., 2004). Several key factors impede the efficiency of oncolytic virus delivery via systemic route.

[0006] First, anti-HSV neutralizing antibodies, which may already be present due to the patient's previous exposure to the virus or may arise de novo from repeated administration of the oncolytic virus during the course of treatment, may bind to the introduced viral particles, preventing the virus from infecting tumor cells and leading to rapid clearance by antibody-dependent cellular phagocytosis (ADCP) in the host (Huber et al., 2001; Tay, Wiehe, and Pollara, 2019). Indeed, animal studies have shown that pre-existing humoral immunity is detrimental to oncolytic HSV infectivity (Fu and Zhang, 2001).

[0007] Anti-HSV neutralizing antibodies have been found to target mainly two virally encoded glycoproteins, glycoprotein D (gD) and gB (Cairns et al., 2015, Cairns et al., 2014). Both gD and gB contain many neutralizing epitopes and are presented in various orders of immunodominance hierarchy in different individuals (Bender et al., 2007). Overall, these epitopes are well conserved among infected individuals and / or different species (Eing, Kuhn, and Braun, 1989). These studies have also shown that HSV-2 is more difficult to neutralize than HSV-1 (Silke Heilingloh et al., 2020). The presence of strong epitopes appears as a major obstacle for systemic administration of HSV-based oncolytic viruses, as neutralizing antibodies may easily recognize them and negate their therapeutic activity.

[0008] Another important limiting factor that reduces the efficacy of systemically delivered oncolytic virotherapy is the host's mononuclear phagocyte system (MPS). It has been found that after systemic delivery, viral particles can be rapidly cleared by the MPS (Ellermann-Eriksen, 2005; Hume, 2006; Van Strijp et al., 1989). Specifically, a study by Fulci et al. found that macrophage depletion can significantly improve the therapeutic efficacy of oncolytic HSV (Fulci et al., 2007).

[0009] Yet another important antiviral mechanism that may reduce the effectiveness of viral delivery by HSV is natural killer (NK) cells (Alvarez-Breckenridge et al., 2012a, Alvarez-Breckenridge et al., 2012b). The viral glycoprotein E (gE) is known to bind to IgG (Dubin et al., 1994). Recent studies have shown that NK cells can recognize HSV or HSV-infected cells through the binding of the surface CD16 activating receptor with the Fc region of IgG bound to gE (Dai and Caligiuri, 2018).

[0010] One way to limit the impact of neutralizing antibodies on oncolytic virus infectivity is to induce mutations in each of the major epitopes. Indeed, recent studies have shown that inducing mutations in two of these epitopes from gD of oncolytic HSV can actually abolish neutralization by the corresponding monoclonal antibody (mAb) (Tuzmen et al., 2020). However, both gB and gD of HSV-1 and HSV-2 are major targets of neutralizing antibodies, each of which contains nearly 12 neutralizing epitopes. In view of the viral genomes that are over 150 kb long, using traditional methods to induce mutations in each of these epitopes from the two glycoproteins is not practical in practice. Moreover, this approach only addresses one of the three main obstacles facing systemic delivery of oncolytic virotherapy, namely neutralizing antibodies, leaving MPS intact.

[0011] US Patent Publication No. 2012 / 0301506 discloses the construction of FusOn-H2, an oncolytic virus derived from HSV-2, and its use in the treatment of malignant tumors. Administration of FusOn-H2 induces the patient's innate immune response against tumor cells via neutrophils. Once neutrophils migrate into the tumor mass, they can efficiently destroy the tumor. Due to the induced innate antitumor immunity, FusOn-H2 is effective in eradicating tumors even when used at very low doses.

[0012] U.S. Patent Nos. 10,039,796 and 8,986,672 disclose compositions and uses of modified herpes simplex virus type 2 (HSV-2) for the treatment of cancer, the modified HSV-2 comprising a modified / mutated ICP10 polynucleotide encoding a polypeptide having ribonucleotide reductase activity and lacking protein kinase activity.

[0013] Fu et al., Oncotarget, 2018, 9(77):34543-34553, describe genetically grafting CD47 onto the membrane envelope of oncolytic HSV, allowing it to escape MPS.

[0014] There remains a need for oncolytic viruses that are suitable for systemic administration. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] U.S. Provisional Application No. 63 / 200,011 [Patent Document 2] U.S. Provisional Application No. 63 / 263,528 [Patent Document 3] U.S. Patent Publication No. 2012 / 0301506 [Patent Document 4] U.S. Patent No. 10,039,796 [Patent Document 5] U.S. Patent No. 8,986,672 [Non-patent literature]

[0016] [Non-Patent Document 1] Greig, 2016 [Non-Patent Document 2] Macedo et al., 2020 [Non-Patent Document 3] Fu et al., 2006 [Non-Patent Document 4] Nakamori et al., 2004

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[0017] The present invention is directed to oncolytic viruses suitable for systemic administration that are resistant to immune clearance in a subject. Described herein are methods for a series of modifications to be introduced into herpes simplex viruses, such as herpes simplex virus-1 (HSV-1) or herpes simplex virus-2 (HSV-2) based oncolytic viruses (such as FusOn-H2), that allow the virus to escape clearance and neutralization in a subject. These modifications include 1) serial passaging of FusOn-H2 in immune serum or serum containing high levels of anti-HSV antibodies, and optionally 2) inserting the extracellular domain of CD47, a molecule that contains the "don't eat me" signal, at the N-terminus of glycoprotein C (gC). As a result, the modified virus, FusOn-SD, becomes much more effective than the parent FusOn-H2 in infecting and lysing tumor cells when administered by systemic routes.

[0018] Detailed analysis of FusOn-SD revealed several novel results. A striking finding is the almost complete absence of gE from the viral particles. This allows the virus to escape from NK cell-mediated antiviral mechanisms. The absence of gE from the viral particles was only seen in CD47-containing viruses (e.g., FusOn-CD47) after serial passage in the presence of antiviral serum, but not in non-CD47-containing viruses (e.g., FusOn-gC-Luc) subjected to similar passage in the presence of antiviral serum. Another novel finding was that the insertion of the extracellular domain of CD47 into the N-terminus of gC also allowed the virus to escape neutralization by anti-HSV antibodies, even without passage. These modifications and the unexpected resulting changes significantly improved the capacity of oncolytic viruses in cancer therapy by systemic delivery, without compromising replication and safety.

[0019] One embodiment is a composition comprising an oncolytic herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), the oncolytic HSV1 or HSV-2 being prepared by passaging the oncolytic HSV1 or HSV-2 at least twice with immune serum having elevated levels of anti-HSV antibodies. In one preferred embodiment, the oncolytic HSV1 or HSV-2 has a membrane envelope comprising glycoproteins, where at least one glycoprotein comprises an extracellular CD47 domain (e.g., an extracellular CD47 domain comprising amino acids 19-141 of CD47 (SEQ ID NO: 21)) inserted at the N-terminus of the glycoprotein. The glycoprotein may be selected from glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, glycoprotein G, glycoprotein L, or any other viral membrane protein. For example, in one embodiment, the glycoprotein is glycoprotein C. Without being bound to any particular theory, the inventors believe that passaging in immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of oncolytic HSV-1 or HSV-2 or other viral genes or forces other changes in these membrane proteins.

[0020] In another embodiment, the immune serum is a mixture of rat serum and human serum with elevated levels of anti-HSV antibodies.

[0021] The composition comprising the oncolytic HSV-2 may be prepared by passage 7 times in the presence of rat serum with elevated levels of anti-HSV antibodies, followed by passage 17 times in the presence of a mixture of rat serum with elevated levels of anti-HSV antibodies and at least one human serum. The oncolytic HSV-2 may be prepared by passage 7 times in the presence of rat serum with elevated levels of anti-HSV antibodies, followed by passage 23 times in the presence of a mixture of rat serum with elevated levels of anti-HSV antibodies and at least one human serum.

[0022] In one embodiment, the oncolytic HSV-1 or oncolytic HSV-2 comprises a modified ICP10 coding region lacking nucleotides 1-1204 of the endogenous ICP10 coding region, the oncolytic HSV-1 or HSV-2 comprises the modified ICP10 operably linked to an endogenous or constitutive promoter and expresses a modified ICP10 peptide that lacks protein kinase (PK) activity but retains ribonucleotide reductase activity. Preferably, the oncolytic HSV-1 or HSV-2 is capable of selectively killing cancer cells.

[0023] In one embodiment of the compositions described herein, the composition comprises an oncolytic HSV-2 prepared by passaging the FusOn-H2 oncolytic virus.

[0024] In another embodiment, the oncolytic HSV1 or HSV-2 having an extracellular CD47 domain is free or substantially free of gE.

[0025] Another embodiment is a method for preparing a composition comprising oncolytic herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), comprising passaging the oncolytic herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2) at least twice with immune serum with elevated levels of anti-HSV antibodies. The oncolytic HSV-1 or HSV-2 to be passaged and the immune serum can be as described herein. The serum with elevated levels of anti-HSV antibodies can be obtained from an animal vaccinated with HSV (e.g., HSV-2). In one embodiment, the method comprises passaging the oncolytic HSV-1 or HSV-2 at least twice in the presence of rat serum, followed by at least one passaging in the presence of a mixture of rat serum with elevated levels of anti-HSV antibodies and at least one human serum. In another embodiment, the method comprises passaging the oncolytic HSV-1 or HSV-2 7 times in the presence of rat serum with elevated levels of anti-HSV antibodies, followed by 17 passagings in the presence of a mixture of rat serum with elevated levels of anti-HSV antibodies and at least one human serum. In yet another embodiment, the method comprises passaging the oncolytic HSV-2 7 times in the presence of rat serum with elevated levels of anti-HSV antibodies, followed by 23 passagings in the presence of a mixture of rat serum with elevated levels of anti-HSV antibodies and at least one human serum.

[0026] In a preferred embodiment of any of the compositions or methods described herein, the oncolytic HSV-2 that is passaged is the FusOn-H2 oncolytic virus.

[0027] In yet another preferred embodiment of the compositions or methods described herein, the oncolytic HSV-2 that is passaged is a FusOn-CD47 oncolytic virus.

[0028] Yet another embodiment is a method of treating cancer (e.g., metastatic cancer) in a patient in need of cancer treatment by HSV-based oncolytic virus therapy, comprising administering a composition described herein or an HSV-1 or HSV-2 oncolytic virus. In one embodiment, an effective amount of the composition is administered. In a preferred embodiment, the composition is administered systemically. In certain embodiments, the composition can be used to treat solid tumors. In one embodiment, the patient is vaccinated with HSV-1 and / or HSV-2. In another embodiment, the patient has HSV-1 and / or HSV-2.

[0029] For a more complete understanding of the present invention, including its features and advantages, reference is made to the following detailed description taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0030] [Figure 1] Figure 1 is a bar graph showing the differential sensitivity of trained and untrained FusOn-H2 to anti-HSV sera. 1x104 untrained FusOn-H2 or trained FusOn-SS9 were mixed with either human or rat anti-HSV-2 sera at a final concentration of 1:5. After 1 hour incubation at 37°C, the solution was plated onto a monolayer of Vero in a 12-well plate. Plaques were counted 48 hours after crystal violet staining. [Figure 2A]Schematic diagram of the construction strategy of FusOn-cd47. It shows that the insertion of the EGFP-Luc gene cassette and CMVp-HAtag-CD47ECD into the backbone of FusOn-H2 was performed via homologous recombination, with the 3' region of gC (transmembrane domain to polyA) as the left flanking (LF) sequence and the right flanking sequence. Details of the individual components of this gene cassette are shown in the figure and labelled accordingly. Abbreviations are as follows: CMVp, cytomegalovirus immediate early promoter; LTR, Rous sarcoma long terminal repeat containing the promoter region of this virus; GFP-Luc, EGFP-luciferase fusion gene; HA, HA tag; CD47, mouse CD47 extracellular domain; gC, complete gC coding region; LF, left flanking region of gC. Recombinant viruses were identified by GFP expression and purified to homogeneity. [Figure 2B] Schematic diagram of the construction strategy of FusOn-cd47. FusOn-Luc, used as a control virus, was constructed in a similar manner, except that it does not contain the CD47 extracellular domain. [Diagram 3] Graph showing a comparison of FusOn-CD47 and FusOn-Luc in their ability to evade clearance by phagocytes. This is a comparison of virus yield with and without phagocytes. Vero cells in 12-well plates were infected with either virus at 0.1 pfu / cell without or with 200,000 splenocytes. Cells were allowed to recover for 48 hours and virus yield was determined by plaque assay. [ka] p<0.05 compared with the other three wells. [Figure 4A]1 is a graph showing the average photon readings from daily IVIS imaging measurements, demonstrating that FusOn-CD47 can be delivered more efficiently than FusOn-Luc by systemic route in CT26 tumor model. Tumors were established in the right flank of Balb / c mice by subcutaneous implantation of CT26 cells. After tumors reached a size of approximately 8 mm in diameter, 2X106 pfu of either FusOn-CD47 or FusOn-Luc was administered systemically. [Figure 4B] Time-lapse images of a typical mouse several days after systemic delivery of FusOn-CD47. The CT26 tumor model shows that FusOn-CD47 can be delivered more efficiently than FusOn-Luc by systemic route. Tumors were established in the right flank of Balb / c mice by subcutaneous implantation of CT26 cells. After tumors reached a size of approximately 8 mm in diameter, 2X106 pfu of either FusOn-CD47 or FusOn-Luc were administered systemically. Animals were imaged daily from day 2 for luciferase expression. [Diagram 5] Figure 1 shows the improved resistance of FusOn-CD47 to the neutralizing effect of anti-HSV serum after culturing. 1x104 uncultivated FusOn-CD47 or cultivated FusOn-CD47-SS24 (7 passages in the presence of rat serum and 17 passages in the presence of rat serum and one human serum) were mixed with either human or rat anti-HSV-2 serum at a final concentration of 1:5. After 1 hour incubation at 37°C, the solution was plated on a monolayer of Vero in a 12-well plate. Plaques were counted 48 hours after crystal violet staining. [Figure 6]Figure 1 shows that cultivated FusOn-CD47 acquired further resistance to anti-HSV immune serum. 5x103 uncultivated FusOn-CD47 or cultivated different stages of the same virus [Serial Selection 24 (FusOn-CD47-SS24), Serial Selection 49 (HR49, N2-N5 and N7-N8)] were mixed at different dilutions with a mixture of 8 human sera. Cultured FusOn-H2 (FusOn-SS9) was also included in this experiment. After 1 h incubation at 37°C, the solution was plated on a monolayer of Vero in a 12-well plate. Plaques were counted 48 h after crystal violet staining. [Figure 7] 1 is a bar graph showing that CD47 insertion can enhance the ability of oncolytic HSV to escape neutralizing HSV antibodies. 500 pfu of FusOn-CD47 and FusOn-Luc were incubated with anti-HSV-2 serum at the indicated dilutions (1:40 or 1:160) for 1 h at 37° C. and then used to infect Vero cells in 6-well plates. As a control, the same number of viruses was incubated with medium alone. To count viral plaques, cells were stained with crystal violet after 48 h. The percentage of plaques was calculated by dividing the number of plaques in wells containing anti-HSV serum by the number of plaques from wells containing the same virus without anti-HSV serum (control). [ka] indicates p<0.05 compared to FusOn-Luc. [Figure 8]Time lapse images of a typical mouse several days after systemic delivery of FusOn-CD47, FusOn-Luc or FusOn-SD. FusOn-CD47, but not FusOn-Luc, was well delivered by the systemic route to tumor-bearing mice with pre-existing anti-HSV immunity, demonstrating that passaging FusOn-CD47 in serum containing anti-HSV (to generate FusOn-SD) further enhanced the ability of the virus to be delivered systemically in these mice. Balb / c mice were vaccinated with FusOn-H2 and then implanted with CT26 tumor cells in the right flank. Separate groups of mice received each of these three viruses at a dose of 1X107 pfu via the tail vein. Mice were imaged using an IVIS imager on the indicated days after virus injection. [Figure 9A] FIG. 1 is a bar graph showing absorbance at 450 nm of FusOn-H2, FusOn-SD, and PBS (negative control) following treatment with mouse anti-HSV-2 gE (1:1000 dilution) and HPR-conjugated rabbit anti-mouse IgG (1:10,000 dilution). [Figure 9B] Western blot showing detection of gE in FusOn-SD and FusOn-H2 samples after treatment with mouse anti-HSV-2 gE (1:1000 dilution) and HPR-conjugated rabbit anti-mouse IgG (1:10,000 dilution). [Figure 9C] FIG. 2 is a schematic diagram of the mechanism of gE-mediated NK cell recognition of HSV or HSV-infected cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] In describing the preferred embodiments of the invention shown in the drawings, specific terms are used for clarity. However, it is understood that the invention is not intended to be limited to the specific terms so selected, and each specific term includes all technical equivalents that operate in a similar manner to accomplish a similar purpose. Although several preferred embodiments of the invention have been described for illustrative purposes, it is understood that the invention may be embodied in other forms not specifically shown in the drawings.

[0032] definition

[0033] As used herein, the term "herpes simplex virus" or "HSV" refers to an enveloped, icosahedral, double-stranded DNA virus that infects mammals, including humans. Wild-type HSV infects and replicates in both terminally differentiated, non-dividing and dividing cells. "HSV-2" refers to a member of the HSV family that contains the ICP10 gene. As used herein, the term "FusOn-H2" refers to an HSV-2 variant that has a modified ICP10 polynucleotide encoding a polypeptide having ribonucleotide reductase activity and lacking protein kinase activity as described herein. FusOn-H2 and modified ICP10 polynucleotides are described in U.S. Patent Nos. 8,986,672 and 10,039,796, and U.S. Patent Publication No. 2015 / 0246086, which are incorporated herein by reference.

[0034] The terms "HSV-2 oncolytic virus" and "HSV-2 mutant" are used interchangeably herein.

[0035] As used herein, the terms "cell membrane fusion" and "fusion" refer to the fusion of the outer membranes of at least two cells, for example, two adjacent cells.

[0036] As used herein, the term "enhanced fusogenic activity" refers to an enhancement, increase, augmentation, enhancement, amplification, or combinations thereof of cell membrane fusion.

[0037] As used herein, the term "oncolytic" refers to the property of an agent that can directly or indirectly bring about the destruction of a malignant cell. In certain embodiments, this property includes fusing the membrane of a malignant cell with another membrane.

[0038] As used herein, the term "vector" refers to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a replicable cell. An inserted nucleic acid sequence is called "exogenous" when it is foreign to the cell into which the vector is introduced, or when it is homologous to a sequence within the cell, but not at a location within the host cell's nucleic acid where the sequence is not normally found. A vector can be either a non-viral DNA vector or a viral vector. A viral vector is packaged in viral proteins and is capable of infecting a cell. Non-limiting examples of vectors include viral vectors, non-viral vectors, naked DNA expression vectors, plasmids, cosmids, artificial chromosomes (e.g., YACS), phage vectors, DNA expression vectors associated with cationic condensing agents, DNA expression vectors packaged in liposomes, or certain eukaryotic cells, e.g., producer cells. Unless otherwise stated, as used herein, "vector" refers to both DNA vectors and viral vectors. Those skilled in the art will be knowledgeable in constructing vectors by standard recombinant techniques. Generally, these include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989), and references cited therein. Virological considerations are also reviewed in Coen DM, Molecular Genetics of Animal Viruses in Virology, 2nd Edition, BN Fields (editor), Raven Press, NY (1990), and references cited therein.

[0039] The term "expression vector" refers to any type of genetic construct that contains a nucleic acid that encodes a transcribable RNA. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences, described below, that also perform other functions.

[0040] A "promoter" is a control sequence, which is a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. It may contain genetic elements to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors that initiate or regulate the temporal and spatial transcription of a nucleic acid sequence. The terms "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control transcription initiation and / or expression of that sequence. Exemplary non-limiting promoters include constitutive promoters, tissue-specific promoters, tumor-specific promoters, or endogenous promoters under the control of exogenous inducible elements.

[0041] As used herein, the term "constitutive promoter" refers to a promoter that drives the expression of a gene or polynucleotide in a continuous temporal manner throughout the cell cycle. A constitutive promoter can be cell or tissue type specific, as long as it operates continuously throughout the cell cycle to drive the expression of the associated gene or polynucleotide. Exemplary non-limiting constitutive promoters include immediate early cytomegalovirus (CMV) promoter, SV40 early promoter, RSV LTR, beta chicken actin promoter, and HSV TK promoter.

[0042] The term "enhancer" refers to a cis-acting regulatory sequence involved in controlling the transcriptional activation of a nucleic acid sequence.

[0043] The phrase "modified ICP10 polynucleotide" refers to an ICP10 polynucleotide that encodes an ICP10 polypeptide that has ribonucleotide reductase (RR) activity but lacks protein kinase activity.

[0044] The phrase "ribonucleotide reductase activity" refers to the ability of the C-terminal domain of the polypeptide encoded by the ICP10 polynucleotide to generate sufficient deoxynucleotide triphosphates (dNTPs) required for viral replication.

[0045] The phrase "protein kinase activity" refers to the ability of the amino-terminal domain of a polypeptide encoded by an ICP10 polynucleotide to phosphorylate serine and threonine residues, which can activate the Ras / MEK / MAPK pathway.

[0046] As used herein, the term "effective" or "therapeutically effective" refers to inhibiting or inhibiting the worsening of a condition, inhibiting, inhibiting, or preventing the onset of a disease, inhibiting, inhibiting, or preventing the spread of a disease, ameliorating at least one symptom of a disease, or a combination thereof.

[0047] The term "patient" or "subject" refers to a mammal, such as a human or a domestic animal (e.g., a dog or cat). In a preferred embodiment, the patient or subject is a human.

[0048] The term "sera" or "serum" refers to the fluid from blood that remains when the blood cells and clotting proteins have been removed.

[0049] As used herein, the term "anti-cancer agent" refers to an agent that can adversely affect cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, slowing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, shrinking tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response to cancer cells or tumors, preventing or inhibiting the progression of cancer, or prolonging the survival of a subject with cancer.

[0050] As used herein, the terms "pharmaceutical" or "pharmaceutical acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal, or a human, as appropriate. The term "pharmaceutical acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0051] The term "unit dose" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen.

[0052] preface Viruses can only replicate in living cells, and their replication usually requires the activation of certain cellular signaling pathways. Many viruses have acquired various strategies over the course of evolution to activate these signaling pathways and aid in their replication. The large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10 or RR1) contains a unique amino-terminal domain with serine / threonine protein kinase (PK) activity. This PK activity has been shown to activate the cellular Ras / MEK / MAPK pathway (Smith, et al., (2000) J Virol 74(22):10417-29). As a result, it has been reported that deletion of this PK domain (ICP10PK) from the ribonucleotide reductase gene severely impairs the ability of the virus to replicate in cells, including those without a pre-existing activated Ras signaling pathway (Smith, et al., (1998) J. Virol. 72(11):9131-9141).

[0053] When the PK domain of HSV-2 is replaced and / or modified so that the protein encoded by the modified ICP10 gene has ribonucleotide reductase activity but lacks protein kinase activity, the virus selectively replicates in and destroys tumor cells (at least tumor cells in which the Ras signaling pathway is constitutively activated by tumorigenesis). Furthermore, the modification of the ICP10 polynucleotide described herein makes the virus essentially fusogenic, i.e., infection of tumor cells by the virus induces extensive cell membrane fusion (syncytium formation). This property enhances the destructive power of the virus against tumor cells. Furthermore, in vivo tests show that the virus is very safe for either local or systemic administration.

[0054] In some embodiments of the invention, modification of the PK domain includes the insertion of a reporter gene, such as one expressing the green fluorescent gene, and / or replacement of the native promoter gene with a constitutive promoter, such as the immediate early cytomegalovirus promoter.

[0055] In some embodiments, the HSV-2 is engineered by inserting a second polynucleotide into the polynucleotide encoding the protein kinase activity domain of the ICP10 gene, or by replacing a portion of the protein kinase domain with the second polynucleotide, so that the polypeptide encoded by the modified polynucleotide has ribonucleotide reductase activity but lacks protein kinase activity. For example, the second polynucleotide can encode a glycoprotein, such as a fusogenic membrane glycoprotein. A preferred glycoprotein for use within the scope of the present invention is a truncated form of gibbon ape leukemia virus envelope fusogenic membrane glycoprotein (GALV.fus). In a particular aspect of the present invention, in the context of the oncolytic virus of the present invention, the expression of GALV.fus significantly enhances the antitumor effect of the virus.

[0056] In some embodiments, the modified HSV-2 of the present invention comprises a mutation, such as a deletion in ICP10, that confers cell fusion properties to the virus. Such mutations may arise randomly during virus screening or may be obtained from nature, and the pool of potential candidates with cell fusion properties is then assayed for the function by means described herein and / or known in the art. The mutation that leads to the fusogenic phenotype may be a point mutation, a frameshift, an inversion, a deletion, a splicing error mutation, a post-transcriptional processing mutation, overexpression of a certain viral glycoprotein, a combination thereof, etc. The mutation may be identified by sequencing a particular HSV-2 and comparing it to a known wild-type sequence.

[0057] The modified HSV-2 of the present invention is useful for treating malignant cells, such as inhibiting their spread, reducing or inhibiting their division, eradicating them, preventing their generation or proliferation, or a combination thereof. The malignant cells can be derived from any form of cancer, such as solid tumors, although other forms can also be treated. The modified HSV-2 of the present invention is useful for treating lung, liver, prostate, ovarian, breast, brain, pancreatic, testicular, colon, head and neck, melanoma, and other types of malignant tumors. The present invention is useful for treating malignant cells at any stage of cancer disease, including the metastatic stage of the disease. The present invention can be used as a sole treatment or in conjunction with other treatment measures, including chemotherapy, surgery, or radiation.

[0058] Modified ICP10 polynucleotides The present invention describes HSV-2 mutants having modified ICP10 polynucleotides, which encode a polypeptide having ribonucleotide reductase activity but lacking protein kinase (PK) activity. The ICP10 polynucleotides can be modified by deleting at least a portion of the sequence required to encode a functional PK domain, or by replacing at least a portion of the sequence encoding the PK domain with a second polynucleotide. Those skilled in the art will recognize that any suitable method can be used to generate the modified ICP10 polynucleotides, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering technique known to those skilled in the art.

[0059] Mutagenesis In a particular embodiment of the invention, the ICP10 sequence of the HSV-2 virus is mutated, such as by deletion, using any of a variety of standard mutagenesis procedures. Mutations can include modifications of nucleotide sequences, single genes, or blocks of genes. Mutations can include single nucleotides (such as point mutations, including removal, addition, or substitution of a single nucleotide base in a DNA sequence) or can include insertions or deletions of multiple nucleotides. Mutations can occur naturally as a result of events such as errors in the fidelity of DNA replication, or can be induced after exposure to chemical or physical mutagens. Mutations can also be site-specific by using specific targeting methods well known to those skilled in the art.

[0060] Genetically modified In other embodiments of the invention, the ICP10 polynucleotide is modified using recombinant genetic techniques to delete or replace at least a portion of the sequence encoding the PK domain. The region of the PK domain that is deleted / replaced can be any suitable region, so long as the polypeptide encoded by the modified ICP10 polynucleotide retains ribonucleotide reductase activity and lacks protein kinase activity. In certain embodiments, however, the modification of the PK domain affects one or more of the eight PK catalytic motifs (amino acid residues 106-445, although the PK activity may also be considered as amino acid residues 1-445), and / or the transmembrane (TM) region, and / or the invariant Lys (Lys176). An exemplary wild-type ICP10 polypeptide sequence is provided in SEQ ID NO: 15 (GenBank database accession number 1813262A at the National Center for Biotechnology Information). An exemplary wild-type polynucleotide encoding an ICP10 polypeptide is provided in SEQ ID NO: 17.

[0061] In certain embodiments, the ICP10 polynucleotide is modified by simply deleting a portion of the sequence encoding the PK domain required for PK activity. An exemplary ICP10 polynucleotide lacking at least some sequence encoding the PK domain is provided in SEQ ID NO: 18. In another exemplary embodiment, the ICP10 polynucleotide is modified to delete the PK domain in its entirety, as provided in SEQ ID NO: 19. Both SEQ ID NO: 18 and SEQ ID NO: 19 encode polypeptides that have ribonucleotide reductase activity but lack protein kinase activity, and are therefore suitable for use in generating the HSV-2 mutants described herein. In certain embodiments of the invention, the modified ICP10 polynucleotide disclosed in SEQ ID NO: 18 or SEQ ID NO: 19 may be under the control of an endogenous HSV-2 promoter or may be operably linked to a constitutive promoter, such as the immediate early cytomegalovirus promoter set forth in SEQ ID NO: 20.

[0062] In yet another embodiment of the invention, the ICP10 polynucleotide is modified by replacing at least a portion of the sequence encoding the PK domain with a second polynucleotide, such as green fluorescent protein, which is positioned in frame with the sequence encoding the RR domain of the ICP10 polynucleotide. This construct may be under the control of an endogenous HSV-2 promoter or a constitutive promoter, such as the CMV promoter (SEQ ID NO: 20).

[0063] In another embodiment of the present invention, the polynucleotide that replaces at least a part of the protein kinase activity domain of endogenous ICP10 in HSV-2 may encode at least a fusogenic portion of a cell membrane fusion-inducing polypeptide, such as a viral fusogenic membrane glycoprotein (FMG), which is preferably capable of inducing cell membrane fusion at, for example, substantially neutral pH (e.g., about pH 6 to 8).

[0064] In certain embodiments, the FMG comprises at least the fusogenic domain from a C-type retroviral envelope protein, such as MLV (e.g., SEQ ID NO: 6) or GALV (e.g., SEQ ID NO: 5). Retroviral envelope proteins with some, most, or all of the cytoplasmic domain deleted are useful because such manipulations result in hyperfusogenic activity in human cells. In some embodiments, certain modifications are introduced into viral membrane glycoproteins to enhance their ability to induce cell membrane fusion. For example, truncation of the cytoplasmic domain of some retroviral and herpesviral glycoproteins has been shown to enhance their fusogenic activity, and may concomitantly reduce the efficiency of incorporation into virions (Rein et al., (1994) J Virol 68(3):1773-81).

[0065] Some examples of cell membrane fusion polypeptides include measles virus fusion protein (SEQ ID NO:7), HIV gp160 (SEQ ID NO:8) and SIV gp160 (SEQ ID NO:9) proteins, retroviral Env protein (SEQ ID NO:10), Ebola virus Gp (SEQ ID NO:11), and influenza virus hemagglutinin (SEQ ID NO:12).

[0066] In other embodiments, a second functional polynucleotide may be inserted into the PK domain or may be used to replace part or all of the PK domain. This second functional polynucleotide may code for an immunomodulatory or other therapeutic agent. These additional agents are intended to affect cell surface receptor and gap junction upregulation, cytostatic and differentiation agents, inhibit cell adhesion, or enhance the susceptibility of malignant cells to apoptosis. Illustrative non-limiting examples of polynucleotides encoding immunomodulatory or other therapeutic agents include tumor necrosis factor, interferon, alpha, beta, gamma, interleukin-2 (IL-2), IL-12, granulocyte macrophage colony stimulating factor (GM-CSF), F42K, MIP-1, MIP-1β, MCP-1, RANTES, herpes simplex virus-thymidine kinase (HSV-tk), cytosine deaminase, and caspase-3.

[0067] In yet other embodiments of the invention, the ICP10 polynucleotide is modified by the insertion of a polynucleotide encoding a reporter protein. Exemplary, non-limiting polynucleotides encoding reporter proteins include green fluorescent protein, enhanced green fluorescent protein, β-galactosidase, luciferase, and HSV-tk.

[0068] Ribonucleotide reductase activity assay Biological activity of RR can be detected as previously described (Averett, et al., J. Biol. Chem. 258:9831-9838 (1983) and Smith et al., J. Virol. 72:9131-9141 (1998)) with the following modifications. BHK cells are first grown to confluence in complete GMEM (containing 10% FBS) and then incubated in 0.5% FBS EMEM for 3 days, followed by infection with 20 pfu of wild-type HSV, HSV-2 mutants, or mock infection. Cells are harvested 20 hours post-infection, resuspended in 500 μl HD buffer [100 mM HEPES buffer (pH 7.6), 2 mM dithiothreitol (DTT)], and incubated on ice for 15 minutes, followed by 30 seconds of sonication. The cell debris is removed by centrifugation (16,000 g, 20 min, 4°C) and the supernatant is precipitated with 45% saturated crystalline ammonium sulfate (0.258 g / ml). After a second centrifugation (16,000 g, 30 min), the pellet is dissolved in 100 μl of HD buffer, from which 50 μl is removed and diluted with an equal volume of 2x reaction buffer (400 mM HEPES buffer (pH 8.0), 20 mM DTT and 0.02 mM [ 3 The solution is then mixed with [H]-CDP (24 Ci / mmol, Amersham, Chicago, Ill.). The reaction is terminated by adding 100 mM hydroxyurea containing 10 mM EDTA (pH 8.0) and boiling for 3 min. 1 ml of Crotalux atrox venom (Sigma, St. Louis, Mo.) is then added and incubated at 37°C for 30 min, followed by boiling for an additional 3 min. The solution is then passed through a 0.5 ml Dowex-1 borate column and the sample is eluted with 2 ml of water and collected in four elution fractions for scintillation counting after mixing with Biofluor (New England Nuclear, Boston, Mass.). Ribonucleotide reductase activity is expressed as units / mg protein, with 1 unit representing 1 nmol of [ 3 H]CDP to dCDP / hour / mg protein.

[0069] Protein kinase activity assay To determine whether the modified ICP10 polynucleotide encodes a polypeptide lacking protein kinase activity, extracts of cells infected with HSV-2 carrying the modified ICP10 polynucleotide or wild-type HSV-2 (moi=200, 16 hours post-infection) are immunoprecipitated with anti-LA-1 antibody and subjected to the PK assay described in Chung et al. J. Virol. 63:3389-3398, 1998 and U.S. Patent No. 6,013,265. Generally, immunoprecipitates of cell extracts are standardized for protein concentration using a BCA protein assay kit (PIERCE, Rockford Ill.), washed with TS buffer containing 20 mM Tris-HCL (pH 7.4), 0.15 M NaCl, and resuspended in 20 mM Tris-HCL (pH 7.4), 5 mM MgCl2, 2 mM MnCl2, 10 μCi of [ 32 The beads are suspended in 50 μl of kinase reaction buffer consisting of [p]ATP (3000 Ci / mmol, DuPont, New England Research Prod.) and incubated at 30° C. for 15 minutes. The beads are washed once with 1 ml of TS buffer, resuspended in 100 μl of denaturing solution, and boiled for 5 minutes. The proteins are then separated by SDS-PAGE on a 7% polyacrylamide gel. The proteins are then electrophoretically transferred onto nitrocellulose membranes as previously described (see Aurelian et.al., Cancer Cells 7:187-191 1989) and immunoblotted by incubating with specific antibodies followed by protein A-peroxidase (Sigma, St. Louis, Mo.) for 1 hour at room temperature. Detection can be performed using ECL reagents (Amersham, Chicago, Ill.) as described in Smith et al., Virol. 200:598-612, (1994).

[0070] Vector construction The present invention is directed to HSV-2 vectors that contain a substitution or deletion of at least a portion of the ICP10 sequence such that the protein encoded by the modified ICP10 polynucleotide has ribonucleotide reductase activity but lacks protein kinase activity, and in certain embodiments, further contain regulatory sequences, such as a constitutive promoter. In some embodiments, the composition is a naked (non-viral) DNA vector that contains the modified ICP10 gene, and in other embodiments, the composition is a recombinant HSV-2 carrying the modified ICP10 gene. Both the naked DNA vector and the recombinant virus may further be comprised of some or all of the following components:

[0071] vector Vectors as defined above include, but are not limited to, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Methods for the construction of engineered viruses and DNA vectors are known in the art. In general, these include Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989), and references cited therein. Virological considerations are also reviewed in Coen DM, Molecular Genetics of Animal Virus in Virology, 2.sup.nd Edition, BN Fields (editor), Raven Press, NY (1990), and references cited therein.

[0072] Expression vectors can contain a variety of "control sequences," which refers to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, DNA vectors, expression vectors, and viruses can contain nucleic acid sequences, described below, that serve other functions as well.

[0073] 1. Promoters and enhancers Promoters generally contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters that lack a TATA box (e.g., the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter), separate elements that overlap the start site themselves help determine the start location. Additional promoter elements regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but some promoters have been found to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. This "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[0074] Spacing between promoter elements is often flexible, so that elements can be inverted or moved relative to one another while still retaining promoter function. In the tk promoter, promoter elements can be spaced as much as 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription. Promoters may or may not be used in conjunction with enhancers.

[0075] A promoter may be one that is naturally associated with a nucleic acid sequence, as may be obtained by isolating 5' non-coding sequences located upstream of a coding segment and / or exon. Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence located either downstream or upstream of that sequence. Alternatively, certain advantages are obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or from prokaryotic or eukaryotic cells, and promoters or enhancers that are not "naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. For example, the most commonly used promoters in recombinant DNA construction include the β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in conjunction with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0076] Of course, it is important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, as is advantageous in large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0077] Furthermore, any promoter / enhancer combination may be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional gene expression construct.

[0078] The identity of tissue-specific promoters or elements, and assays to characterize their activity, are well known to those of skill in the art. Non-limiting examples of such regions include the human LIMK2 gene (Nomoto et al. (1999) Gene 236(2):259-271), the somatostatin receptor-2 gene (Kraus et al., (1998) FEBS Lett. 428(3):165-170), the mouse epididymal retinoic acid binding gene (Lareyre et al., (1999) J. Biol. Chem. 274(12):8282-8290), human CD4 (Zhao-Emonet et al., (1998) Biochem. Biophys. Acta, 1442(2-3):109-119), mouse alpha-2(XI) collagen (Tsumaki, et al., (1999) J. Biol. Chem. 274(12):8282-8290), and the human 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) (IL-19)). al., (1998), J. Biol. Chem. 273(36):22861-4), D1A dopamine receptor gene (Lee, et al., (1997), DNA Cell Biol. 16(11):1267-1275), insulin-like growth factor II (Vu et al., (1997) Biophys Biochem Res. Comm. 233(1):221-226), and human platelet endothelial cell adhesion molecule-1 (Almendro et al., (1996) J. Immunol. 157(12):5411-5421).

[0079] 2. Initiation signals and internal ribosome binding sites Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. It may also be necessary to provide exogenous translational control signals, including the ATG initiation codon. One of ordinary skill in the art would be readily able to determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. The exogenous translational control signals and initiation codons may be natural or synthetic. The efficiency of expression may be improved by including appropriate transcriptional enhancer elements.

[0080] In certain embodiments of the invention, the use of internal ribosome entry site (IRES) elements is used to create multigene or polycistronic messengers. IRES elements can bypass the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at an internal site. IRES elements can link heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating a polycistronic messenger. The IRES element allows each open reading frame access to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single messenger (see U.S. Patent Nos. 5,925,565 and 5,935,819).

[0081] 3. Termination signal The vector or construct of the present invention generally comprises at least one termination signal. A "termination signal" or "terminator" is composed of a DNA sequence that is involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary to achieve a desired level of messaging in vivo.

[0082] In eukaryotic systems, the terminator region may also contain specific DNA sequences that allow site-specific cleavage of the new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail are likely to be more stable and are translated more efficiently. Therefore, in other embodiments involving eukaryotes, it is contemplated that the terminator contains a signal for RNA cleavage, and that the terminator signal promotes polyadenylation of the messenger. The terminator and / or polyadenylation site elements may help to increase messenger levels and minimize read-through from the cassette to other sequences.

[0083] Terminators contemplated for use in the present invention include any known transcription terminator described herein or known to one of skill in the art, including, but not limited to, a genetic termination sequence, such as the bovine growth hormone terminator, or a viral termination sequence, such as the SV40 terminator, etc. In certain embodiments, the termination signal may be the absence of a transcribable or translatable sequence, for example, due to sequence truncation.

[0084] 4. Polyadenylation Signal In expression, particularly in eukaryotic expression, polyadenylation signals are usually included to ensure proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful implementation of the present invention, and any such sequence may be used. Preferred embodiments include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, both of which are easy to use and known to function well in a variety of target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

[0085] 5. Selectable and Screenable Markers In certain embodiments of the present invention, cells containing the nucleic acid constructs of the present invention can be identified in vitro or in vivo by including a marker in the expression vector. Such a marker confers a distinguishable change to the cell that facilitates the identification of cells containing the expression vector. In general, a selection marker is one that confers a property that allows for selection. A positive selection marker is one whose presence allows for its selection, and a negative selection marker is one whose presence prevents its selection. An example of a positive selection marker is a drug resistance marker.

[0086] Typically, the inclusion of a drug selection marker aids in the cloning and identification of transformants. For example, genes that confer neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol resistance are useful selection markers. In addition to markers that confer a phenotype that allows for the identification of transformants based on the implementation of conditions, other types of markers are also contemplated, including screenable markers such as GFP based on colorimetry. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used. Those skilled in the art will also know how to use immunological markers, perhaps in conjunction with fluorescence activated cell sorting (FACS) analysis. The marker used is not believed to be critical, so long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selectable and screenable markers are well known to those skilled in the art.

[0087] The vector is introduced into the originally infected cell by an appropriate method. Such methods for nucleic acid delivery for transformation of an organelle, cell, tissue or organism for use in the present invention are intended to include virtually any method described herein or known to one of skill in the art by which a nucleic acid (e.g., an HSV vector) can be introduced into an organelle, cell, tissue or organism. Non-limiting exemplary methods include: direct delivery of DNA by ex vivo transfection, injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859), microinjection (U.S. Pat. No. 5,789,215), electroporation (U.S. Pat. No. 5,384,253), calcium phosphate precipitation, DEAE dextran followed by polyethylene glycol, direct ultrasound loading, liposome-mediated transfection, receptor-mediated transfection, particle bombardment (PCB), and the like. Nos. WO 94 / 09699 and 95 / 06128, U.S. Patent Nos. 5,610,042, 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880), agitation with silicon carbide fibers (U.S. Patent Nos. 5,302,523 and 5,464,765), Agrobacterium mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055), PEG mediated transformation of protoplasts (U.S. Patent Nos. 4,684,611 and 4,952,500), desiccation / inhibition mediated DNA uptake, and any combination of these methods or other methods known to those of skill in the art. The composition can also be delivered to mammalian cells by administering it systemically, for example, intravenously, in a pharma- ceutically acceptable vehicle.

[0088] Methods for delivering DNA vectors into cells 1. Ex Vivo Transformation Methods for transfecting cells and tissues removed from an organism in an ex vivo setting are known to those skilled in the art. Thus, it is contemplated in the present invention that cells or tissues may be removed and transfected ex vivo using the nucleic acids and compositions described herein. In certain aspects, the transplanted cells or tissues may be placed in an organism. In some embodiments, the nucleic acid is expressed in the transplanted cells or tissues.

[0089] 2. Injection In certain embodiments, the nucleic acid can be delivered to an organelle, cell, tissue or organism via one or more injections (i.e., needle injections), such as subcutaneous, intradermal, intramuscular, intravenous, intraperitoneal, etc. Methods of injection are well known to those skilled in the art (e.g., injection of a composition containing saline). Further embodiments of the invention include the introduction of nucleic acid by direct microinjection. The amount of the composition of the invention used can vary depending on the nature of the affected cell, tissue or organism.

[0090] 3. Electroporation In certain embodiments of the invention, nucleic acids are introduced into organelles, cells, tissues or organisms via electroporation. Electroporation involves exposing a suspension of cells and DNA to a high voltage electrical discharge. In some variations of this method, certain cell wall degrading enzymes, e.g., pectin degrading enzymes, are used to make the target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Patent No. 5,384,253). Alternatively, recipient cells can be made susceptible to transformation by mechanical wounding.

[0091] 4. Liposome-mediated Transfection In further embodiments of the present invention, the compositions described herein, e.g., vectors carrying modified ICP10 polynucleotides, can be incorporated into lipid complexes, such as liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form naturally when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-reorganization to form a closed structure, incorporating water and dissolved solutes between the lipid bilayers. Also contemplated are nucleic acids complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen).

[0092] In certain embodiments of the invention, the liposome may be complexed with hemagglutinating virus (HVJ), which has been shown to promote fusion with cell membranes and facilitate cellular import of liposome-encapsulated DNA (Kaneda et al., (1989) Science 20;243(4889):375-8). In other embodiments, the liposome may be complexed or combined with nuclear non-histone chromosomal proteins (HMG1) (Kato et al., (1991) J Biol Chem. (1991) February 25;266(6):3361-4). In further embodiments, the liposome may be complexed or combined with both HVJ and HMG1. In other embodiments, the delivery vehicle may include a ligand and a liposome.

[0093] 5. Receptor-mediated Transfection Nucleic acids can be delivered to target cells via receptor-mediated delivery vehicles. This approach takes advantage of the selective uptake of macromolecules by receptor-mediated endocytosis. Given the cell type-specific distribution of various receptors, this delivery method adds another degree of specificity to the present invention.

[0094] In certain embodiments, the receptor-mediated gene targeting vehicle comprises a receptor-specific ligand and a nucleic acid binding agent. Other embodiments comprise a receptor-specific ligand operably linked to the nucleic acid to be delivered. Several ligands have been used in receptor-mediated gene transfer, including epidermal growth factor (EGF), which has been used to deliver genes to squamous cell carcinoma cells, as described in European Patent No. EPO 0 273 085.

[0095] In other embodiments, the nucleic acid delivery vehicle component of the cell-specific nucleic acid targeting vehicle may include a specific binding ligand in combination with liposome. The nucleic acid(s) to be delivered are contained within the liposome, and the specific binding ligand is functionally incorporated into the liposome membrane. Thus, the liposome specifically binds to the receptor(s) of the target cell and delivers the contents to the cell.

[0096] In a further embodiment, the nucleic acid delivery vehicle component of the targeted delivery vehicle may be liposome itself, which preferably comprises one or more lipids or glycoproteins that direct cell-specific binding.For example, lactosylceramide, a galactose-terminal asialoganglioside, was incorporated into liposomes, and increased uptake of insulin gene by hepatocytes was observed (Nicolau et al., (1987) Methods Enzymol.149:157-76).It is contemplated that the tissue-specific transformation construct of the present invention can be specifically delivered to target cells in a similar manner.

[0097] 6. Microparticle gun Using microprojectile bombardment technology, nucleic acids can be introduced into at least one organelle, cell, tissue or organism (US Pat. No. 5,550,318, US Pat. No. 5,538,880, US Pat. No. 5,610,042, and PCT Application WO94 / 09699). This method relies on the ability to accelerate microprojectiles coated with or containing DNA to high velocities, allowing them to break through cell membranes and enter cells without killing them. The microprojectiles can be composed of any biologically inert material, such as tungsten, platinum, or gold. For bombardment, the cells contained in the suspension are concentrated on a filter or solid medium. Alternatively, immature embryos or other target cells may be placed on solid medium. The cells to be bombarded are placed on a stopping plate at an appropriate distance below the microprojectile bombardment device. A wide variety of microprojectile bombardment technologies useful in the practice of the present invention will be known to those skilled in the art.

[0098] host cell As used herein, the terms "cell", "cell line" and "cell culture" may be used interchangeably. All of these terms include their progeny, any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or unintended mutations. In the context of expressing heterologous nucleic acid sequences, a "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector and / or expressing a heterologous gene encoded by the vector. A host cell can be used and has been used as a recipient of a vector. A host cell may be "transfected" or "transformed", which refers to the process by which an exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence, e.g., a vector, has been introduced. Thus, a recombinant cell is distinguishable from a naturally occurring cell that does not contain a recombinantly introduced nucleic acid.

[0099] The tissue may include host cells or cells transformed with cell membrane fusion-producing HSV-2 mutants. The tissue may be part of an organism or may be separated from an organism. In certain embodiments, the tissue may include, but is not limited to, adipocytes, alveoli, ameloblasts, nerve cells, basal cells, blood (e.g., lymphocytes), blood vessels, bone, bone marrow, glial cells, breast, cartilage, cervix, colon, cornea, embryo, endometrium, endothelium, epithelium, esophagus, fascia, fibroblasts, follicles, ganglion cells, glial cells, goblet cells, kidney, liver, lung, lymph nodes, muscle, neurons, ovaries, pancreas, peripheral blood, prostate, skin, small intestine, spleen, stem cells, stomach, testes, and all cancers thereof.

[0100] In certain embodiments, the host cell or tissue can be contained in at least one organism.In certain embodiments, the organism can be, but is not limited to, a prokaryote (e.g., eubacteria, archaea) or a eukaryote, as will be understood by those skilled in the art.

[0101] Numerous cell lines and cultures are available as host cells and are commercially available through organizations such as the American Type Culture Collection (ATCC). Appropriate hosts can be identified by one of skill in the art based on the vector backbone and the desired results. Exemplary non-limiting cell types available for vector replication and / or expression include bacteria, such as E. coli (e.g., E. coli strains RR1, LE392, B, X 1776 (ATCC No. 31537), W3110, F, lambda, DH5α, JM109, and KC8), bacilli, such as Bacillus subtilis, other enterobacteria, such as Salmonella typhimurium, Serratia marcescens, as well as several commercially available bacterial hosts and competent cells, such as SURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla, Calif.). Non-limiting examples of eukaryotic host cells for replication and / or expression of vectors include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12.

[0102] Some vectors may use control sequences that allow them to replicate and / or be expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the above host cells are incubated to maintain them and allow replication of the vectors. Also understood and known are the techniques and conditions that allow for large-scale production of vectors, as well as the production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides.

[0103] Packaging and propagation of viral vectors 1. Viral Packaging In a specific embodiment of the present invention, the ICP10 gene is modified and then inserted into the virus by homologous recombination. Typically, this is done by co-transfecting Vero cells with plasmid DNA containing the modified ICP10 gene and purified HSV-2 genomic DNA using lipofectamine. The recombinant virus is then identified (usually by screening viral plaques for the presence of a selection marker) and plaques containing the modified ICP10 polynucleotide are selected. The selected recombinant virus is then characterized in vitro to confirm that the modified ICP10 gene is correctly inserted into the HSV-2 genome and replaces the original ICP10 gene.

[0104] 2. Viral Stock Preparation After the recombinant HSV-2 mutant virus is selected, virus stocks can be prepared as follows: Vero cells are grown in 10% fetal bovine serum (FBS) and infected with 0.01 plaque-forming units (pfu) per cell. Then, after 2 days, the virus is harvested from the cells by repeated freezing and thawing and sonication. The harvested virus is then purified as described (Nakamori, et al., (2003) Clinical Cancer Res. 9(7):2727-2733). The purified virus is then titered, aliquoted, and stored at -80°C until use.

[0105] Protein Expression Systems Protein expression systems can be used in the production of the DNA vector compositions of the invention, for example, to express the polypeptides encoded by the modified ICP10 polynucleotides for functional testing. Numerous expression systems exist that contain at least some or all of the compositions described above. Prokaryotic and / or eukaryotic based systems can be used in the present invention to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially available and widely available.

[0106] The insect cell / baculovirus system is capable of producing high level protein expression of heterologous nucleic acid segments, as described, for example, in U.S. Pat. Nos. 5,871,986 and 4,879,236, and is commercially available (e.g., CLONTECH, Inc. Mountain View, Calif.).

[0107] Other examples of commercially available expression systems include an inducible mammalian expression system containing a synthetic ecdysone-inducible receptor, or the pET expression system, or an E. coli expression system (STRATAGENE, LaJolla, Calif.), a tetracycline-regulated expression system, an inducible mammalian expression system using the full-length CMV promoter, or a yeast expression system designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica (INVITROGEN, Carlsbad, Calif.).

[0108] It is contemplated that the protein, polypeptide or peptide produced by the method of the invention may be "overexpressed", i.e., expressed at a higher level compared to its native expression in the cell. Such overexpression may be assessed by a variety of methods, including radiolabeling and / or protein purification. However, simple and straightforward methods are preferred, including, for example, SDS / PAGE and protein staining or Western blotting, followed by quantitative analysis such as densitometric scanning of the resulting gel or blot. A specific increase in the level of a recombinant protein, polypeptide or peptide compared to the level in a native cell is indicative of overexpression, as is the relative abundance, visible, for example, on a gel, of the particular protein, polypeptide or peptide with respect to other proteins produced by the host cell.

[0109] Functional roles of HSV-2 mutants The HSV-2 mutants described herein exhibit multiple functional roles as oncolytic agents. For example, the virus can destroy tumor cells by lysis and by inducing syncytium formation and apoptosis in both infected and bystander cells. Furthermore, tumor destruction by HSV-2 mutants induces a strong antitumor immune response, which further contributes to the therapeutic efficacy of the mutant viruses as oncolytic agents for the treatment of malignant diseases.

[0110] HSV-2 mutant viruses exhibit selective replication in cycling but not non-cycling cells. Mutant HSV-2 lacking protein kinase activity exhibits at least a 40-fold decrease in growth in non-cycling cells compared to growth in cycling cells. In contrast, wild-type HSV-2 has only a minor effect on the growth characteristics between cycling and non-cycling cells. Thus, the HSV-2 mutants described herein are suitable for use as oncolytic agents in cycling cells with an activated Ras pathway, such as tumor cells.

[0111] In addition to lytic and fusogenic activity, HSV-2 mutants also have strong apoptosis-inducing activity and can induce strong anti-tumor immune responses. In an in vitro setting, the HSV-2 mutants can induce apoptosis not only in virus-infected cells but also in uninfected bystander cells surrounding the infected cells. Furthermore, the HSV-2 mutants are effective in inducing apoptosis of tumor cells in vivo. Not only are the compositions described herein more effective at killing tumor cells than other oncolytic viruses, but the HSV-2 mutants also show strong therapeutic effects against primary and metastatic tumors in vivo due to the induction of strong anti-tumor immune responses. Adoptively transferred CTLs from FusOn-H2-treated mice can inhibit the growth of the original tumor and effectively prevent the occurrence of metastasis.

[0112] Apoptosis, or programmed cell death, is a process essential for normal embryonic development, maintaining homeostasis in adult tissues, and suppressing carcinogenesis. In some embodiments of the present invention, the modified HSV-2 is a potent inducer of apoptosis in virus-infected tumor cells and uninfected bystander tumor cells. For example, in certain embodiments, tumor cells are infected with an HSV-2 construct in which a portion of the protein kinase domain of the ICP10 gene is replaced with a gene encoding green fluorescent protein (GFP). Infected cells can be identified under a fluorescent microscope by visualizing GFP, and cells undergoing apoptosis were identified by evidence of chromatin condensation. The ratio of cells showing chromatin condensation to GFP expression was 2.6:1, suggesting that a significant number of tumor cells not infected with modified HSV-2 are undergoing apoptosis.

[0113] A strong anti-tumor immune response is useful for the treatment of malignant diseases. The HSV-2 mutant described herein is capable of inducing a strong anti-tumor immune response against primary and metastatic tumors in vivo. In a particular embodiment, the mutant HSV-2 (FusOn-H2) selectively replicates in a mouse mammary tumor model using 4T1 mouse mammary tumor cell line, lyses tumor cells, and shows a strong therapeutic effect against primary and metastatic tumors in vivo by inducing a strong anti-tumor immune response. Specifically, cytotoxic T lymphocytes (CTLs) adoptively transferred from FusOn-H2-treated mice can inhibit the growth of the original tumor in mice not treated with FusOn-H2 and effectively prevent metastasis.

[0114] Pharmaceutical Compositions and Routes of Administration The compositions of the present invention may be administered as pharmaceutical compositions comprising either recombinant HSV-2 mutants with modified ICP10 gene or naked (non-viral) DNA vectors with modified ICP10 gene as described herein. The compositions of the present invention comprise standard pharmaceutical formulations. In general, the compositions of the present invention may be administered as pharmacologically active substances by dissolving or dispersing the compositions in a pharmacologic carrier or aqueous medium. The use of such media and agents for pharmacologic active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compositions of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients, such as other anti-disease agents, may also be incorporated into the pharmaceutical composition. The administration of the composition is via any common route, so long as the target cells are available via that route. Exemplary routes of administration include oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or by direct intratumoral injection. The pharmaceutical formulations, dosages and routes of administration of the compositions of the present invention are described below.

[0115] Formulation of HSV-2 mutants The mutant virus composition of the present invention can be prepared as a pharmacologically acceptable formulation. Usually, the mutant virus is mixed with an excipient that is pharma- ceutically acceptable and compatible with the virus. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if necessary, the preparation may contain small amounts of auxiliary substances that enhance the effectiveness of the virus mutant, such as wetting or emulsifying agents, pH buffers, adjuvants, or immuno-enhancing agents (see Remington's Pharmaceutical Sciences, Gennaro, AR et al., eds., Mack Publishing Co., pub., 18th ed., 1990). For example, a typical pharma-ceutically acceptable carrier for injection purposes may contain 50 mg to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Further non-limiting exemplary non-aqueous solvents suitable for use in the formulation of pharmacologically acceptable compositions include propylene glycol, polyethylene glycol, vegetable oil, sesame oil, peanut oil, and injectable organic esters, such as ethyl oleate.Exemplary non-limiting aqueous carriers include water, aqueous solutions, saline, parenteral vehicles, such as sodium chloride, Ringer's dextrose, and the like.Intravenous vehicles include fluid and nutrient supplements.The determination of pH and exact concentration of various components of the pharmaceutical composition is routine and within the knowledge of those skilled in the art (see Goodman and Gilman's The Pharmacological Basis for Therapeutics, Gilman, AGet al., eds., Pergamon Press, pub., 8th ed., 1990).

[0116] Sterile injectable solutions are prepared by incorporating the required amount of the active compound in the appropriate solvent with various other sterile ingredients as required above. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium above and the required other ingredients.

[0117] Route and dose of administration of HSV-2 mutants The mutant virus composition can be delivered by any route that provides access to the target tissue.Exemplary non-limiting administration routes may include oral, nasal, oral, rectal, vaginal, topical, or by injection (including orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or direct intratumoral injection).Usually, the virus mutant is prepared as an injectable material, either as a solution or suspension, and solid forms suitable for dissolving or suspending in liquid before injection can also be prepared.The preparation may be emulsified.

[0118] For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered if necessary, and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection fluid or injected at the intended injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject.

[0119] Those skilled in the art will recognize that the best treatment regimen for using the compositions of the present invention to provide therapy can be determined directly. This is not a matter of experimentation, but rather an optimization problem routinely performed in the medical field. For example, in vivo testing in mice provides a starting point to begin optimizing the dose and delivery regime. The frequency of injections can be once a week initially. However, this frequency may be optimally adjusted from once a day to every two weeks to monthly, depending on the results obtained from the initial clinical trials and the needs of the particular patient. The human dosage can be determined by extrapolating from the amount of composition initially used in mice.

[0120] dose The amount of viral vector delivered will depend on several factors, including the number of treatments, the subject being treated, the capacity of the subject's immune system to synthesize anti-viral antibodies, the target tissue to be destroyed, and the degree of protection desired. The precise amount of viral composition to be administered will depend on the physician's judgment and will be unique to each individual. However, appropriate doses may range from 10 to 20 mg / kg. 5 Plaque forming units (pfu) ~10 10 In certain embodiments, the dose of viral DNA ranges from about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 It may be less than or equal to pfu.

[0121] Non-viral DNA vector formulations In addition to the above-mentioned preparation for viral formulation, non-viral DNA vector can also be prepared as sterile powder for preparing pharmacologically acceptable sterile solution.The representative method for preparing sterile powder includes vacuum drying and freeze-drying technology, which allows the powder of active ingredient and any additional desired ingredient to be obtained from its solution that has been previously sterile filtered.

[0122] Route and dosage of non-viral DNA vectors Several methods are contemplated for the delivery of non-viral vectors for the transfer of the polynucleotide of the present invention into mammalian cells.These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene gun using high-velocity microprojectiles, and the aforementioned receptor-mediated transfection.Some of these techniques can be well adapted for in vivo or ex vivo use.

[0123] In some embodiments of the present invention, the expression vector can simply be composed of naked recombinant DNA or plasmid containing the polynucleotide.The transfer of the construct can be carried out by any of the methods described herein, which physically or chemically permeabilize cell membrane.This is particularly applicable to transfer in vitro, but may also be applied to in vivo use.

[0124] In other embodiments, the delivery vehicle may include a ligand and a liposome. For example, Nicolau et al. used lactosylceramide, a galactose-terminal asialoganglioside, incorporated into liposomes and observed increased uptake of the insulin gene by liver cells (Nicolau et al., (1987) Methods Enzymol. 149:157-76). Thus, it is possible that nucleic acids encoding specific genes can be similarly delivered to cell types in a specific manner by a number of receptor-ligand systems, with or without liposomes. For example, epidermal growth factor (EGF) may be used as a receptor for mediated delivery of nucleic acids to cells that show upregulation of EGF receptors (as described in European Patent No. EP 0 273 085), and mannose can be used to target the mannose receptor of liver cells.

[0125] In certain embodiments, DNA transfer may be more easily performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of nucleic acid to the cells in vitro, and then returning the modified cells to the animal. This may involve surgical removal of tissues / organs from an animal or primary culture of cells and tissues.

[0126] dose In certain embodiments, the dose is about 10 3 pfu / kg body weight ~ approx. 10 8 It is contemplated that the dose may vary between about 10 pfu / kg body weight. In certain embodiments, the dose is about 10 3 , 10 4 , 10 5 , 10 6 , 10 7, 10 8 The dosage may be up to or including pfu / kg body weight. Of course, this dosage may be adjusted upwards or downwards depending on the results of initial clinical trials and the needs of the particular patient, as is routinely done in such treatment protocols.

[0127] Combination treatment To enhance the effectiveness of the methods and compositions of the present invention, it may be desirable to combine the methods and compositions disclosed herein with other anti-cancer drugs. This process may include contacting the cancer cells with the compositions of the present invention in conjunction with at least one other anti-cancer drug. This may be accomplished by contacting the cells with a single composition or pharmacological formulation that includes both drugs, or by contacting the cells with two separate compositions or formulations. When two separate formulations are used, the cancer cells may be contacted with both formulations at the same time, or one formulation may precede the other (e.g., the composition of the present invention is administered before or after the administration of another anti-cancer drug), or any combination or repeated cycle thereof. In embodiments in which the composition of the present invention and the other drug are administered separately, there is usually a long period of time between each delivery time that does not expire, so that the composition of the present invention and the other drug can still exert a beneficial combined effect on the cancer cells. This time interval between the administration of the two formulations may range from a few minutes to a few weeks.

[0128] Non-limiting examples of anti-cancer agents that may be used in conjunction with the compositions or methods of the invention include chemotherapeutic agents (e.g., cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosoureas, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, and the like. These may include combination drugs, taxol, gemcitabine, navelbine, farnesyltransferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or analog or derivative variants of any of the foregoing, radiotherapy drugs (e.g., gamma radiation, X-ray, microwave and UV radiation, and / or directed delivery of radioisotopes to tumor cells), immunotherapy and immunomodulatory agents, gene therapy drugs, pro-apoptotic agents, and other cell cycle regulators known to those of skill in the art.

[0129] Immunotherapy can also be used in conjunction with the compositions and methods described herein as a combination therapy for the treatment of malignant diseases. Immunotherapy generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector can be, for example, an antibody specific for some marker on the surface of tumor cells. The antibody can function alone as an effector of therapy or can recruit other cells (e.g., cytotoxic T cells or NK cells) to actually effect cell killing. The antibody can also be conjugated to a drug or toxin (e.g., chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply function as a targeting agent. In some embodiments, the effector can be a lymphocyte carrying a surface molecule that directly or indirectly interacts with the tumor cell target. In other embodiments, the tumor cell must bear some marker suitable for targeting. Non-limiting exemplary tumor markers suitable for targeting may include carcinoembryonic antigen (CEA), prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155.

[0130] In one preferred embodiment, checkpoint blockade immunotherapy is used in combination with the compositions and methods described herein. Checkpoint blockade immunotherapy can be performed by administering checkpoint inhibitors such as PD-L1 inhibitors (such as atezolizumab, avelumab, and durvalumab), PD-1 inhibitors (such as pembrolizumab, nivolumab, and cemiplimab), or CTLA-4 inhibitors (such as ipilimumab), or by adoptive T cell transfer.

[0131] Gene therapy can also be used in conjunction with the compositions and methods described herein as a combination therapy for the treatment of malignant diseases. Gene therapy as a combination therapy relies on the delivery and expression of a therapeutic gene separate from the mutant HSV-2 described herein. The gene therapy can be administered before, after, or simultaneously with the HSV-2 mutants described herein. Exemplary non-limiting targets for gene therapy include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiating agents, inhibitors of cell adhesion, or agents that induce or increase the susceptibility of target cells to apoptosis. Exemplary non-limiting immunomodulatory genes that can be used as part of gene therapy in combination with the present invention include tumor necrosis factor, interferon alpha, beta, and gamma, IL-2 and other cytokines, F42K and other cytokine analogs, or MIP-1, MIP-1 beta, MCP-1, RANTES, and other chemokines.

[0132] An example of an inhibitor of cell proliferation is p16. The major transitions of the eukaryotic cell cycle are triggered by cyclin-dependent kinases, i.e., CDKs. One of the CDKs, cyclin-dependent kinase 4 (CDK4), regulates the progression of G1. The activity of this enzyme may be to phosphorylate Rb in late G1. The activity of CDK4 is controlled by the activating subunit, D-type cyclin, and by the inhibitory subunit, p16INK4, which specifically binds and inhibits CDK4, and thus may regulate Rb phosphorylation. The p16INK4 gene belongs to a newly described class of CDK inhibitor proteins that also includes p16B, p19, p21WAF1, and p27KIP1. Homozygous deletions and mutations of the p16INK4 gene are commonly found in human tumor cell lines. Since p16INK4 protein is a CDK4 inhibitor, deletion of this gene may increase the activity of CDK4 and lead to hyperphosphorylation of Rb protein.Other genes that can be used in gene therapy to inhibit cell proliferation include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1 / PTEN, DBCCR-1, FCC, rsk-3, p27, p27 / p16 fusion, p21 / p27 fusion, antithrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, frns, trk, ret, gsp, hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.

[0133] It is further contemplated that upregulation of cell surface receptors or their ligands, such as Fas / Fas ligand, DR4 or DR5 / TRAIL, will enhance the apoptosis-inducing ability of the present invention by establishing autocrine or paracrine effects on hyperproliferative cells. Increasing intercellular signaling by increasing the number of gap junctions enhances the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiating agents can be used in combination with the present invention to improve the anti-hyperproliferative effect of the treatment. It is contemplated that inhibitors of cell adhesion will improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, can be used in combination with the present invention to improve the efficacy of the treatment.

[0134] Hormonal therapy can also be used in conjunction with the present invention.The use of hormones is used in the treatment of certain cancers, such as breast cancer, prostate cancer, ovarian cancer or cervical cancer, and the level or action of certain hormones, such as testosterone or estrogen, is reduced or blocked.This treatment is often used in combination with at least one other cancer treatment, as a treatment option or to reduce the risk of metastasis.

[0135] Development of resistant oncolytic viruses Herein, we describe an advanced process for generating FusOn-H2-based oncolytic viruses that can circumvent all the limiting factors in the bloodstream that reduce the efficacy of oncolytic virus therapy. This disclosure also describes the design of a novel virus, FusOn-SD.

[0136] The native HSV-2 virus contains an ICP10 polynucleotide (sometimes referred to as an RR1 polynucleotide) that encodes a polypeptide having a protein kinase (PK) activity, e.g., an amino-terminal domain with serine / threonine protein kinase activity, and a c-terminal domain with ribonucleotide reductase activity. In certain aspects of the invention, the endogenous PK domain is modified to include an activity that renders the virus fusogenic or fusogenic in that it contains selective replication activity in tumor cells (and thus tumor cell destruction activity) and / or the virus contains membrane fusion (syncytium formation) activity. In some embodiments, the ICP10 polynucleotide is modified by deleting at least a portion of the endogenous sequence encoding the protein kinase domain, such that the encoded polypeptide lacks protein kinase activity.

[0137] Viruses can only replicate in living cells, and their replication usually requires the activation of certain cellular signaling pathways. Many viruses have acquired various strategies over the course of evolution to activate these signaling pathways and aid in their replication. The large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10 or RR1) contains a unique amino-terminal domain with serine / threonine protein kinase (PK) activity. This PK activity has been shown to activate the cellular Ras / MEK / MAPK pathway (Smith, et al., (2000) J Virol 74(22):10417-29). As a result, it has been reported that deletion of this PK domain (ICP10PK) from the ribonucleotide reductase gene severely impairs the ability of the virus to replicate in cells, including those without a pre-existing activated Ras signaling pathway (Smith, et al., (1998) J. Virol. 72(11):9131-9141).

[0138] In certain embodiments, the ICP10 sequence of the HSV-2 virus is mutated, such as by deletion, using any of a variety of standard mutagenesis procedures. Mutations can include modifications of nucleotide sequences, single genes, or blocks of genes. Mutations can include single nucleotides (such as point mutations, including the removal, addition, or substitution of a single nucleotide base in a DNA sequence), or can include the insertion or deletion of multiple nucleotides. Mutations can occur naturally as a result of events such as errors in the fidelity of DNA replication, or can be induced after exposure to chemical or physical mutagens. Mutations can also be site-specific by using specific targeting methods well known to those skilled in the art.

[0139] The ICP10 polynucleotide is modified using recombinant gene technology to delete or replace at least a portion of the sequence encoding the PK domain. The region of the PK domain to be deleted / replaced can be any suitable region, as long as the polypeptide encoded by the modified ICP10 polynucleotide retains ribonucleotide reductase activity and lacks protein kinase activity. The ICP10 polynucleotide can also be modified by simply deleting a portion of the sequence encoding the PK domain required for PK activity. The ICP10 polynucleotide can also be modified by replacing at least a portion of the sequence encoding the PK domain with a second polynucleotide, such as green fluorescent protein, which can be placed in frame with the sequence encoding the RR domain of the ICP10 polynucleotide.

[0140] The polynucleotide that replaces at least a portion of the protein kinase activity domain of endogenous ICP10 in HSV-2 may encode at least the fusogenic portion of a cell membrane fusion-inducing polypeptide, for example, a viral fusogenic membrane glycoprotein (FMG). The polypeptide is preferably capable of inducing cell membrane fusion, for example, at substantially neutral pH (e.g., pH about 6-8). Retroviral envelope proteins with some, most, or all of the cytoplasmic domain deleted are useful because such manipulation results in super-fusogenic activity in human cells. In some embodiments, specific modifications are introduced into viral membrane glycoproteins to enhance their ability to induce cell membrane fusion. For example, truncation of the cytoplasmic domain of some retroviral and herpesviral glycoproteins has been shown to enhance their fusogenic activity, and may concomitantly reduce the efficiency of incorporation into virions (Rein et al., (1994) J Virol 68(3):1773-81). EXAMPLES

[0141] The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to work well in carrying out the invention, and thus may be considered to constitute the most commonly used methods for carrying it out. However, it should be understood by those skilled in the art in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results, without departing from the spirit and scope of the invention.

[0142] Example 1: Construction of FusOn-H2 First, the HSV genomic region containing the ICP10 left flanking region (corresponding to nucleotides 85994-86999 of the HSV-2 genome) was amplified with the following exemplary primer pair: 5'-TTGGTCTTCACCTACCGACA (SEQ ID NO: 1), and 3'-GACGCGATGAACGGAAAC (SEQ ID NO: 2). The RR domain and right flanking region (corresponding to nucleotides 88228-89347 of the HSV-2 genome) was amplified with the following exemplary primer pair: 5'-ACACGCCCTATCATCTGAGG (SEQ ID NO: 13), and 5'-AACATGATGAAGGGGCTTCC (SEQ ID NO: 14). These two PCR products were cloned into pNeb193 by EcoRI-NotI-XbaI ligation to generate pNeb-ICP10-deltaPK. A DNA sequence containing the CMV promoter-EGFP gene was then amplified by PCR from pSZ-EGFP using the following exemplary primer pair: 5'-ATGGTGAGCAAGGGCGAG (SEQ ID NO: 3) and 3'-CTTGTACAGCTCGTCCATGC (SEQ ID NO: 4). This PCR-amplified DNA was then cloned into the deleted PK locus of pNeb-ICP10-deltaPK by BglII and NotI ligation to generate pNeb-PKF-2. During the design of the PCR amplification strategy, these primers were designed such that the EGFP gene is fused in frame with the remaining RR domain of this ICP gene, so that the new protein product of this fusion gene contains an intact functional EGFP, which facilitates the selection of recombinant viruses in the following experimental steps.

[0143] The modified ICP10 gene was inserted into the virus via homologous recombination by co-transfecting pNeb-PKF-2 plasmid DNA and purified HSV-2 genomic DNA (strain 186) into Vero cells by lipofectamine. The recombinant virus was screened and identified by selecting GFP-positive virus plaques. During this screening process, all of the GFP-positive plaques were found to show clear syncytium formation of infected cells, indicating that in a specific embodiment of the present invention, the modified virus induces extensive cell membrane fusion. A total of six plaques were picked, of which one, designated FusOn-H2, was selected for further characterization and all subsequent experiments.

[0144] Example 2 To systematically mutate most of the neutralizing epitopes of both gB and gD and to enhance the complement antagonism ability of gC, FusOn-H2 was subjected to a series of selections in the presence of anti-HSV-2 serum. First, FusOn-H2 was subjected to selection in cell culture in the presence of a mixture of sera collected from five rats vaccinated with HSV and with high anti-HSV antibodies in their blood. One of the main reasons for starting this selection with rat serum is that it contains both native immunoglobulins and mannan-binding lectin (MBL) that can activate complement against HSV, whereas mouse and human serum contains only one of these two activation mechanisms (MBL in mouse and native immunoglobulin in human, respectively) (Wakimoto et al., 2002). After seven consecutive rounds of selection under this rat serum mixture, one human serum containing high levels of anti-HSV-2 antibodies was added to this rat serum and selection continued.

[0145] The infectivity of the virus in the presence of anti-HSV serum was monitored periodically during this selection process, and the results showed that its resistance to neutralizing antibodies was continuously improved over the unselected FusOn-H2. One of the test results is shown in Figure 1. This was done after the virus had undergone a total of nine rounds of selection (i.e., seven rounds of selection with rat serum and two rounds of selection with a mixture of rat and human serum). As a result, the cultivated virus (designated FusOn-SS9) showed 28.5-fold and 27.2-fold resistance to the neutralizing effect of human and rat anti-HSV-2 serum, respectively.

[0146] To enable FusOn-H2 with the ability to escape host clearance by the mononuclear phagocyte system (MPS) in blood during systemic delivery, we explored the possibility of genetically grafting the “don’t eat me” signal molecule CD47 onto the viral membrane envelope. Macrophages are the main phagocytes involved in the rapid clearance of HSV particles, and it has been reported that macrophage depletion can significantly improve the therapeutic efficacy of oncolytic HSV (Fulci et al., 2007). The phagocytic activity of macrophages is controlled by both positive and negative regulatory mechanisms. The interaction of CD47 with its receptor, SIRPα, provides a strong negative regulatory signal (“don’t eat me signal”) to macrophages (Kinchen and Ravichandran, 2008).

[0147] HSV encodes several glycoproteins that assemble at the surface of the viral envelope. They include glycoproteins C (gC), gB, gD, gH, and gL. Each of them can serve as a candidate molecule for incorporating the extracellular domain (ECD) of mouse CD47 (mCD47) and thus engraft on the surface of the viral envelope. Glycoprotein C (gC) was chosen here because, unlike the other glycoproteins mentioned, it is not essential for the infectivity of the virus. Therefore, modifying it to incorporate mCD47 does not risk altering the natural tropism of the oncolytic virus. The ECD (aa19-141) of mCD47 (SEQ ID NO: 16) (shown in the table below) was first inserted at the N-terminus of gC, creating a chimeric form of gC (cgC), the expression of which is driven by the CMV IE promoter. [Table 1]

[0148] A HA (hemagglutinin) tag was included in cgC to allow easy detection of the chimeric molecule. To facilitate identification and in vivo imaging of the recombinant virus, cgC was linked to another gene cassette containing an EGFP-luciferase gene. These two gene cassettes were inserted together into the backbone of FusOn-H3, which was derived from an HSV-2-based oncolytic virus (FusOn-H2) by deleting the GFP gene from the virus. FusOn-H2 was constructed by deleting the N-terminal region of the ICP10 gene and replacing it with GFP, which gave it the ability to selectively replicate in and kill tumor cells. Recombinant viruses were identified by picking GFP-positive plaques. Each individually picked virus was concentrated to homogeneous GFP positivity by multiple rounds of plaque purification. The newly generated virus is designated FusOn-CD47-Luc (Figure 2A). A control virus was also constructed by inserting only the EGFP-Luc gene cassette into the backbone of FusOn-H3, FusOn-Luc (Figure 2B). All selected viruses maintained the fusion properties of the parent FusOn-H2.

[0149] An interesting recent study found that coating nanoparticles with CD47 mimetic peptides helps them escape phagocytic clearance by MPS (Rodriguez et al., 2013). We explored the possibility of genetically grafting the extracellular domain of the CD47 molecule onto the membrane envelope of FusOn-H2, allowing it to escape from MPS in case of systemic delivery. This virus construction strategy is illustrated in Figure 2. In vitro assays show that coating viral particles with the extracellular domain of CD47 via recombinant gC makes the virus more resistant to phagocytosis (Figure 3). Tested in vivo in a mouse colon cancer model, FusOn-CD47 was shown to be 15 times more effective than FusOn-Luc to be delivered to the tumor site by the systemic route. Moreover, the data also showed that FusOn-CD47 can persist significantly longer than FusOn-Luc at the tumor site after systemic delivery.

[0150] First, transgene expression was examined by either flow cytometry or Western blot analysis. For flow cytometry analysis, 293 cells were infected with either FusOn-CD47-Luc or FusOn-Luc at 1 pfu / cell. After 24 h, cells were labeled with either PE-conjugated anti-mCD47 antibody or rabbit anti-HA tag antibody. For HA tag detection, goat anti-rabbit antibody conjugated with FITC was added. Both CD47 and HA tag labeled cells were subjected to flow cytometry analysis. The results in Figure 2A show that both anti-mCD47 and anti-HA tag antibodies could easily detect cells infected with FusOn-CD47-Luc but could not detect cells infected with FusOn-Luc. For Western blot analysis, 293 cells were infected with these two viruses in the same way or transfected with a plasmid expressing cgC or a plasmid expressing wild-type gC without the HA tag. Cell lysates were prepared 24 hours later and subjected to Western blot analysis. The results included in Figure 2B show that cgC is abundantly expressed by FusOn-CD47-Luc, but not by FusOn-Luc.

[0151] We next compared the quantification of Luc gene expression from these two viruses. Vero, CT26 and 4T1 cells were infected with either FusOn-CD47-Luc or FusOn-Luc at 1 pfu / cell. Cells were harvested 24 hours later for measurement of luciferase activity. The results contained in Figure 3A showed that luciferase activity from cells infected with these two viruses was at nearly identical levels. Next, another experiment was performed to determine in an in vivo setting whether the transfer of the ECD of mCD47 to oncolytic HSV could allow the virus to evade phagocytosis and clearance by phagocytes. Mouse splenocytes were used as a source of fresh phagocytes since the spleen is the largest unit of the mononuclear phagocyte system. Vero cells were infected with FusOn-CD47-Luc or FusOn-Luc in the presence or absence of mouse splenocytes. Cells were harvested 48 hours later for quantitative measurement of virus yield by plaque assay. The results contained in Figure 3B showed that in wells without splenocytes, both FusOn-CD47-Luc and FusOn-Luc replicated well and the virus yields were similar in these two wells. However, in wells with splenocytes, the FusOn-Luc yield was significantly reduced, while the replication of FusOn-CD47-Luc was only slightly affected. These results indicate that the presence of mCD47 ECD in the viral particles endows the virus with the ability to resist the influence of macrophages and possibly some other immune cells during the course of viral infection.

[0152] To measure the ability of the incorporated mCD47 ECD to enable systemic delivery of the virus, CD26 murine colon carcinoma cells were implanted into the right flank of immune-protected Balb / c mice. After the tumors reached a size of about 8 mm in diameter, we systemically injected each mouse with 2×106 pfu of either FusOn-CD47-Luc or FusOn-Luc. The luciferase activity of the mice was monitored by IVIS Spectrum System from day 2 and every day thereafter until the signal completely disappeared. The results included in FIG. 4 show that on day 2 after virus administration, the image signal from FusOn-CD47-Luc was about 1.5 logs stronger than that from FusOn-Luc. This indicates that the former was delivered to the tumor site more efficiently by the systemic route than the latter. Furthermore, FusOn-CD47-Luc appears to remain in the tumor tissue for a significantly longer period than FusOn-Luc. By day 5, imaging signals were barely detectable in tumors from mice administered FusOn-Luc, whereas signals in tumors from FusOn-CD47-Luc were detectable until day 7. Nevertheless, this HSV-2-based oncolytic virus barely grows in CT26 tumor cells, so neither virus showed significant amplification in tumor tissue. Interestingly, in some cases where several mice were imaged on day 1 after virus delivery, significant imaging signals were transiently detected in the liver. These signals completely disappeared by day 2.

[0153] To determine whether FusOn-CD47-Luc is superior to FusOn-Luc for systemic delivery in other tumor models, we repeated the above experiments and found that FusOn-H2 could grow moderately in mice bearing tumors established by implantation of 4T1 mouse mammary tumor cells in the right flank. Furthermore, 4T1 tumor cells secrete macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF), which may promote invasion and phagocytosis by macrophages. This allows us to more vigorously investigate CD47-mediated evasion strategies. Indeed, the IVIS imaging results included in Figure 5A showed that, in general, 4T1 tumors contained lower signals than those detected in CT26 tumors (as shown in Figure 4). Nevertheless, these results again showed that FusOn-CD47-Luc could be delivered to local tumors more efficiently than FusOn-Luc by the systemic route. By day 2, the difference in image signal intensity between these two viruses was approximately 1.5 logs. The maximum difference was recorded on day 4, when the imaging signal from FusOn-Luc had dropped to nearly background levels, while that from FusOn-CD47-Luc reached its highest value. These data again demonstrated that CD47 modification enabled the virus to be delivered more efficiently by the systemic route and, once it reached tumor tissue, to persist there longer than the control virus.

[0154] To generate a FusOn-H2 product that can resist all three major limiting factors in the bloodstream, i.e., neutralizing antibodies, complement, and phagocytes, we subjected FusOn-CD47 to the anti-HSV-2 serum selection described above. It was subjected to 23 successive passages in the presence of seven successive sections of anti-HSV rat serum and then one human serum containing extremely high levels of anti-HSV-2 antibodies, as described above. Again, several tests were performed during the selection process to determine whether the virus had acquired the ability to resist the neutralizing action of these immune sera. One of the results of such a test is shown in Figure 5, which was performed after FusOn-CD47 was first subjected to seven sections of rat serum and 17 sections of rat and human serum. These results clearly show that the cultivated FusOn-CD47 (designated FusOn-CD47-SS24), like FusOn-SS9, has acquired a substantial ability to maintain infectivity in the presence of immune anti-HSV-2 serum.

[0155] To ensure more complete resistance to neutralizing antibodies in humans, we subjected the virus to three additional rounds of sectioning using a mixture of HSV-2 positive sera from 12 individuals, four at a time. The selection rounds were 8 rounds for batch 1, 6 rounds for batch 2, and 4 rounds for batch 3. After these extensive selections, the resulting viruses were plaque purified and six plaques were picked and grown for further analysis. We designate them HR49-N2-5, HR49-N7, and HR49-N8. First, they were examined for their ability to resist neutralization from a mixture of the eight human sera used for selection. This is the most stringent neutralization test we have performed. For comparison, both FusOn-SS9 and FusOn-CD47-SS24 were included in this experiment. The anti-HSV serum mixture was used at various dilutions and incubated with the indicated viruses, after which the infectivity of the viruses was measured. The results contained in Figure 6 show that under these stringent neutralization conditions (including a mixture of antiviral sera from multiple individuals), the selected viruses were still able to produce a significant number of plaques, while the unselected viruses did not produce plaques until the serum mixture was diluted 80-fold. The data also showed that subsequent three additional rounds of selection with the human serum mixture further enhanced the selected viruses' ability to resist neutralization. The data also showed that some of the collected viruses had a better ability than others to resist neutralization by the mixture of human antiviral sera.

[0156] Example 3 The ability of oncolytic virus FusOn-H2, with and without insertion of the extracellular domain of CD47 into the N-terminus of gC, to escape neutralization by anti-HSV antibodies was evaluated. In this in vitro experiment, 500 plaque-forming units (pfu) of either FusOn-CD47 or FusOn-Luc, mixed or not with diluted anti-HSV-2 serum (1:40 or 1:160 dilution), was incubated at 37°C, and then plated onto a monolayer of Vero cells for infectivity assays and quantification of plaque formation. The results included in Figure 7 showed that at a 1:40 dilution, this serum was able to almost completely neutralize the viral infectivity of FusOn-Luc. Even at a 1:160 dilution, this serum was able to significantly reduce the infection (and therefore the number of plaques formed) of FusOn-Luc. In contrast, FusOn-CD47 was much less neutralized in the presence of the same dilution of anti-HSV serum. In the presence of a 1:40 dilution of serum, FusOn-CD47 produced a significant number of plaques (approximately 15% of wells that did not receive anti-HSV serum), whereas FusOn-Luc showed almost no viral plaques.

[0157] In vivo delivery by systemic route was evaluated for FusOn-CD47, FusOn-Luc, and FusOn-SD in Balb / c mice vaccinated with HSV-2 and implanted with CT26 mouse colon carcinoma cells in the right flank. 7Either FusOn-CD47, FusOn-Luc, or FusOn-SD was delivered systemically by tail vein injection into different groups of mice. The mice were imaged with an IVIS imager on the days shown in Figure 8. These results indicated that FusOn-Luc was not delivered to tumors by systemic route in the presence of anti-HSV immunity. In contrast, FusOn-CD47 was readily detected in tumor tissues by the same delivery route in the presence of anti-HSV immunity. Furthermore, FusOn-CD47 remained in tumor tissues for nearly 8 days after reaching the tumor site, despite the presence of anti-viral immunity. CD47 unexpectedly assisted the oncolytic virus to escape neutralization by anti-HSV antibodies, contributing to the overall ability of FusOn-SD to escape neutralizing antibodies for systemic delivery. Thus, FusOn-SD performed the best in systemic delivery to tumor sites. FusOn-SD is therefore useful in patients with HSV-1 and / or HSV-2 antibodies, e.g., patients who have been vaccinated against HSV-1 and / or HSV-2 or patients who have HSV-1 and / or HSV-2.

[0158] Unexpectedly, we found that gE was absent from FusOn-SD viral particles, as shown in Figures 9A-9C.

[0159] Figure 9A is a bar graph showing the absorbance of FusOn-H2, FusOn-SD, and PBS (negative control) after treatment with mouse anti-HSV-2gE (1:1000 dilution) and HPR-conjugated rabbit anti-mouse IgG (1:10,000 dilution). More specifically, in Figure 9A, 1X10 6pfu of virus (parental FusOn-H2 and FusOn-SD) were coated onto 96-well plates for ELISA assays. Wells coated with PBS served as negative controls. The first antibody used was mouse anti-HSV-2 gE (1:1000 dilution) and the second antibody was HPR-conjugated rabbit anti-mouse IgG (1:10,000 dilution). Significant measurements were detected with FusOn-H2 but not with FusOn-SD, indicating the presence of gE in FusOn-H2 viral particles but not in FusOn-SD.

[0160] FIG. 9B is a Western blot showing detection of gE for FusOn-SD and FusOn-H2. Proteins were diluted at 1×10 6 pfu of virus (FusOn-H2 and FusOn-SD) were run on an acrylamide gel for Western blot analysis. The same antibodies, mouse anti-HSV-2 gE and HPR-conjugated rabbit anti-mouse IgG, used for Figure 9A were used for this Western blot. The results showed a clear gE band for FusOn-H2, but not in the lane loaded with protein from FusOn-SD. This result confirms the findings in Figure 9A.

[0161] Figure 9C is a schematic diagram of the mechanism of NK cell recognition of HSV or HSV-infected cells via gE. The diagram shows that gE binds to IgG (either virus-specific or non-virus-specific). Then, CD16a, one of the most important NK cell activation receptors, binds to the Fc region of IgG, leading to NK cell activation and clearance of virus particles or virus-infected cells. This reduces the delivery and replication of oncolytic viruses, thus minimizing the therapeutic effect of virotherapy. FusOn-SD are not recognized by this mechanism, so NK cells do not remove them, and the therapeutic effect of FusOn-SD persists.

[0162] References Bender,F.C.,Samanta,M.,Heldwein,E.E.,de Leon,M.P.,Bilman,E.,Lou,H.,Whitbeck,J.C.,Eisenberg,R.J.and Cohen,G.H.,2007. Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions. J Virol 81,3827-41.

[0163] Cairns,T.M.,Huang,Z.Y.,Gallagher,J.R.,Lin,Y.,Lou,H.,Whitbeck,J.C.,Wald,A.,Cohen,G.H.and Eisenberg,R.J.,2015. Patient-Specific Neutralizing Antibody Responses to Herpes Simplex Virus Are Attributed to Epitopes on gD,gB,or Both and Can Be Type Specific.J Virol 89,9213-31.

[0164] Cairns,T.M.,Huang,Z.Y.,Whitbeck,J.C.,Ponce de Leon,M.,Lou,H.,Wald,A.,Krummenacher,C.,Eisenberg,R.J.and Cohen,G.H.,2014. Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans.J Virol 88,12612-22.

[0165] Eing,B.R.,Kuhn,J.E.and Braun,R.W.,1989. Neutralizing activity of antibodies against the major herpes simplex virus type 1 glycoproteins.J Med Virol 27,59-65.

[0166] Ellermann-Eriksen,S.,2005. Macrophages and cytokines in the early defence against herpes simplex virus.Virol J 2,59.

[0167] Fu,X.,Tao,L.,Li,M.,Fisher,W.E.and Zhang,X.,2006.Effective treatment of pancreatic cancer xenografts with a conditionally replicating virus derived from type 2 herpes simplex virus.Clin Cancer Res 12,3152-3157.

[0168] Fu,X.and Zhang,X.,2001. Delivery of herpes simplex virus vectors through liposome formulation.Mol Ther 4,447-53.

[0169] Fulci,G.,Dmitrieva,N.,Gianni,D.,Fontana,E.J.,Pan,X.,Lu,Y.,Kaufman,C.S.,Kaur,B.,Lawler,S.E.,Lee,R.J.,Marsh,C.B.,Brat,D.J.,van Rooijen,N.,Stemmer-Rachamimov,A.O.,Hochberg,F.H.,Weissleder,R.,Martuza,R.L.and Chiocca,E.A.,2007.Depletion of peripheral macrophages and brain microglia increases brain tumor titers of oncolytic viruses.Cancer Res 67,9398-406.

[0170] Greig,S.L.,2016. Talimogene Laherparepvec:First Global Approval.Drugs 76,147-54.

[0171] Huber,V.C.,Lynch,J.M.,Bucher,D.J.,Le,J.and Metzger,D.W.,2001. Fc Receptor-Mediated Phagocytosis Makes a Significant Contribution to Clearance of Influenza Virus Infections.The Journal of Immunology 166,7381-7388.

[0172] Hume,D.A.,2006. The mononuclear phagocyte system.Curr Opin Immunol 18,49-53.

[0173] Kinchen,J.M.and Ravichandran,K.S.,2008. Phagocytic signaling: you can touch,but you can’t eat.Curr Biol 18,R521-4.

[0174] Macedo,N.,Miller,D.M.,Haq,R.and Kaufman,H.L.,2020.Clinical landscape of oncolytic virus research in 2020. Journal for ImmunoTherapy of Cancer 8,e001486.

[0175] Nakamori,M.,Fu,X.,Pettaway,C.A.and Zhang,X.,2004. Potent antitumor activity after systemic delivery of a doubly fusogenic oncolytic herpes simplex virus against metastatic prostate cancer.Prostate 60,53-60.

[0176] Rodriguez,P.L.,Harada,T.,Christian,D.A.,Pantano,D.A.,Tsai,R.K.and Discher,D.E.,2013. Minimal “Self” peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles.Science 339,971-5.

[0177] Silke Heilingloh,C.,Lull,C.,Kleiser,E.,Alt,M.,Schipper,L.,Witzke,O.,Trilling,M.,Eis-Hubinger,A.M.,Dittmer,U.and Krawczyk,A.,2020. Herpes Simplex Virus Type 2 Is More Difficult to Neutralize by Antibodies Than Herpes Simplex Virus Type 1.Vaccines(Basel)8.

[0178] Tay,M.Z.,Wiehe,K.and Pollara,J.,2019. Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses.Frontiers in immunology 10.

[0179] Tuzmen,C.,Cairns,T.M.,Atanasiu,D.,Lou,H.,Saw,W.T.,Hall,B.L.,Cohen,J.B.,Cohen,G.H.and Glorioso,J.C.,2020.Point Mutations in Retargeted gD Eliminate the Sensitivity of EGFR / EGFRvIII-Targeted HSV to Key Neutralizing Antibodies.Molecular Therapy-Methods & Clinical Development 16,145-154.

[0180] Van Strijp,J.A.,Van Kessel,K.P.,van der Tol,M.E.,Fluit,A.C.,Snippe,H.and Verhoef,J.,1989. Phagocytosis of herpes simplex virus by human granulocytes and monocytes.Arch Virol 104,287-98.

[0181] Wakimoto,H.,Ikeda,K.,Abe,T.,Ichikawa,T.,Hochberg,F.H.,Ezekowitz,R.A.,Pasternack,M.S.and Chiocca,E.A.,2002. The complement response against an oncolytic virus is species-specific in its activation pathways.Mol Ther 5,275-82.

[0182] Alvarez-Breckenridge,C.A.,Yu,J.,Price,R.,Wei,M.,Wang,Y.,Nowicki,M.O.,Ha,Y.P.,Bergin,S.,Hwang,C.,Fernandez,S.A.,Kaur,B.,Caligiuri,M.A.and Chiocca,E.A.,2012a. The histone deacetylase inhibitor valproic acid lessens NK cell action against oncolytic virus-infected glioblastoma cells by inhibition of STAT5 / T-BET signaling and generation of gamma interferon.J Virol 86,4566-77(“Alvarez-Breckenridge et al.,2012a”).

[0183] Alvarez-Breckenridge,C.A.,Yu,J.,Price,R.,Wojton,J.,Pradarelli,J.,Mao,H.,Wei,M.,Wang,Y.,He,S.,Hardcastle,J.,Fernandez,S.A.,Kaur,B.,Lawler,S.E.,Vivier,E.,Mandelboim,O.,Moretta,A.,Caligiuri,M.A.and Chiocca,E.A.,2012b.NK cells impede glioblastoma virotherapy through NKp30 and NKp46 natural cytotoxicity receptors.Nat Med 18,1827-34(“Alvarez-Breckenridge et al.,2012b”).

[0184] Bender,F.C.,Samanta,M.,Heldwein,E.E.,de Leon,M.P.,Bilman,E.,Lou,H.,Whitbeck,J.C.,Eisenberg,R.J.and Cohen,G.H.,2007.Antigenic and mutational analyses of herpes simplex virus glycoprotein B reveal four functional regions.J Virol 81,3827-41.

[0185] Cairns,T.M.,Huang,Z.Y.,Gallagher,J.R.,Lin,Y.,Lou,H.,Whitbeck,J.C.,Wald,A.,Cohen,G.H.and Eisenberg,R.J.,2015.Patient-Specific Neutralizing Antibody Responses to Herpes Simplex Virus Are Attributed to Epitopes on gD,gB,or Both and Can Be Type Specific.J Virol 89,9213-31.

[0186] Cairns,T.M.,Huang,Z.Y.,Whitbeck,J.C.,Ponce de Leon,M.,Lou,H.,Wald,A.,Krummenacher,C.,Eisenberg,R.J.and Cohen,G.H.,2014.Dissection of the antibody response against herpes simplex virus glycoproteins in naturally infected humans.J Virol 88,12612-22.

[0187] Dai,H.-S.and Caligiuri,M.A.,2018.Molecular Basis for the Recognition of Herpes Simplex Virus Type 1 Infection by Human Natural Killer Cells.Frontiers in immunology 9,183-183.

[0188] Dubin,G.,Basu,S.,Mallory,D.L.,Basu,M.,Tal-Singer,R.and Friedman,H.M.,1994. Characterization of domains of herpes simplex virus type 1 glycoprotein E involved in Fc binding activity for immunoglobulin G aggregates.J Virol 68,2478-85.

[0189] Eing,B.R.,Kuhn,J.E.and Braun,R.W.,1989. Neutralizing activity of antibodies against the major herpes simplex virus type 1 glycoproteins. J Med Virol 27,59-65.

[0190] Ellermann-Eriksen,S.,2005. Macrophages and cytokines in the early defence against herpes simplex virus. Virol J 2,59.

[0191] Fu,X.,Tao,L.,Li,M.,Fisher,W.E.and Zhang,X.,2006.Effective treatment of pancreatic cancer xenografts with a conditionally replicating virus derived from type 2 herpes simplex virus.Clin Cancer Res 12,3152-3157.

[0192] Fu,X.and Zhang,X.,2001. Delivery of herpes simplex virus vectors through liposome formulation.Mol Ther 4,447-53.

[0193] Fulci,G.,Dmitrieva,N.,Gianni,D.,Fontana,E.J.,Pan,X.,Lu,Y.,Kaufman,C.S.,Kaur,B.,Lawler,S.E.,Lee,R.J.,Marsh,C.B.,Brat,D.J.,van Rooijen,N.,Stemmer-Rachamimov,A.O.,Hochberg,F.H.,Weissleder,R.,Martuza,R.L.and Chiocca,E.A.,2007.Depletion of peripheral macrophages and brain microglia increases brain tumor titers of oncolytic viruses.Cancer Res 67,9398-406.

[0194] Greig,S.L.,2016. Talimogene Laherparepvec:First Global Approval.Drugs 76,147-54.

[0195] Huber,V.C.,Lynch,J.M.,Bucher,D.J.,Le,J.and Metzger,D.W.,2001.Fc Receptor-Mediated Phagocytosis Makes a Significant Contribution to Clearance of Influenza Virus Infections.The Journal of Immunology 166,7381-7388.

[0196] Hume,D.A.,2006. The mononuclear phagocyte system.Curr Opin Immunol 18,49-53.

[0197] Kinchen,J.M.and Ravichandran,K.S.,2008. Phagocytic signaling: you can touch,but you can’t eat.Curr Biol 18,R521-4.

[0198] Macedo,N.,Miller,D.M.,Haq,R.and Kaufman,H.L.,2020.Clinical landscape of oncolytic virus research in 2020.Journal for ImmunoTherapy of Cancer 8,e001486.

[0199] Nakamori,M.,Fu,X.,Pettaway,C.A.and Zhang,X.,2004. Potent antitumor activity after systemic delivery of a doubly fusogenic oncolytic herpes simplex virus against metastatic prostate cancer.Prostate 60,53-60.

[0200] Rodriguez,P.L.,Harada,T.,Christian,D.A.,Pantano,D.A.,Tsai,R.K.and Discher,D.E.,2013.Minimal “Self” peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles.Science 339,971-5.

[0201] Silke Heilingloh,C.,Lull,C.,Kleiser,E.,Alt,M.,Schipper,L.,Witzke,O.,Trilling,M.,Eis-Hubinger,A.M.,Dittmer,U.and Krawczyk,A.,2020.Herpes Simplex Virus Type 2 Is More Difficult to Neutralize by Antibodies Than Herpes Simplex Virus Type 1.Vaccines(Basel)8.

[0202] Tay,M.Z.,Wiehe,K.and Pollara,J.,2019. Antibody-Dependent Cellular Phagocytosis in Antiviral Immune Responses.Frontiers in immunology 10.

[0203] Tuzmen,C.,Cairns,T.M.,Atanasiu,D.,Lou,H.,Saw,W.T.,Hall,B.L.,Cohen,J.B.,Cohen,G.H.and Glorioso,J.C.,2020.Point Mutations in Retargeted gD Eliminate the Sensitivity of EGFR / EGFRvIII-Targeted HSV to Key Neutralizing Antibodies.Molecular Therapy-Methods & Clinical Development 16,145-154.

[0204] Van Strijp, JA, Van Kessel, KP, van der Tol, ME, Fluit, AC, Snippe, H. and Verhoef, J., 1989. Phagocytosis of herpes simplex virus by human granulocytes and monocytes. Arch Virol 104, 287-98.

[0205] Wakimoto, H., Ikeda, K., Abe, T., Ichikawa, T., Hochberg, FH, Ezekowitz, RA, Pasternack, MS and Chiocca, EA, 2002. The complement response against an oncolytic virus is species-specific in its activation pathways.Mol Ther 5,275-82.

[0206] All publications, patents, and patent applications cited herein are incorporated herein by reference as if set forth in their entirety. Although the present invention has been described with reference to exemplary embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of the exemplary embodiments, as well as other embodiments of the present invention, will be apparent to those skilled in the art after reference to the description. Accordingly, it is intended that the appended claims encompass such modifications and enhancements.

Claims

1. A composition comprising an oncolytic herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), wherein the oncolytic HSV1 or HSV-2 is prepared by passaging the oncolytic HSV1 or HSV-2 at least twice with immune serum having elevated levels of anti-HSV antibodies.

2. The composition of claim 1, wherein the oncolytic HSV1 or HSV-2 has a membrane envelope comprising glycoproteins, at least one glycoprotein comprising an extracellular CD47 domain inserted at the N-terminus of the glycoprotein.

3. A composition comprising oncolytic herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), wherein the oncolytic HSV1 or HSV-2 has a membrane envelope comprising glycoproteins, at least one glycoprotein comprising an extracellular CD47 domain inserted at the N-terminus of the glycoprotein, and the oncolytic HSV1 or HSV-2 is substantially free of gE.

4. The composition of claim 2 or 3, wherein the glycoprotein is selected from glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, and glycoprotein L.

5. The composition of any one of claims 1, 2 and 4, wherein passaging with the immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of the oncolytic HSV-1 or HSV-2.

6. The composition of any one of claims 2 to 5, wherein the extracellular CD47 domain comprises amino acids 19 to 141 of CD47.

7. The composition of any one of claims 2 and 4 to 6, wherein the oncolytic HSV1 or HSV-2 having an extracellular CD47 domain is free or substantially free of gE.

8. The composition of any one of claims 1, 2 and 4-6, wherein the immune serum is a mixture of rat serum and human serum having elevated levels of anti-HSV antibodies.

9. 9. The composition of any one of claims 1 to 8, comprising oncolytic HSV-2, the oncolytic HSV-2 having been prepared by passaging the oncolytic HSV-2 7 times in the presence of rat serum having elevated levels of anti-HSV antibodies, followed by (a) 17 or (b) 22 passaging in the presence of a mixture of rat serum and at least one human serum having elevated levels of anti-HSV antibodies.

10. 10. The composition of any one of claims 1 to 9, wherein the oncolytic HSV-1 or oncolytic HSV-2 comprises a modified ICP10 coding region lacking nucleotides 1 to 1204 of the endogenous ICP10 coding region, the oncolytic HSV-1 or HSV-2 comprises the modified ICP10 operably linked to an endogenous or constitutive promoter and expresses a modified ICP10 polypeptide that lacks protein kinase (PK) activity but retains ribonucleotide reductase activity, and the oncolytic HSV-1 or HSV-2 is capable of selectively killing cancer cells.

11. The composition according to any one of claims 1, 2 and 4 to 10, comprising an oncolytic HSV-2 and wherein the oncolytic HSV-2 to be passaged is (a) a FusOn-H2 oncolytic virus or (b) a FusOn-CD47 oncolytic virus.

12. A method for preparing a composition comprising an oncolytic herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2), comprising passaging the oncolytic herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2) at least twice with immune serum having elevated levels of anti-HSV antibodies.

13. 13. The method of claim 12, wherein the oncolytic HSV1 or HSV-2 has a membrane envelope comprising glycoproteins, at least one glycoprotein comprising an extracellular CD47 domain inserted at the N-terminus of the glycoprotein.

14. 14. The method of claim 13, wherein the glycoprotein is selected from glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, and glycoprotein L.

15. The method of any one of claims 12 to 14, wherein passaging with the immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of the oncolytic HSV-1 or HSV-2.

16. The method of any one of claims 12 to 15, wherein the extracellular domain comprises amino acids 19 to 141 of CD47.

17. The method of any one of claims 12 to 16, wherein the immune serum comprises mammalian anti-HSV antibodies.

18. 18. The method of any one of claims 12 to 17, comprising passage of the oncolytic HSV-2 at least twice in the presence of rat serum with elevated levels of anti-HSV antibodies, followed by at least one passage in the presence of a mixture of rat serum and at least one human serum with elevated levels of anti-HSV antibodies.

19. 19. The method of claim 18, comprising passage 7 times of the oncolytic HSV-2 in the presence of rat serum with elevated levels of anti-HSV antibodies, followed by passage 17 times in the presence of a mixture of rat serum and at least one human serum with elevated levels of anti-HSV antibodies.

20. 19. The method of claim 18, comprising passage 7 times of the oncolytic HSV-2 in the presence of rat serum with elevated levels of anti-HSV antibodies, followed by 23 times of passage in the presence of a mixture of rat serum and at least one human serum with elevated levels of anti-HSV antibodies.

21. The method according to any one of claims 12 to 20, wherein the composition comprises an oncolytic HSV-2 and the oncolytic HSV-2 subjected to passage is a FusOn-H2 oncolytic virus.

22. 22. A HSV-based oncolytic virus therapy for the treatment of cancer in a patient in need thereof comprising a composition according to any one of claims 1 to 11 or a composition prepared by the method according to any one of claims 12 to 21.

23. 23. The HSV-based oncolytic virus therapy of claim 22, wherein the composition is administered systemically, the cancer is metastatic, and / or the patient has been vaccinated against or has HSV-1 and / or HSV-2.

24. 24. The HSV-based oncolytic virus therapy of claim 22 or 23, further comprising a checkpoint inhibition immunotherapy, optionally wherein said checkpoint inhibition immunotherapy is selected from: (a) a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA-4 inhibitor; or (b) adoptive T cell transfer.

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