CN116782917A - Oncolytic viruses and enhanced anti-tumor activity for systemic delivery - Google Patents

Abstract

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

Description

Oncolytic viruses and enhanced anti-tumor activity for systemic delivery

The application claims the benefit of U.S. provisional application Ser. No. 63/200,011, filed on 9/2/2021, and U.S. provisional application Ser. No. 63/263,528, filed on 4/11/2021, each of which is hereby incorporated by reference.

Statement of federally sponsored research or development

The present application was carried out with government support under grant No. G110207 awarded by the national institutes of health. The government has certain rights in this application.

Technical Field

The present application relates to oncolytic viruses that are more resistant to clearance and neutralization of the immune system, methods of making the same, and methods of using the same in the treatment of conditions and diseases such as cancer, including metastatic cancer.

Background

Cancer viral therapy is based on a practical approach with a simple and practical therapeutic mechanism, namely the use of the inherent infection/cytolytic activity of the virus to selectively destroy malignant cells. Significant progress has been made in recent years in the development of cancer viral therapies. Currently, an oncolytic virus, imlygic or T-VEC, derived from herpes simplex virus type I (HSV-1) has been approved for clinical use in the treatment of unresectable skin, subcutaneous and lymph node lesions (Greig, 2016) in patients with melanoma that recurs after primary surgery. In addition, a number of ongoing clinical trials are testing the efficacy of a variety of oncolytic viruses constructed from different viruses (Macedo et al 2020).

Currently, oncolytic viral therapies are administered primarily by the intratumoral route. However, systemic delivery is ideal for treating patients with many malignant tumors, especially for those who have developed metastatic disease. In fact, the vast majority of patients receiving viral therapy may suffer from metastatic disease. Although this route of delivery is important, optimal systemic delivery strategies have not been developed. Studies have shown that, although therapeutic effects were detected in some experiments following delivery of oncolytic HSV by the systemic route, overall therapeutic efficacy was significantly lower than those observed in intratumoral delivery (Fu et al, 2006; nakamori et al, 2004). Several key components hinder the efficiency of oncolytic virus delivery via the systemic route.

First, anti-HSV neutralizing antibodies may bind to the introduced viral particles, either pre-existing due to previous exposure of the patient to the virus or newly generated by repeated administration of oncolytic virus during treatment. This prevents the virus from infecting tumor cells and results in their rapid clearance by Antibody Dependent Cellular Phagocytosis (ADCP) of the host (Huber et al, 2001; tay, wiehe and Polara, 2019). In fact, animal studies have demonstrated that preexisting humoral immunity is detrimental to the infectivity of oncolytic HSV (Fu and Zhang, 2001).

anti-HSV neutralizing antibodies were found to target mainly two virally encoded glycoproteins, glycoprotein D (gD) and gB (Cairns et al 2015; cairns et al 2014). gD and gB each contain many neutralizing epitopes and are presented in different orders of immunodominant order in different individuals (Bender et al, 2007). In summary, these epitopes are well conserved in infected individuals and/or in different species (ing, kuhn and Braun, 1989). 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 is a major obstacle to systemic administration of HSV-based oncolytic viruses, as neutralizing antibodies can readily recognize them and eliminate therapeutic activity.

Another important limiting factor that reduces the effectiveness of systemic delivery of oncolytic viral therapies is the host's Mononuclear Phagocyte System (MPS). It has been demonstrated that viral particles can be rapidly cleared by MPS after systemic delivery (Ellermann-Eriksen, 2005;Hume,2006;Van Strijp et al, 1989). In particular, studies by Fulci et al have demonstrated that depletion of macrophages can significantly improve the therapeutic effect of oncolytic HSV (Fulci et al, 2007).

Another important antiviral mechanism that can reduce the effectiveness of viral delivery of HSV is Natural Killer (NK) cells (Alvarez-Brecken ridge et al, 2012a; alvarez-Brecken ridge et al, 2012 b). Viral glycoprotein E (gE) is known to bind IgG (Dubin et al, 1994). Recent studies have shown that NK cells can recognize HSV or HSV-infected cells via binding of the surface CD16 activating receptor to the Fc region in IgG bound to gE (Dai and Caligiuri, 2018).

One way to limit the impact of neutralizing antibodies on oncolytic virus infectivity is to mutagenize each major epitope. Indeed, recent studies have shown that mutagenesis of two of these epitopes from gD of oncolytic HSV does eliminate neutralization of the corresponding monoclonal antibodies (mAbs) (Tuzmen et al 2020). However, both gB and gD of HSV-1 and HSV-2 are primary targets of neutralizing antibodies, and each of them contains almost twelve neutralizing epitopes. For viral genomes longer than 150kb, the traditional approach of using mutagenesis of each of these epitopes from both glycoproteins is not practical. Furthermore, this approach only addresses one of the three major hurdles faced by systemic delivery of oncolytic viral therapies, namely neutralizing antibodies, and leaving MPS unaffected.

U.S. patent publication No. 2012/0301506 discloses the construction of an HSV-2-derived oncolytic virus FusOn-H2 and its use in the treatment of malignant tumors. Administration of FusOn-H2 induces a patient's innate immune response to tumor cells via neutrophils that are capable of effectively destroying the tumor as it migrates to the tumor mass. FusOn-H2 is effective in eradicating tumors using induced innate anti-tumor immunity, even when used at very low doses.

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 treating cancer. Modified HSV-2 comprises a modified/mutated ICP10 polynucleotide encoding a polypeptide having ribonucleotide reductase activity and lacking protein kinase activity.

Fu et al, oncostarget, 2018,9 (77): 34543-34553 describe the transplantation of the CD47 gene onto the envelope of oncolytic HSV to enable it to escape from the MPS.

Thus, there remains a need for oncolytic viruses suitable for systemic administration.

Disclosure of Invention

The present invention relates to oncolytic viruses suitable for systemic administration that are resistant to immune clearance in a subject. Described herein are methods for introducing a series of modifications to a herpes simplex virus, such as an oncolytic virus based on herpes simplex virus-1 (HSV-1) or herpes simplex virus-2 (HSV-2), such as FusOn-H2, that enable the virus to escape from clearance and neutralization occurring in a subject. These modifications include: 1) FusOn-H2 is serially passaged in immune serum containing high levels of anti-HSV antibodies, and optionally 2) the extracellular domain of molecule CD47 containing a "do not eat me" signal is inserted into the N-terminus of glycoprotein C (gC). Thus, when administered by the systemic route, the modified virus FusOn-SD is significantly more effective in infecting and lysing tumor cells than the parent FusOn-H2.

A detailed analysis of FusOn-SD revealed several new findings. One prominent finding is that the virus particles have almost no gE present. This allows the virus to escape from NK cell mediated antiviral mechanisms. The absence of gE is found in CD 47-containing viruses (e.g., fusOn-CD 47) only after serial passage in the presence of antiviral serum, whereas it is not in non-CD 47-containing viruses (e.g., fusOn-gC-Luc) that undergo similar passage in the presence of antiviral serum. Another new finding is that insertion of the extracellular domain of CD47 into the N-terminus of gC was also found to enable the virus to escape neutralization of anti-HSV antibodies, even if not passaged. These modifications and the unexpected consequent changes significantly increase the ability of oncolytic viruses to treat cancer by systemic delivery without compromising their replication and safety.

One embodiment is a composition comprising an oncolytic herpes simplex virus type 1 (HSV-1) or 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 an immune serum having elevated levels of anti-HSV antibodies. In a preferred embodiment, the oncolytic HSV1 or HSV-2 has an envelope comprising glycoproteins, wherein at least one glycoprotein comprises an extracellular CD47 domain inserted at the N-terminus of the glycoprotein, such as an extracellular CD47 domain comprising amino acids 19-141 (SEQ ID NO: 21) of CD 47. 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 by any particular theory, the inventors believe that passage 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.

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

Compositions comprising oncolytic HSV-2 may be prepared by passaging oncolytic HSV-2 seven times in the presence of a rat serum having elevated levels of anti-HSV antibodies, then passaging seventeen times in the presence of a mixture of a rat serum having elevated levels of anti-HSV antibodies and at least one human serum. Oncolytic HSV-2 may be prepared by passaging oncolytic HSV-2 seven times in the presence of a rat serum having elevated levels of anti-HSV antibodies, then passaging twenty-three times in the presence of a mixture of a rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

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

In one embodiment of the compositions described herein, the composition comprises oncolytic HSV-2, which is prepared by passaging FusOn-H2 oncolytic virus.

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

Another embodiment is a method of preparing a composition comprising an oncolytic herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) comprising passaging an oncolytic herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) at least twice with an immune serum having an elevated level of anti-HSV antibodies. Oncolytic HSV-1 or HSV-2 to be passaged and immune serum may be as described herein. Serum with elevated levels of anti-HSV antibodies may be obtained from animals vaccinated with HSV (e.g., HSV-2). In one embodiment, the method comprises passaging oncolytic HSV-1 or HSV-2 at least twice in the presence of rat serum, followed by passaging at least once in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum. In another embodiment, the method comprises passaging oncolytic HSV-1 or HSV-2 seven times in the presence of rat serum having elevated levels of anti-HSV antibodies, followed by seventeen times in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum. In another embodiment, the method comprises passaging oncolytic HSV-2 seven times in the presence of rat serum having elevated levels of anti-HSV antibodies, followed by passaging twenty-three times in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

In a preferred embodiment of any of the compositions or methods described herein, the oncolytic HSV-2 to be passaged is FusOn-H2 oncolytic virus.

In yet another preferred embodiment of the compositions or methods described herein, the oncolytic HSV-2 to be passaged is FusOn-CD47 oncolytic virus.

Another embodiment is a method of treating cancer (e.g., metastatic cancer) in a patient in need thereof with an HSV-based oncolytic virus therapy, the method 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 compositions are useful for treating solid tumors. In one embodiment, the patient is vaccinated against HSV-1 and/or HSV-2. In another embodiment, the patient has HSV-1 and/or HSV-2.

Drawings

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

FIG. 1 is a bar graph showing differential sensitivity of trained and untrained FusOn-H2 to anti-HSV serum. Will be 1X 10 ⁴ The untrained Fuson-H2 or trained FusOn-SS9 was mixed with human or rat anti-HSV-2 serum at a final concentration of 1:5. After incubation for 1 hour at 37 ℃, the solution was applied to Vero monolayers on 12-well plates. After 48 hours of crystal violet staining, plaques were counted.

FIG. 2A is a schematic diagram of the FusOn-cd47 construction strategy. It shows that EGFP-Luc gene cassette and CMVp-HAtag-CD47ECD are inserted into the backbone of FusOn-H2 by homologous recombination, the Left Flanking (LF) sequence and the 3' region of gC as the right flanking sequence (from transmembrane domain to polyA). Details of the individual components of the gene cassette are depicted in the figures and labeled accordingly. Abbreviated as: CMVp, cytomegalovirus immediate early promoter; LTR, rous sarcoma long terminal repeat sequence containing promoter region of the virus; GFP-Luc, EGFP-luciferase fusion gene; HA, HA tag; CD47, murine CD47 extracellular domain; gC, complete gC coding region. Left flanking region of LF, gC. Recombinant viruses were identified by GFP expression and purified to homogeneity.

FIG. 2B is a schematic diagram of the FusOn-cd47 construction strategy. It shows that FusOn-Luc, used as a control virus, was constructed in a similar manner except that it did not contain the CD47 extracellular domain.

FIG. 3 is a graph showing a comparison of the ability of FusOn-CD47 and FusOn-Luc to evade phagocyte clearance. This is a comparison of viral yields in the presence or absence of phagocytes. Vero cells in 12-well plates were infected with 0.1 pfu/cell virus in the absence or presence of 200,000 spleen cells. Cells were harvested for 48 hours and virus yield was determined by plaque assay. Compared to the other three wells, +.p <0.05.

Fig. 4A is a graph showing average photon readings measured by daily IVIS imaging, demonstrating that FusOn-CD47 can be delivered more effectively by the systemic route than FusOn-Luc in a CT26 tumor model. Tumors were established in the right flank of Balb/c mice by subcutaneous implantation of CT26 cells. Once the tumor reaches a large diameter of about 8mmSmall, systemic administration of 2X 10 ⁶ FusOn-CD47 or FusOn-Luc for each pfu.

Fig. 4B is a time-lapse image of the number of days in a typical mouse after systemic delivery of FusOn-CD 47. It shows that in the CT26 tumor model, fusOn-CD47 can be delivered more efficiently by the systemic route than FusOn-Luc. Tumors were established in the right flank of Balb/c mice by subcutaneous implantation of CT26 cells. Once the tumor reached a size of about 8mm in diameter, 2 x 10 was administered systemically ⁶ FusOn-CD47 or FusOn-Luc for each pfu. Animals were imaged for luciferase expression daily starting on day 2.

FIG. 5 is a graph showing enhanced resistance of FusOn-CD47 to neutralizing effects of HSV serum after training. Will be 1X 10 ⁴ The individual untrained FusOn-CD47 or trained FusOn-CD47-SS24 (passaged 7 times in the presence of rat serum and 17 times in the presence of rat serum plus one human serum) was mixed with human or rat anti-HSV-2 serum at a final concentration of 1:5. After incubation for 1 hour at 37 ℃, the solution was applied to Vero monolayers on 12-well plates. After 48 hours of crystal violet staining, plaques were counted.

Figure 6 is a graph showing additional resistance obtained by trained FusOn-CD47 against HSV immune serum. Will be 5X10 ³ Untrained Fuson-CD47 or the same virus trained at different stages [ selection 24 series (FusOn-CD 47-SS 24), selection 49 series (HR 49, N2-N5 and N7-N8)]Mixed with different dilutions of the octahuman serum mixture. Trained FusOn-H2 (FusOn-SS 9) was also included in this experiment. After incubation for 1 hour at 37 ℃, the solution was applied to Vero monolayers on 12-well plates. After 48 hours of crystal violet staining, plaques were counted.

Fig. 7 is a bar graph showing that insertion of CD47 can enhance the ability of oncolytic HSV to escape neutralizing HSV antibodies. Five hundred 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 hour at 37℃and then used to infect Vero cells in 6-well plates. The same amount of virus was incubated with medium alone as a control. After 48 hours, cells were stained with crystal violet to count viral plaques. The percentage of plaques was calculated by dividing the number of plaques in wells containing anti-HSV serum by the number of plaques in wells containing the same virus but no anti-HSV serum (control). The expression of p <0.05 compared to FusOn-Luc.

FIG. 8 is a time-lapse image of typical mouse days after systemic delivery of FusOn-CD47, fusOn-Luc or FusOn-SD. It shows that FusOn-CD47, but not FusOn-Luc, can be delivered by systemic route to tumor-bearing mice with pre-existing anti-HSV immunity, and that passage of FusOn-CD47 in serum containing anti-HSV antibodies (to generate FusOn-SD) further enhances 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. Via the tail vein at 1X 10 ⁷ The dose of pfu was administered to mice in different groups for each of these three viruses. Mice were imaged using an IVIS imager after a specified number of days of virus injection.

FIG. 9A is a bar graph showing absorbance at 450nm of FusOn-H2, fusOn-SD, and PBS (negative control) after treatment with mouse anti-HSV-2 gE (1:1000 dilution) and HPR conjugated rabbit anti-mouse IgG (1:10,000 dilution).

FIG. 9B is a 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).

Fig. 9C is a schematic diagram of NK cell mechanisms that recognize HSV or HSV infected cells via gE.

Detailed Description

In describing the preferred embodiments of the present invention illustrated in the drawings, specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is to be understood that each specific term includes all technical equivalents that operate in a similar manner to accomplish a similar purpose. Several preferred embodiments of the invention have been described for purposes of illustration, it being understood that the invention may be embodied in other forms not specifically shown in the drawings.

Definition of the definition

The term "herpes simplex virus" or "HSV" as used herein refers to enveloped icosahedral double stranded DNA viruses that infect mammals, including humans. Wild-type HSV infects terminally differentiated non-dividing cells and replicates therein. "HSV-2" refers to a member of the HSV family that contains the ICP10 gene. The term "FusOn-H2" as used herein refers to HSV-2 mutants having a modified ICP10 polynucleotide encoding a polypeptide having ribonucleotide reductase activity but lacking protein kinase activity as described herein. FusOn-H2 and modified ICP10 polynucleotides are described in U.S. Pat. Nos. 8,986,672 and 10,039,796 and U.S. patent publication No. 2015/0246086, which are hereby incorporated by reference.

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

The terms "cell membrane fusion" and "fusion" as used herein refer to the fusion of the outer membranes of at least two cells, such as two adjacent cells.

The term "enhanced fusion activity" as used herein refers to enhancement, increase, fortification, potentiation, amplification or a combination thereof of cell membrane fusion.

The term "oncolytic" as used herein refers to the property of an agent that can directly or indirectly cause destruction of malignant cells. In a specific embodiment, the property comprises causing fusion of the malignant cell membrane with another membrane.

The term "vector" as used herein refers to a vector nucleic acid molecule into which a nucleic acid sequence may be inserted for introduction into a cell in which it may be replicated. An inserted nucleic acid sequence is said to be "exogenous" when it is foreign to the cell into which the vector is introduced, or when it is homologous to a sequence in the cell but in a position where the sequence is not normally present in the host cell nucleic acid. The vector may be a non-viral DNA vector or a viral vector. The viral vector is encapsulated in a viral protein and is capable of infecting cells. Non-limiting examples of carriers include: viral vectors, non-viral vectors, naked DNA expression vectors, plasmids, cosmids, artificial chromosomes (e.g., YACS), phage vectors, DNA expression vectors in combination with cationic condensing agents, DNA expression vectors encapsulated in liposomes, or certain eukaryotic cells such as producer cells. As used herein, "vector" refers to DNA vectors and viral vectors unless otherwise indicated. The person skilled in the art will be able to construct the vector by standard recombinant techniques. Generally, these include Sambrook et al, molecular Cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory Press (1989), and references cited therein. Coen D.M, molecular Genetics of Animal Viruses in Virology, 2 nd edition, b.n. fields (editions), raven Press, n.y. (1990), and references cited therein, also review virologic considerations.

The term "expression vector" refers to any type of genetic construct comprising a nucleic acid encoding an RNA capable of being transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example in the preparation of antisense molecules or ribozymes. Expression vectors may contain a variety of "control sequences," which refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that also serve other functions, as described below.

A "promoter" is a control sequence, which is a region of a nucleic acid sequence that controls transcription initiation and rate. It may contain genetic elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors, to initiate or regulate the temporal and spatial transcription of nucleic acid sequences. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional position and/or orientation relative to the nucleic acid sequence to control transcription initiation and/or expression of the sequence. Exemplary non-limiting promoters include: constitutive promoters, tissue-specific promoters, tumor-specific promoters or endogenous promoters under the control of exogenous inducible elements.

The term "constitutive promoter" as used herein refers to a promoter that drives expression of a gene or polynucleotide in a continuous time fashion throughout the cell cycle. Constitutive promoters may be cell or tissue type specific as long as they operate in a continuous fashion throughout the cell cycle to drive expression of the gene or polynucleotide with which they are associated. Exemplary non-limiting constitutive promoters include: an immediate early Cytomegalovirus (CMV) promoter, an SV40 early promoter, an RSV LTR, a beta chicken actin promoter, and an HSV TK promoter.

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

The phrase "modified ICP10 polynucleotide" refers to an ICP10 polynucleotide encoding an ICP10 polypeptide having Ribonucleotide Reductase (RR) activity but lacking protein kinase activity.

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

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 that are capable of activating the Ras/MEK/MAPK pathway.

The term "effective" or "therapeutically effective" as used herein refers to suppressing or inhibiting the worsening of symptoms, inhibiting, suppressing or preventing the onset of a disease, inhibiting, suppressing or preventing the spread of a disease, ameliorating at least one symptom of a disease, or a combination thereof.

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.

The term "serum (sera)" or "serum (serum)" refers to the blood fluid that remains when blood cells and coagulin are removed.

The term "anti-cancer agent" as used herein refers to an agent that can adversely affect a cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply of tumor or cancer cells, promoting an immune response against cancer cells or tumor, preventing or inhibiting the progression of cancer, or increasing the longevity of a subject with cancer.

The phrase "pharmaceutically" or "pharmacologically acceptable" as used herein refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human. The phrase "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

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

Introduction to the invention

Viruses can only replicate within living cells, and their replication often requires activation of certain cell signaling pathways. Many viruses have acquired various strategies during their evolution to activate these signaling pathways to benefit their replication. The large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP 10 or RR 1) comprises a unique amino-terminal domain with serine/threonine Protein Kinase (PK) activity. This PK activity has been found to activate the cellular Ras/MEK/MAPK pathway (Smith et al, (2000) J Virol 74 (22): 10417-29). Thus, deletion of this PK domain (ICP 10 PK) from the ribonucleotide reductase gene has been reported to severely impair the ability of the virus to replicate in cells, such as those in which there is no pre-existing activated Ras signaling pathway (Smith et al, (1998) J.Virol.72 (11): 9131-9141).

When the PK domain of HSV-2 is replaced and/or modified such 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 those in which the Ras signaling pathway is constitutively activated due to tumorigenesis. Furthermore, modifications of ICP10 polynucleotides as described herein allow the virus to fuse inherently, i.e. infection of tumor cells with the virus induces extensive cell membrane fusion (syncytia formation). This property increases the destructive power of the virus on tumor cells. In addition, in vivo studies have shown that this virus is extremely safe for local or systemic administration.

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

In some embodiments, HSV-2 is genetically engineered by inserting a second polynucleotide into a polynucleotide encoding a protein kinase activity domain of the ICP10 gene, or by replacing a portion of the protein kinase domain with a second polynucleotide such that the polypeptide encoded by the modified polynucleotide has ribonucleotide reductase activity but lacks protein kinase activity. For example, the second polynucleotide may encode a glycoprotein, such as a fusion membrane glycoprotein. A preferred glycoprotein for use within the scope of the present invention is a truncated form of the gibbon leukemia virus envelope fusion membrane glycoprotein (GALV. Fus). In certain aspects of the invention, expression of galv.fus significantly enhances the anti-tumor effect of the virus in the context of the oncolytic viruses of the invention.

In some embodiments, the modified HSV-2 of the present invention comprises a mutation, such as a deletion, in ICP10 that provides cell fusion properties to the virus. Such mutations may be randomly generated during viral screening or obtained from nature and then assayed for function of a pool of potential candidates having cell fusion properties by means described herein and/or known in the art. Mutations that result in a fusion phenotype may be point mutations, frameshifts, inversions, deletions, splice error mutations, post-transcriptional processing mutations, overexpression of certain viral glycoproteins, combinations thereof, and the like. Mutations can be identified by sequencing a particular HSV-2 and comparing it to known wild-type sequences.

The modified HSV-2 of the present invention may be used to treat malignant cells, such as to inhibit their spread, reduce or inhibit their division, eradicate them, prevent their production or proliferation, or a combination thereof. Malignant cells may be from any form of cancer, such as a solid tumor, but other forms are treatable as well. The modified HSV-2 of the present invention may be used to treat lung, liver, prostate, ovary, breast, brain, pancreas, testis, colon, head and neck, melanoma, and other types of malignancies. The invention is useful for treating malignant cells at any stage of a cancer disease, including the metastatic stage of the disease. The present invention may be used as stand alone therapy or in combination with other therapeutic means including chemotherapy, surgery or radiation therapy.

Modified ICP10 polynucleotides

The present invention describes HSV-2 mutants having a modified ICP10 polynucleotide, wherein the modified ICP10 polynucleotide encodes a polypeptide having ribonucleotide reductase activity but lacking Protein Kinase (PK) activity. The ICP10 polynucleotide may be modified by deleting at least some of the sequence required to encode a functional PK domain or replacing at least a portion of the sequence encoding a PK domain with a second polynucleotide. One of skill in the art will recognize that any suitable method may be used to generate the modified ICP10 polynucleotide, including mutagenesis, polymerase chain reaction, homologous recombination, or any other genetic engineering technique known to those of skill in the art.

Mutagenesis

In particular embodiments of the invention, the ICP10 sequence of HSV-2 virus is mutated, such as by deletion, using any of a number of standard mutagenesis methods. Mutations may involve modification of the nucleotide sequence, individual genes or gene blocks. Mutations may involve single nucleotides (such as point mutations, which involve the removal, addition or substitution of a single nucleotide base within a DNA sequence), or they may involve the insertion or deletion of a large number of nucleotides. Mutations may occur spontaneously as a result of events such as DNA replication fidelity errors, or may be induced upon exposure to chemical or physical mutagens. Mutations can also be targeted by using specific targeting methods well known to those skilled in the art.

Gene recombination

In other embodiments of the invention, ICP10 polynucleotides are modified using genetic recombination 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/substituted may be any suitable region as long as the polypeptide encoded by the modified ICP10 polynucleotide retains ribonucleotide reductase activity and lacks protein kinase activity. However, in certain embodiments, modifications to the PK domain affect one or more of the eight PK catalytic motifs (amino acid residues 106-445, although PK activity may be considered amino acid residues 1-445) and/or Transmembrane (TM) regions and/or constant Lys (Lys 176). SEQ ID NO. 15 (national center for Biotechnology information GenBank database accession No. 1813262A) provides an exemplary wild-type ICP10 polypeptide sequence. SEQ ID NO. 17 provides an exemplary wild type polynucleotide encoding an ICP10 polypeptide.

In certain embodiments, the ICP10 polynucleotide is modified by deleting only a portion of the sequence encoding a PK domain necessary for PK activity. SEQ ID NO. 18 provides exemplary ICP10 polynucleotides lacking at least some of the PK domain encoding sequences. In another exemplary embodiment, the ICP10 polynucleotide is modified such that the PK domains are all deleted as provided in SEQ ID NO. 19. Both SEQ ID NO. 18 and SEQ ID NO. 19 are suitable for generating HSV-2 mutants as described herein, since both sequences encode polypeptides having ribonucleotide reductase activity but lacking protein kinase activity. 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 described in SEQ ID NO. 20.

In other embodiments 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 a green fluorescent protein, in frame with the sequence encoding the RR domain of the ICP10 polynucleotide. The construct may be under the control of an endogenous HSV-2 promoter, or under the control of a constitutive promoter such as a CMV promoter (SEQ ID NO: 20).

In another aspect of the invention, a polynucleotide that replaces at least a portion of the protein kinase activity domain of endogenous ICP10 in HSV-2 may encode at least a fusion portion of a cell membrane fusion inducing polypeptide, such as a viral Fusion Membrane Glycoprotein (FMG). For example, the polypeptide is preferably capable of inducing cell membrane fusion at a substantially neutral pH (such as about pH 6-8).

In particular embodiments, FMG comprises at least a fusion domain derived from a retroviral envelope protein of type C, such as MLV (e.g., SEQ ID NO: 6) or GALV (e.g., SEQ ID NO: 5). Retroviral envelope proteins lacking some, most or all cytoplasmic domains are useful because such manipulation results in fusion activity of human cells that is too high. In some embodiments, specific modifications are introduced into the viral membrane glycoprotein to enhance its function of inducing cell membrane fusion. For example, truncations of cytoplasmic domains of various retroviral and herpesvirus glycoproteins have been shown to increase their fusion activity, sometimes while simultaneously reducing their efficiency of incorporation into virions (Rein et al, (1994) J Virol 68 (3): 1773-81).

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, retrovirus Env protein (SEQ ID NO: 10), ebola virus Gp (SEQ ID NO: 11) and influenza virus hemagglutinin (SEQ ID NO: 12).

In other embodiments, the second functional polynucleotide may be inserted into the PK domain, or used to replace part or all of the PK domain. The second functional polynucleotide may encode an immunomodulatory or other therapeutic agent. It is contemplated that these additional agents will affect the upregulation of cell surface receptors and GAP junctions, inhibit cell growth and differentiation agents, inhibit cell adhesion or increase the sensitivity of malignant cells to apoptosis. Illustrative non-limiting examples of polynucleotides encoding immunomodulators 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.

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

Ribonucleotide reductase Activity assay

The biological activity of RR can be tested as described previously (Averett et al, J.biol. Chem.258:9831-9838 (1983) and Smith et al, J.Virol.72:9131-9141 (1998)) modified as follows. BHK cells were initially grown to confluence in complete GMEM (containing 10% FBS), then incubated in 0.5% FBS EMEM for three days, followed by infection with 20 pfu of wild-type HSV, HSV-2 mutants or mock infectious agents. Cells were harvested 20 hours after infection and resuspended in 500. Mu.l of HD buffer [100mM HEPES buffer (pH 7.6), 2mM Dithiothreitol (DTT)]And incubated on ice for 15 minutes, followed by sonication for 30 seconds. Cell debris was removed by centrifugation (16,000 g,20 min, 4 ℃) and the supernatant was precipitated with 45% saturation (0.258 g/ml) of crystalline ammonium sulfate. After a second centrifugation (16,000 g,30 min), the pellet was dissolved in 100. Mu.l of HD buffer, from which 50. Mu.l of 2 Xreaction buffer (400 mM HEPES buffer (pH 8.0), 20mM DTT and 0.02mM [ were removed in equal volumes) ³ H]CDP (24 Ci/mmol, amersham, chicago, ill.) were mixed. The reaction was stopped by adding 100mM hydroxyurea containing 10mM EDTA (pH 8.0) and boiling for 3 minutes. 1ml of a western sidetrack rattle venom (Sigma, st.louis, mo.) was then added and incubated at 37 ℃ for 30 minutes, followed by boiling for an additional 3 minutes. The solution was then passed through a 0.5ml Dowex-1 borate column, eluting the sample with 2ml water, and four eluted fractions were collected for scintillation counting after mixing with Biofluor (New England Nuclear, boston, mass.). Ribonucleotide reductase activity is expressed as units per mg protein, wherein 1 unit represents 1nmol [ ³ H]CDP was converted to dCDP/hr/mg protein.

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 or wild-type HSV-2 having the modified ICP10 polynucleotide (moi=200, 16 hours post infection) were immunoprecipitated with an anti-LA-1 antibody and PK assays were performed as described in Chung et al, j. Virol.63:3389-3398,1998 and U.S. patent No. 6,013,265. Generally, immunoprecipitates of cell extracts were normalized to protein concentration using BCA protein assay kit (PIERCE, rockford ill.) washed with TS buffer containing 20mM Tris-HCL (pH 7.4), 0.15M NaCl, suspended in 50 μl of a solution consisting of 20mM Tris-HCL (pH 7.4), 5mM MgCl ₂ 、2mM Mn Cl ₂ 、10μCi[ ³² p]ATP (3000 Ci/mmol, duPont, new England Research Prod.) in kinase reaction buffer and incubated at 30℃for 15 min. The beads were washed once with 1ml TS buffer, resuspended in 100. Mu.l denaturing solution and boiled for 5 minutes. The proteins were then resolved by SDS-PAGE on a 7% polyacrylamide gel. Proteins were then electrotransferred onto nitrocellulose membranes as previously described (see Aurelian et al, cancer Cells 7:187-191 1989) and immunoblotted by incubation with specific antibodies followed by incubation with 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).

Vector construction

The present invention relates to HSV-2 vectors comprising substitutions or deletions 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 particular embodiments also comprises regulatory sequences, such as a constitutive promoter. In some embodiments, the composition is a naked (non-viral) DNA vector comprising a modified ICP10 gene, while in other embodiments, the composition is a recombinant HSV-2 having a modified ICP10 gene. Both the naked DNA vector and the recombinant virus may also comprise some or all of the following components.

Carrier body

Vectors as defined above include, but are not limited to, plasmids, cosmids, viruses (phage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Methods of construction of engineered viruses and DNA vectors are known in the art. Generally, these include Sambrook et al, molecular Cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory Press (1989), and references cited therein. Coen D.M, molecular Genetics of Animal Viruses in Virology, 2 nd edition, b.n. fields (editions), raven Press, n.y. (1990), and references cited therein, also review virologic considerations.

Expression vectors may contain a variety of "control sequences," which refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that control transcription and translation, DNA vectors, expression vectors, and viruses may contain nucleic acid sequences that also serve other functions, as described below.

1.Promoters and enhancers

Promoters typically comprise sequences for locating the start site of RNA synthesis. The most well known example is the TATA box, but in some promoters lacking a TATA box (e.g., promoters of mammalian terminal deoxynucleotidyl transferase genes and promoters of SV40 late genes), discrete elements covering the initiation site itself help to fix the initiation position. Additional promoter elements regulate the frequency of transcription initiation. Typically, they are located in a region 30 to 110bp upstream of the start site, but many promoters have been shown to contain functional elements downstream of the start site as well. In order to place the coding sequence "under the control of" the promoter, the 5 'end of the transcription initiation site of the transcriptional reading frame is positioned "downstream" (i.e., 3') of the selected promoter. An "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

The spacing between promoter elements is typically flexible so that promoter function is maintained when the elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50bp before activity begins to decrease. Depending on the promoter, it appears that individual elements may act synergistically or independently to activate transcription. Promoters may or may not be used in combination with enhancers.

The promoter may be one naturally associated with the nucleic acid sequence, which may be obtained by isolation of 5' non-coding sequences located upstream of the coding segments and/or exons. Similarly, an enhancer may be one naturally associated with a nucleic acid sequence located downstream or upstream of the sequence. Alternatively, certain advantages will be 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. 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, as well as promoters or enhancers isolated from any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally occurring" (i.e., contain different elements of different transcriptional regulatory regions and/or mutations that alter expression). For example, the promoters most commonly used in recombinant DNA construction include the beta lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to synthetically producing nucleic acid sequences of promoters and enhancers, recombinant cloning and/or nucleic acid amplification techniques (including PCR) can be used in conjunction with the compositions disclosed herein to produce sequences (see U.S. Pat. nos. 4,683,202 and 5,928,906). Furthermore, it is contemplated that control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like may also be employed.

Of course, it is important to employ promoters and/or enhancers which are effective to direct the expression of a DNA fragment in the organelle, cell type, tissue, organ or organism selected for expression. The promoters employed may be constitutive, tissue-specific, inducible, and/or may be useful under appropriate conditions to direct high level expression of the introduced DNA fragments, such as is advantageous in large-scale production of recombinant proteins and/or peptides. Promoters may be heterologous or endogenous.

In addition, 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 may support cytoplasmic transcription from certain bacterial promoters, either as part of a delivery complex or as an additional gene expression construct, if provided with an appropriate bacterial polymerase.

The identification of tissue-specific promoters or elements and assays for characterizing their activity are well known to those skilled in the art. Non-limiting examples of such regions include the human LIMK2 Gene (N omoto et al, (1999) Gene 236 (2): 259-271), the somatostatin receptor-2 Gene (Kraus et al, (1998) FEBS Lett.428 (3): 165-170), the murine epididymal retinoic acid binding Gene (Lareyr et al, (1999) J.biol.chem.274 (12): 8282-8290), human CD4 (Zhao-Emonet et al, (1998) Biochem. Acta,1442 (2-3): 109-119), mouse alpha-2 (XI) collagen (Tsumaki et al, (1998), J.biol. Chem.273 (36): 22861-4), the D1A dopamine receptor Gene (Lee et al, (1997), DNA Cell l.16 (11): 1277-5), insulin-like growth factor II (U et al, (1997) Biohem Biochem.226 (1996) and human endothelial Cell (1-157) adhesion (1996) 1) and small human endothelial Cell (54-157) (human blood Cell) Cell (54-157).

2.Initiation signal and internal ribosome binding site

Efficient translation of the coding sequence may also require a specific initiation signal. These signals include the ATG initiation codon or adjacent sequences. It may be desirable to provide exogenous translational control signals, including the ATG initiation codon. One of ordinary skill in the art will be readily able to determine this and provide the necessary signals. It is well known that the initiation codon must be in "the same reading frame" as the desired coding sequence to ensure translation of the entire insert. Exogenous translational control signals and initiation codons can be natural or synthetic. Expression efficiency can be increased by the inclusion of appropriate transcriptional enhancer elements.

In certain embodiments of the invention, internal Ribosome Entry Site (IRES) elements are used to generate polygenic or polycistronic information. IRES elements are able to bypass the ribosome scanning pattern of 5' methylated Cap dependent translation and begin translation at internal sites. IRES elements may be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, to produce polycistronic information. Due to the IRES element, each open reading frame can access the ribosome for efficient translation. A single promoter/enhancer can be used to transcribe a single message to efficiently express multiple genes (see U.S. patent nos. 5,925,565 and 5,935,819).

3.Termination signal

The vectors or constructs of the invention generally comprise at least one termination signal. A "stop signal" or "terminator" consists of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. Thus, in certain embodiments, a termination signal that terminates the production of an RNA transcript may be envisaged. Terminators may be necessary in vivo to achieve the desired information level.

In eukaryotic systems, the terminator region may also comprise a specific DNA sequence that allows site-specific cleavage of the new transcript to expose the polyadenylation site. This marks that a specific endogenous polymerase adds a fragment of about 200A residues (polyA) to the 3' end of the transcript. RNA molecules modified with such polyA tails appear to be more stable and more efficient in translation. Thus, in other embodiments involving eukaryotic cells, it is contemplated that the terminator comprises a signal for RNA cleavage, and that the terminator signal promotes polyadenylation of the information. Terminator and/or polyadenylation site elements may be used to enhance information levels and minimize read-through from the cassette to other sequences.

It is contemplated that the terminator used in the present invention includes any known transcription terminator described herein or known to one of ordinary skill in the art, including, but not limited to, termination sequences of genes, such as bovine growth hormone terminator or viral termination sequences, such as SV40 terminator, for example. In certain embodiments, the termination signal may be lack of a transcribable or translatable sequence, such as due to sequence truncation.

4.Polyadenylation signal

In expression, particularly eukaryotic expression, polyadenylation signals are typically included to effect proper polyadenylation of the transcript. It is believed that the nature of the polyadenylation signal is not critical to the successful practice of the present invention, and that any such sequence may be used. Preferred embodiments include SV40 polyadenylation signals or bovine growth hormone polyadenylation signals, both of which are convenient and are known to function well in a variety of target cells. Polyadenylation may increase the stability of the transcript or may promote cytoplasmic translocation.

5.Selectable and screenable markers

In certain embodiments of the invention, cells containing the nucleic acid constructs of the invention can be identified in vitro or in vivo by including a marker in the expression vector. Such markers will confer a recognizable change to the cells, allowing for easy identification of the cells containing the expression vector. Generally, a selectable marker is one that confers a property that allows for selection. A positive selection marker is a marker in which the presence of the marker allows its selection, whereas a negative selection marker is a marker in which the presence of the marker prevents its selection. An example of a positive selection marker is a drug resistance marker.

In general, the inclusion of a drug selectable marker aids in the cloning and identification of transformants, for example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, bleomycin (zeocin) and histidinol are useful selectable markers. In addition to conferring markers that allow the phenotype of transformants to be distinguished based on the implementation of conditions, other types of markers are contemplated, including screenable markers such as GFP, the basis of which is colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or Chloramphenicol Acetyl Transferase (CAT) may be utilized. The skilled person also knows how to use an immunological marker, possibly in combination with Fluorescence Activated Cell Sorting (FACS) analysis. It is believed that the marker used is not critical, as long as it is capable of simultaneous expression with the nucleic acid encoding the gene product. Other examples of selectable and screenable markers are well known to those of skill in the art.

The vector is introduced into the initially infected cells by a suitable method. It is believed that such nucleic acid delivery methods for transforming an organelle, cell, tissue or organism for use in the invention include virtually any method by which a nucleic acid (e.g., an HSV vector) can be introduced into an organelle, cell, tissue or organism, as described herein or as known to one of ordinary skill in the art. 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. patent No. 5,789,215); electroporation (U.S. Pat. No. 5,384,253); precipitating calcium phosphate; DEAE dextran then polyethylene glycol; direct acoustic loading; liposome-mediated transfection; receptor-mediated transfection; microprojectile bombardment (PCT application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042;5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880); stirring with silicon carbide fibers (U.S. Pat. nos. 5,302,523 and 5,464,765); agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055); PEG-mediated protoplast transformation (U.S. patent nos. 4,684,611 and 4,952,500); drying/inhibiting mediated DNA uptake, as well as any combination of these methods, or other methods known to those of skill in the art. The composition may also be delivered to cells of the mammal by systemic administration, such as intravenous administration, in a pharmaceutically acceptable excipient.

Method for delivering DNA vectors to cells

1.In vitro transformation

Methods for transfecting cells and tissues removed from organisms in an ex vivo environment are known to those skilled in the art. Thus, it is contemplated that in the present invention, the nucleic acids and compositions described herein can be used to remove and transfect cells or tissues ex vivo. In particular aspects, the transplanted cells or tissues may be placed in an organism. In some embodiments, the nucleic acid is expressed in the transplanted cell or tissue.

2.Injection of

In certain embodiments, the nucleic acid may be delivered to the 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 of ordinary skill in the art (e.g., injection of a composition comprising a saline solution). Other embodiments of the invention include introducing nucleic acids by direct microinjection. The amount of the composition of the invention may vary depending on the nature of the cell, tissue or organism affected.

3.Electroporation method

In certain embodiments of the invention, the nucleic acid is introduced into the organelle, cell, tissue, or organism via electroporation. Electroporation involves exposing a suspension of cells and DNA to a high voltage discharge. In some variations of this method, certain cell wall degrading enzymes, such as pectin degrading enzymes, are employed to render target recipient cells more susceptible to transformation by electroporation than untreated cells (U.S. Pat. No. 5,384,253). Alternatively, the recipient cells may be more easily transformed by mechanical trauma.

4.Liposome-mediated transfection

In another embodiment of the invention, a composition as described herein, such as a vector with a modified ICP10 polynucleotide, can be embedded in a lipid complex, such as a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. Which spontaneously forms when phospholipids are suspended in an excess of aqueous solution. The lipid component undergoes self-rearrangement prior to forming a closed structure and entraps water and dissolved solutes between the lipid bilayers. Nucleic acids complexed with Lipofectamine (Gibco BRL) or Superfect (Qiagen) are also contemplated.

In certain embodiments of the invention, the liposomes can be complexed with Hemagglutinin Virus (HVJ). This has been shown to promote fusion with cell membranes and promote cellular entry of liposome-encapsulated DNA (Kaneda et al, (1989) Science 20;243 (4889): 375-8). In other embodiments, liposomes can be complexed or used in combination with nuclear non-histone chromosomal proteins (HMG 1) (Kato et al, (1991) J Biol chem. (1991) February 25;266 (6): 3361-4). In other embodiments, the liposomes may be complexed or used in combination with HVJ and HMG 1. In other embodiments, the delivery vehicle may comprise a ligand and a liposome.

5.Receptor-mediated transfection

Nucleic acids can be delivered to target cells via a receptor-mediated delivery vehicle. The method utilizes receptor-mediated endocytosis for selective uptake of macromolecules. This delivery method additionally increases the degree of specificity of the invention in view of the cell type specific distribution of the various receptors.

In certain embodiments, the receptor-mediated gene targeting vehicle comprises a receptor-specific ligand and a nucleic acid binding agent. Other embodiments include receptor-specific ligands to which the nucleic acid to be delivered has been operably linked. Several ligands have been used for receptor-mediated gene transfer, including Epidermal Growth Factor (EGF), which has been used to deliver genes to squamous cancer cells, as described in european patent No. EPO 0 273 085.

In other embodiments, the nucleic acid delivery vehicle component of the cell-specific nucleic acid targeting vehicle can comprise a specific binding ligand in combination with a liposome. The nucleic acid to be delivered is contained in a liposome and the specific binding ligand is functionally incorporated into the liposome membrane. Thus, the liposome will specifically bind to the receptor of the target cell and deliver the contents to the cell.

In further embodiments, the nucleic acid delivery vehicle component of the targeted delivery vehicle may be the liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding. For example, lactose ceramide, a galactose-terminated asialoglycoside, has been incorporated into liposomes, and an increase in insulin gene uptake by hepatocytes has been observed (Nicolau et al, (1987) Methods enzymol. 149:157-76). It is contemplated that tissue-specific transformation constructs of the invention may be specifically delivered into target cells in a similar manner.

6.Microprojectile bombardment

Microprojectile bombardment techniques can be used to introduce nucleic acids into at least one organelle, cell, tissue or organism (U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880; U.S. Pat. No. 5,610,042; and PCT application No. WO 94/09699). The method relies on the ability to accelerate DNA-coated or DNA-containing microparticles to a high velocity that allows them to penetrate cell membranes and enter cells without killing them. The particles may be composed of any biologically inert substance, such as tungsten, platinum or gold. For bombardment, the cells in suspension are concentrated on a filter or solid medium. Alternatively, immature embryos or other target cells can be arranged on a solid medium. The cells to be bombarded are placed at a suitable distance below the microprojectile bombardment device on the stopper plate. A variety of microprojectile bombardment techniques for practicing the invention will be known to those skilled in the art.

Host cells

As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably. All these terms also include their progeny, which are any and all offspring. It is understood that all progeny may not be identical, due to deliberate or inadvertent mutation. In the context of expressing a heterologous nucleic acid sequence, "host cell" refers to a prokaryotic or eukaryotic cell and includes any transformable organism capable of replicating the vector and/or expressing the heterologous gene encoded by the vector. Host cells may and have been used as receptors for vectors. The host cell may be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid is transferred or introduced into the host cell. Transformed cells include primary test cells and their progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells refer to cells into which an exogenous nucleic acid sequence (such as a vector) has been introduced. Thus, recombinant cells are distinguishable from naturally occurring cells that do not contain the recombinantly introduced nucleic acid.

The tissue may comprise one or more host cells transformed with an HSV-2 mutant that produces cell membrane fusion. The tissue may be part of or separate from the organism. In certain embodiments, the tissue may include, but is not limited to, adipocytes, alveoli, enameloblasts, nerves, basal cells, blood (e.g., lymphocytes), blood vessels, bones, bone marrow, glial cells, breast, cartilage, cervix, colon, cornea, embryos, endometrium, endothelium, epithelium, esophagus, fascia (fasciae), fibroblasts, follicles, ganglion cells, glial cells, goblet cells, kidneys, liver, lungs, lymph nodes, muscles, neurons, ovaries, pancreas, peripheral blood, prostate, skin, small intestine, spleen, stem cells, stomach, testes, and all cancers thereof.

In certain embodiments, the host cell or tissue may be comprised in at least one organism. In certain embodiments, the organism may be, but is not limited to, a prokaryote (e.g., eubacterium, archaebacteria) or a eukaryote, as understood by one of ordinary skill in the art.

Many cell lines and cultures are available as host cells and are commercially available through tissues such as the American Type Culture Collection (ATCC). Suitable hosts can be determined by one skilled in the art based on the vector backbone and the desired results. Exemplary non-limiting cell types that can be used for vector replication and/or expression include bacteria, such as E.coli (e.g., E.coli strains RR1, LE392, B, X1776 (ATCC No. 31537), W3110, F, λ, DH5 a, JM109, and KC 8); bacillus, such as bacillus subtilis (Bacillus subtilis); other enterobacteriaceae, such as salmonella typhimurium (Salmonella typhimurium), serratia marcescens (Serratia marcescens), and a variety of commercially available bacterial hosts and competent cells, such as Competent cells and SOLOPACK ^TM Gold cell [ ]La Jolla, calif.). Non-limiting examples of eukaryotic host cells for vector replication and/or expression include HeLa, NIH3T3, jurkat, 293, cos, CHO, saos, and PC12.

Some vectors may employ control sequences that allow replication and/or expression in prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the above-described host cells are incubated to maintain them and allow the vector to replicate. It is also understood and known that techniques and conditions allow for large scale production of vectors and production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins or peptides.

Viral vector packaging and propagation

1.Virus package

In a specific embodiment of the invention, the ICP10 gene is inserted into the virus by homologous recombination after modification. Typically, this is done by co-transfecting plasmid DNA containing the modified ICP10 gene with purified HSV-2 genomic DNA into Vero cells using Lipofectamine. Recombinant viruses are then identified (typically by screening for the presence of a selectable marker in the viral plaques) and plaques containing the modified ICP10 polynucleotide are selected. The selected recombinant virus was then characterized in vitro to confirm that the modified ICP10 gene had been correctly inserted into the HSV-2 genome to replace the original ICP10 gene.

2.Preparation of virus stock

Once the recombinant HSV-2 mutant virus is selected, a virus stock may be prepared as follows. Vero cells were grown in 10% Fetal Bovine Serum (FBS) and infected at 0.01 plaque forming units (pfu) per cell. The virus was harvested from the cells after 2 days by repeated freeze thawing and sonication. The harvested virus is then purified as described in (Nakamori et al, (2003) Clinical Cancer Res.9 (7): 2727-2733). Purified virus was then titrated, aliquoted and stored at-80 ℃ until use.

Protein expression system

Protein expression systems can be used to generate DNA vector compositions of the invention, for example, to express polypeptides encoded by modified ICP10 polynucleotides for functional studies. There are many expression systems comprising at least part or all of the above-described compositions. Prokaryotic and/or eukaryotic based systems may be used in the present invention to produce nucleic acid sequences or their cognate polypeptides, proteins and peptides. Many such systems are widely available commercially.

Insect cell/baculovirus systems can produce high levels of protein expression of heterologous nucleic acid segments, such as described in U.S. Pat. nos. 5,871,986 and 4,879,236, and are commercially available (e.g., CLONTECH, inc.Mountain View, calif.).

Other examples of commercially available expression systems include inducible mammalian expression systems, which involve synthetic ecdysone inducible receptors, or pET expression systems, or e.coli expression systems (STRATAGENE, laJolla, calif.); tetracycline regulated expression systems, which are inducible mammalian expression systems using the full length CMV promoter, or yeast expression systems designed for high level production of recombinant proteins in the methylotrophic yeast pichia methanolica (Pichia methanolica) (INVITROGEN, carlsbad, calif.).

It is contemplated that the protein, polypeptide or peptide produced by the methods of the invention may be "overexpressed," i.e., expressed at an increased level relative to its native expression in a cell. Such overexpression can be assessed by a variety of methods, including radiolabeling and/or protein purification. However, simple and straightforward methods are preferred, such as those involving SDS/PAGE and protein staining or Western blotting, followed by quantitative analysis, such as optical density scanning or blotting of the resulting gel. An increase in the specificity of the level of a recombinant protein, polypeptide or peptide compared to the level in a native cell is indicative of overexpression, such as the relative abundance of a particular protein, polypeptide or peptide relative to other proteins produced by the host cell, and is visible, for example, on a gel.

Functional roles of HSV-2 mutants

HSV-2 mutants as described herein exhibit a variety of functional roles as oncolytic agents. For example, viruses can destroy tumor cells by lysis and syncytial formation, and induce apoptosis in infected cells as well as in neighboring cells. In addition, tumor destruction of HSV-2 mutants induces an effective anti-tumor immune response, which further contributes to the therapeutic efficacy of the mutant virus as an oncolytic agent for the treatment of malignant diseases.

HSV-2 mutant viruses exhibit selective replication in circulating cells but not non-circulating cells. The mutant HSV-2 lacking protein kinase activity has reduced growth in non-circulating cells to at least 1/40 as compared to growth in circulating cells. In contrast, the growth characteristics of wild-type HSV-2 between circulating and non-circulating cells were only marginally affected. Thus, HSV-2 mutants as described herein are well suited for use as oncolytic agents in circulating cells, such as tumor cells, having an activated Ras pathway.

In addition to lytic and fusion activity, HSV-2 mutants also have potent apoptosis-inducing activity and are capable of inducing potent anti-tumor immune responses. In an in vitro setting, HSV-2 mutants may induce apoptosis in virus-infected cells as well as uninfected bystander cells surrounding the infected cells. In addition, HSV-2 mutants are effective in inducing apoptosis of tumor cells in vivo. The compositions described herein not only kill tumor cells more effectively than other oncolytic viruses, but HSV-2 mutants also exhibit a strong therapeutic effect on primary and metastatic tumors in vivo by inducing a strong anti-tumor immune response. Adoptive transfer CTLs from FusOn-H2 treated mice inhibited the growth of primary tumors and effectively prevented the development of metastasis.

Apoptosis or programmed cell death is a necessary process for normal embryonic development, maintenance of adult tissue homeostasis, and suppression of carcinogenesis. In some embodiments of the invention, the modified HSV-2 is an apoptosis-inducing agent effective in both virus-infected tumor cells and uninfected, bypass tumor cells. For example, in one particular embodiment, 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 by visualizing GFP under fluorescent microscopy, and cells undergoing apoptosis are identified by their chromatin condensation. Cells exhibiting chromatin condensation to GFP expression at a ratio of 2.6:1 indicate the presence of a large number of tumor cells that underwent apoptosis that were not infected with modified HSV-2.

A strong anti-tumor immune response can be used to combat malignant diseases. The HSV-2 mutants described herein are capable of inducing potent anti-tumor immune responses against primary and metastatic tumors in vivo. In a particular embodiment, mutant HSV-2 (FusOn-H2) replicates and lyses selectively in tumor cells in a mouse mammary tumor model using a 4T1 mouse mammary tumor cell line and shows a strong therapeutic effect against primary and metastatic tumors in vivo by inducing a strong anti-tumor immune response. In particular, adoptive transfer Cytotoxic T Lymphocytes (CTLs) from FusOn-H2 treated mice can inhibit the growth of primary tumors and effectively prevent metastasis in mice not treated with FusOn-H2.

Pharmaceutical compositions and routes of administration

The compositions of the invention may be administered as pharmaceutical compositions comprising recombinant HSV-2 mutants having a modified ICP10 gene or naked (non-viral) DNA vectors having a modified ICP10 gene, as described herein. The compositions of the present invention include classical pharmaceutical formulations. Generally, the compositions of the present invention may be administered as a pharmacological agent by dissolving or dispersing the composition in a pharmaceutically acceptable carrier or aqueous medium. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional medium 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 compositions. Administration of the composition will be via any common route, provided that the target cells are available via that route. Exemplary routes of administration include oral, nasal, buccal, rectal, vaginal, or topical. Alternatively, administration may be by in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous or direct intratumoral injection. Pharmaceutical formulations, dosages and routes of administration of the compositions of the present invention are described below.

Pharmaceutical formulations of HSV-2 mutants

The mutant virus compositions of the invention may be formulated as pharmaceutically acceptable formulations. Typically, the mutant virus is admixed with a pharmaceutically acceptable and virus compatible excipient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In addition, if desired, the formulations may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, adjuvants or immunopotentiators which enhance the efficacy of the viral mutants (see Remington's Pharmaceutical Sciences, gennaro, A.R. et al, published Mack Publishing Co., 18 th edition, 1990). For example, a typical pharmaceutically acceptable carrier for injection purposes may comprise 50mg to about 100mg of human serum albumin per milliliter of phosphate buffered saline. Additional non-limiting exemplary non-aqueous solvents suitable for formulation of the pharmaceutically acceptable composition include propylene glycol, polyethylene glycol, vegetable oils, sesame oil, peanut oil, and injectable organic esters such as ethyl oleate. Exemplary non-limiting aqueous carriers include water, aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, ringer's dextrose, and the like. Intravenous vehicles include fluids and nutritional supplements. Determination of the pH and exact concentration of the various components of the pharmaceutical composition is conventional and within the knowledge of one of ordinary skill in the art (see Goodman and Gilman's The Pharmacological Basis for Therapeutics, gilman, A.G. et al, pergamon Press publication, 8 th edition, 1990).

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

Routes and dosages of administration of HSV-2 mutants

The mutant virus composition may be delivered by any route that provides access to the target tissue. Exemplary non-limiting routes of administration may include oral, nasal, buccal, rectal, vaginal, topical, or by injection (including in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, or direct intratumoral injection). Typically, the viral mutants will be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid prior to injection may also be prepared. The formulation may also be emulsified.

For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if desired, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that may be employed will be known to those of skill in the art in light of the present disclosure. For example, a dose may be dissolved in 1ml of isotonic NaCl solution and added to 1000ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, e.g., remington's Pharmaceutical Sciences, "15 th edition, pages 1035-1038 and 1570-1580). Depending on the condition of the subject being treated, some variation in dosage will necessarily occur. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject.

Those skilled in the art will recognize that the optimal treatment regimen to provide a treatment using the compositions of the present invention can be determined directly. This is not an experimental problem, but one of the optimizations conventionally performed in the medical field. For example, in vivo studies in mice provide a starting point for starting optimized dose and delivery regimens. The injection frequency may initially be once a week. However, depending on the results obtained from the initial clinical trial and the needs of the particular patient, this frequency may be optimally adjusted from one day to every two weeks to every month. The human dose can be initially determined by extrapolation from the amount of composition used in the mice.

Dosage of

The amount of viral vector delivered will depend on several factors, including the number of treatments, the subject to be treated, the ability of the subject's immune system to synthesize antiviral antibodies, the target tissue to be destroyed, and the degree of protection desired. The precise amount of the viral composition to be administered depends on the discretion of the practitioner and is specific to each individual. However, a suitable dosage is at 10 ⁵ From plaque forming units (pfu) to 10 ¹⁰ The pfu range. In certain embodiments, the dose of viral DNA may be about 10 ⁵ 、10 ⁶ 、10 ⁷ 、10 ⁸ 、10 ⁹ At most and include 10 ¹⁰ Pfu.

Non-viral DNA vector formulations

In addition to the formulations described above for viral pharmaceutical formulations, the non-viral DNA vectors can also be prepared as sterile powders for the preparation of pharmaceutically acceptable sterile solutions. Typical methods for preparing sterile powders include vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Routes and dosages for administration of non-viral DNA vectors

Several methods of delivering non-viral vectors to transfer the polynucleotides of the invention into mammalian cells are contemplated. These methods include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment with high-speed microparticles, and receptor-mediated transfection as previously described. Some of these techniques may be successfully adapted for in vivo or ex vivo use.

In some embodiments of the invention, the expression vector may simply consist of naked recombinant DNA or a plasmid comprising the polynucleotide. The transfer of the construct may be performed by any of the methods mentioned herein for permeabilizing the cell membrane, either physically or chemically. This is particularly applicable to in vitro transfer, but may also be used in vivo.

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, nicolau et al incorporate lactose-ceramide (a galactose-terminated asialoglycoside) into liposomes and observed an increase in insulin gene uptake by hepatocytes (Nicolau et al, (1987) Methods enzymol.149:157-76). Thus, it is possible to specifically deliver a nucleic acid encoding a particular gene into a cell type through any number of receptor-ligand systems, with or without liposomes. For example, epidermal Growth Factor (EGF) can be used as a receptor for the mediated delivery of nucleic acids into cells exhibiting upregulation of the EGF receptor (as described in european patent No. EP 0 273 085), and mannose can be used to target mannose receptors on hepatocytes.

In certain embodiments, DNA transfer may be more readily performed under ex vivo conditions. Ex vivo gene therapy refers to the isolation of cells from animals, the in vitro delivery of nucleic acids into the cells, and the return of modified cells to the animal. This may involve surgical removal of tissue/organs from animals or primary cultures of cells and tissue.

Dosage of

In certain embodiments, the expected dosage may be at about 10 ³ The pfu/kg body weight is about 10 ⁸ The individual pfu/kg body weight. In certain embodiments, the dosage may be about 10 ³ 、10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ At most and include 10 ⁸ The pfu/kg body weight. Of course, depending on the results of the initial clinical trial and the needs of the particular patient, the dose may be adjusted up or down as is conventional in such treatment regimens.

Combination therapy

To increase 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 agents. The method may comprise contacting the cancer cell with a composition of the invention and at least one additional anti-cancer agent. This can be accomplished by contacting the cells with a single composition or pharmaceutical formulation comprising both agents, or by contacting the cells with two different compositions or formulations. When two different agents are used, the cancer cells may be contacted by both agents simultaneously, or one agent before the other (e.g., wherein the composition of the invention is administered before or after the administration of the other anticancer agent), or any combination or repeated cycling thereof. In embodiments where the composition of the invention and the other agent are administered separately, it will generally be ensured that no significant period of time is exceeded between each delivery, so that the composition of the invention and the other agent will still be able to exert a beneficial combined effect on cancer cells. The time interval between administration of the two formulations may range from a few minutes to a few weeks.

Non-limiting examples of anticancer agents that may be used in combination with the compositions or methods of the invention may include chemotherapeutic agents (e.g., cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecins, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel, gemcitabine, novelte, farnesyl protein transferase inhibitors, antiplatin, 5-fluorouracil, vincristine, vinblastine, and methotrexate, or any analog or derivative variant of the foregoing); radiotherapeutic agents (e.g., gamma rays, X-rays, microwaves, and UV radiation, and/or targeted delivery of radioisotopes to tumor cells); immunotherapeutic agents and immunomodulators; a gene therapeutic agent; pro-apoptotic agents and other cell cycle modulators known to those skilled in the art.

Immunotherapy may also be used in combination with the compositions and methods described herein as a combination therapy for the treatment of malignant diseases. Immunotherapeutic agents generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may be used as an effector of therapy, or it may recruit other cells (e.g., cytotoxic T cells or NK cells) to actually effect cell killing. Antibodies may also be conjugated to drugs or toxins (e.g., chemotherapeutic agents, radionuclides, ricin a chain, cholera toxin, pertussis toxin, etc.), and used only as targeting agents. In some embodiments, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target. In other embodiments, the tumor cells must bear some suitable marker for targeting. Non-limiting exemplary tumor markers suitable for targeting can include carcinoembryonic antigen (CEA), prostate-specific antigen, urinary system tumor-associated antigen, embryonic antigen, tyrosinase (p 97), gp68, TAG-72, HMFG, sialyl lewis antigen, mucA, mucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155.

In a preferred embodiment, checkpoint blocking immunotherapy is used in combination with the compositions and methods described herein. Checkpoint blocking immunotherapy may be performed by administration of checkpoint blocking agents such as PD-L1 inhibitors (such as atilizumab, avistuzumab and Dewaruzumab), PD-1 inhibitors (such as pembrolizumab, nivolumab and cimetidine Li Shan antibodies), or CTLA-4 inhibitors (such as ipilimumab), or by adoptive T cell transfer.

Gene therapy may also be used in combination 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 therapeutic genes that are separate from the mutant HSV-2 described herein. Gene therapy may be administered before, after, or simultaneously with the HSV-2 mutants described herein. Exemplary non-limiting targets for gene therapy include immunomodulators, agents that affect upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cytostatic agents, or agents that induce or increase the sensitivity of target cells to apoptosis. Exemplary non-limiting immunomodulatory genes that may be used in combination with the present invention as part of gene therapy include tumor necrosis factor; interferons α, β, and γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1 beta, MCP-1, RANTES and other chemokines.

An exemplary inhibitor of cell proliferation is p16. The major transition of the eukaryotic cell cycle is triggered by cyclin dependent kinases or CDKs. A CDK, cyclin dependent kinase 4 (CDK 4), regulates progression through the G1 process. The activity of the enzyme may be to phosphorylate Rb in the late G1 phase. The activity of CDK4 is controlled by the activation subunit D-type cyclin and the inhibitory subunit p16.sup.INK4, which specifically binds to and inhibits CDK4 and thus modulates Rb phosphorylation. The p16.sup.INK4 gene belongs to a newly described class of CDK inhibitor proteins, which also includes p16B, p19, p21WAF1 and p27KIP 1. Homozygous deletions and mutations of the p16.sup.INK4 gene are common in human tumor cell lines. Since the p16.sup.INK4 protein is a CDK4 inhibitor, deletion of this gene increases CDK4 activity, leading to hyperphosphorylation of Rb protein. Other genes that may 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/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, p, genes involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or their receptors), and MCC.

It is further contemplated that upregulation of cell surface receptors or ligands thereof, such as Fas/Fas ligand, DR4 or DR5/TRAIL, will enhance the apoptosis-inducing capacity of the invention by establishing an autocrine or paracrine effect on hyperproliferative cells. Increasing intercellular signaling by increasing the number of GAP junctions will increase the anti-hyperproliferative effect on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiating agents may be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are expected to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are Focal Adhesion Kinase (FAK) inhibitors and lovastatin. It is also contemplated that other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with the present invention to improve therapeutic efficacy.

Hormone therapy may also be used in conjunction with the present invention. The use of hormones may be used to treat certain cancers such as breast, prostate, ovarian or cervical cancer to reduce the level of or block the effects of certain hormones such as testosterone or estrogen. Such treatment is typically used in combination with at least one other cancer therapy, either as a treatment option or to reduce the risk of metastasis.

Development of resistant oncolytic viruses

Described herein are complex methods of generating FusOn-H2 based oncolytic viruses that can escape all the limiting factors in the blood stream that reduce the effectiveness of oncolytic virus therapies. The present disclosure also describes the design of a novel virus FusOn-SD.

The native HSV-2 virus comprises an ICP10 polynucleotide (which may also be referred to as an RR1 polynucleotide) encoding a polypeptide containing an amino terminal domain having Protein Kinase (PK) activity, such as serine/threonine protein kinase activity, and a C-terminal domain having ribonucleotide reductase activity. In a particular aspect of the invention, the endogenous PK domain is modified such that the virus has selective replication activity in tumor cells (and thus activity to destroy tumor cells) and/or such that the virus fuses or has enhanced fusion activity because it has membrane fusion (syncytial formation) activity. In some embodiments, the ICP10 polynucleotide is modified by deleting at least a portion of the endogenous sequence encoding a protein kinase domain such that the encoded polypeptide lacks protein kinase activity.

Viruses can only replicate within living cells, and their replication often requires activation of certain cell signaling pathways. Many viruses have acquired various strategies during their evolution to activate these signaling pathways to benefit their replication. The large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP 10 or RR 1) comprises a unique amino-terminal domain with serine/threonine Protein Kinase (PK) activity. This PK activity has been found to activate the cellular Ras/MEK/MAPK pathway (Smith et al, (2000) J Virol74 (22): 10417-29). Thus, deletion of this PK domain (ICP 10 PK) from the ribonucleotide reductase gene has been reported to severely impair the ability of the virus to replicate in cells, such as those in which there is no pre-existing activated Ras signaling pathway (Smith et al, (1998) J.Virol.72 (11): 9131-9141).

In certain embodiments, the ICP10 sequence of HSV-2 virus is mutated using any of a variety of standard mutagenesis methods, such as by deletion. Mutations may involve modification of the nucleotide sequence, individual genes or gene blocks. Mutations may involve single nucleotides (such as point mutations, which involve the removal, addition or substitution of a single nucleotide base within a DNA sequence), or they may involve the insertion or deletion of a large number of nucleotides. Mutations may occur spontaneously as a result of events such as DNA replication fidelity errors, or may be induced upon exposure to chemical or physical mutagens. Mutations can also be targeted by using specific targeting methods well known to those skilled in the art.

ICP10 polynucleotides are modified using genetic recombination 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/substituted may be any suitable region as long as the polypeptide encoded by the modified ICP10 polynucleotide retains ribonucleotide reductase activity and lacks protein kinase activity. ICP10 polynucleotides can also be modified by deleting only a portion of the sequence encoding a PK domain necessary for PK activity. The ICP10 polynucleotide may also be modified by replacing at least a portion of the sequence encoding the PK domain with a second polynucleotide, such as a green fluorescent protein, placed in frame with the sequence encoding the RR domain of the ICP10 polynucleotide.

Polynucleotides that replace at least a portion of the protein kinase activity domain of endogenous ICP10 in HSV-2 may encode at least a fusion portion of a cell membrane fusion inducing polypeptide, such as a viral Fusion Membrane Glycoprotein (FMG). For example, the polypeptide is preferably capable of inducing cell membrane fusion at a substantially neutral pH (such as about pH 6-8). Retroviral envelope proteins lacking some, most or all cytoplasmic domains are useful because such manipulation results in fusion activity of human cells that is too high. In some embodiments, specific modifications are introduced into the viral membrane glycoprotein to enhance its function of inducing cell membrane fusion. For example, truncations of cytoplasmic domains of various retroviral and herpesvirus glycoproteins have been shown to increase their fusion activity, sometimes while simultaneously reducing their efficiency of virion incorporation (Rein et al, (1994) J Virol68 (3): 1773-81).

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1: construction of FusOn-H2

Initially, the HSV genomic region comprising the left flanking region of ICP10 (corresponding to nucleotide numbers 85994-86999 of the HSV-2 genome) was amplified with the following exemplary primer pairs: 5' -TTGGTCTTCACCTACCGACA (SEQ ID NO: 1); and 3' -GACGCGATGAACGGAAAC (SEQ ID NO: 2). The RR domain and right flanking region (corresponding to HSV-2 genomic nucleotide sequence numbers 88228-89347) were amplified with the following exemplary primer pairs: 5' -ACACGCCCTATCATCTGAGG (SEQ ID NO: 13); and 5' -AACATGATGAAGGGGCTTCC (SEQ ID NO: 14). Both PCR products were cloned into pNeb 193 by EcoRI-NotI-XbaI ligation to generate pNeb-ICP10-deltaPK. Then, a DNA sequence containing the CMV promoter-EGFP gene was PCR amplified from pSZ-EGFP using the following exemplary primer pair: 5' -ATGGTGAGCAAGGGCGAG (SEQ ID NO: 3); and 3' -CTTGTACAGCTCGTCCATGC (SEQ ID NO: 4). The PCR amplified DNA was then cloned into the deleted PK locus of pNeb-ICP10-deltaPK by BglII and NotI ligation, yielding pNeb-PKF-2. In the course of designing the PCR amplification strategy, primers were designed such that the EGFP gene was fused in-frame with the remaining RR domains of the ICP10 gene, such that the novel protein product of the fused gene contained a fully functional EGFP, which would facilitate selection of recombinant viruses in the following experimental steps.

The pNeb-PKF-2 plasmid DNA was co-transfected with purified HSV-2 genomic DNA (strain 186) into Vero cells by lipofectamine, and the modified ICP10 gene was inserted into the virus by homologous recombination. Recombinant viruses were screened and identified by selecting GFP positive virus plaques. In particular embodiments of the invention, during the screening process, it was noted that all GFP-positive plaques showed clear syncytial formation of infected cells, indicating that this modified virus induced extensive cell membrane fusion. A total of 6 plaques were picked. One of them (called FusOn-H2) was selected for further characterization and for all subsequent experiments.

Example 2

To systematically mutate most of the neutralizing epitopes on gB and gD and enhance the complement antagonistic capacity of gC, a series of selections were made for FusOn-H2 in the presence of anti-HSV-2 serum. FusOn-H2 was initially selected in cell culture in the presence of a serum mixture collected from 5 rats that had been vaccinated with HSV and had high anti-HSV antibodies in the blood. One of the main reasons for starting this selection with rat serum is that they contain native immunoglobulins and mannan-binding lectin (MBL) which activates complement against HSV, whereas mouse and human serum contain only one of these two activation mechanisms (MBL for mice and native immunoglobulins for humans) (Wakimoto et al, 2002). After 7 consecutive rounds of selection in the rat serum mixture, a human serum containing high levels of anti-HSV-2 antibodies was added to the rat serum and selection continued.

Viral infectivity in the presence of anti-HSV serum was monitored periodically during selection, and the results showed that the resistance to neutralizing antibodies was continuously improved compared to unselected FusOn-H2. One of the test results is shown in fig. 1, which is performed after the virus underwent a total of 9 rounds of selection (i.e., 7 rounds of selection with rat serum and 2 rounds of selection with a mixture of rat serum and human serum). The results showed 28.5-fold and 27.2-fold resistance of the trained virus (designated FusOn-SS 9) to neutralization by human and rat anti-HSV-2 serum, respectively.

In order to enable FusOn-H2 to escape the host during systemic delivery to be cleared by the Mononuclear Phagocyte System (MPS) in the blood, the possibility of transplanting the CD47 (a "me-fudo-it-yourself" signaling molecule) gene onto the envelope of the virus was explored. Macrophages are the primary phagocytes responsible for rapid clearance of HSV particles, and depletion of macrophages has been reported to significantly improve the therapeutic effect of oncolytic HSV (Fulci et al, 2007). Phagocytic activity of macrophages is controlled by positive and negative regulatory mechanisms. The interaction between CD47 and its receptor sirpa provides a strong negative regulatory signal to macrophages ("do you eat me signal") (kinehen and ravichandoran, 2008).

HSV encodes several glycoproteins that assemble on the surface of the viral envelope. They include glycoproteins C (gC), gB, gD, gH and gL. Each of them can be incorporated as a candidate molecule into the extracellular domain (ECD) of murine CD47 (mCD 47) so that it can be transplanted onto the surface of the viral envelope. Glycoprotein C (gC) is selected here, which is not necessary for viral infectivity, unlike the other glycoproteins described. Thus, modification of it to incorporate mCD47 does not carry the risk of altering the natural tropism of oncolytic viruses. ECD of mCD47 (aa 19-141) (SEQ ID NO: 16) (shown in the following Table) was initially inserted into the N-terminus of gC to form chimeric form of gC (cgC), and its expression was driven by CMV IE promoter.

HA (hemagglutinin) markers are included in cgC to allow for convenient detection of chimeric molecules. To facilitate identification of recombinant viruses and in vivo imaging, cgC was ligated with another gene cassette containing the EGFP-luciferase gene. These two cassettes were inserted together into the backbone of FusOn-H3, which was derived from HSV-2-based oncolytic virus (FusOn-H2) by deleting the GFP gene from the virus. FusOn-H2 was constructed as follows: deletion of the N-terminal region of the ICP10 gene and substitution with GFP confers the ability to selectively replicate and kill tumor cells. Recombinant viruses were identified by picking GFP positive plaques. Each individually picked virus was enriched to homogeneous GFP positivity by multiple rounds of plaque purification. The newly generated virus was designated FusOn-CD47-Luc (FIG. 2A). Control viruses were also constructed in which only the EGFP-Luc gene cassette alone was inserted into the backbone of FusOn-H3, fusOn-Luc (FIG. 2B). All selected viruses maintained the fusion properties of the parental FusOn-H2.

A recent interesting study found that coating nanoparticles with CD47 mimetic peptides could help them escape phagocytic clearance of MPS (Rodriguez et al, 2013). The possibility of gene-transplanting the extracellular domain of CD47 molecule onto the envelope of FusOn-H2 was investigated in order to enable its escape from MPS for systemic delivery. The virus construction strategy is shown in figure 2. In vitro assays showed that coating of viral particles with CD47 extracellular domain by recombinant gC made the virus more resistant to phagocytosis (fig. 3). When tested in vivo in a murine colon cancer model, the results showed that FusOn-CD47 was 15-fold more effective at delivering to the tumor site via the systemic route than FusOn-Luc. Furthermore, the data also show that after systemic delivery, the residence time of FusOn-CD47 at the tumor site is significantly longer than FusOn-Luc.

Expression of the transgene was initially checked by flow cytometry or western blot analysis. For flow cytometry analysis, 293 cells were infected with 1 pfu/cell of FusOn-CD47-Luc or FusOn-Luc. Twenty-four hours later, cells were labeled with PE conjugated anti-mCD 47 antibodies or rabbit anti-HA tag antibodies. Goat anti-rabbit antibody conjugated with FITC was added for HA tag detection. Flow cytometry analysis was performed on CD47 and HA-tagged cells. The results in FIG. 2A show that both anti-mCD 47 and anti-HA tag antibodies can easily detect FusOn-CD47-Luc infected cells, but not FusOn-Luc infected cells. For western blot analysis, 293 cells were similarly infected with both viruses or transfected with plasmids expressing cgC or wild-type gC without HA tag. Cell lysates were prepared after 24 hours and subjected to western blot analysis. The results in FIG. 2B show that cgC is abundantly expressed by FusOn-C D47-Luc, but not FusOn-Luc.

Next, the Luc gene expression levels from these two viruses were compared. Vero, CT26 and 4T1 cells were infected with 1 pfu/cell of FusOn-CD47-Luc or FusOn-Luc. Cells were harvested after 24 hours to measure luciferase activity. The results in FIG. 3A show that the levels of luciferase activity from cells infected with both viruses are nearly identical. Another experiment was then performed to determine if the implantation of oncolytic HSV with ECD of mCD47 allowed the virus to evade phagocytosis and clearance of phagocytes in an in vitro environment. Mouse spleen cells were used as a source of fresh phagocytes, as the spleen is the largest unit of the mononuclear phagocyte system. Vero cells were infected with FusOn-CD47-Luc or FusOn-Luc, with or without mouse spleen cells. Cells were collected 48 hours later and virus yield was quantitatively measured by plaque assay. The results in FIG. 3B show that FusOn-CD47-Luc and FusOn-Luc are well replicated in wells without splenocytes, and that the viral yields in both wells are similar. 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 mCD47ECD on the viral particles enables the virus to resist the effects of macrophages and possibly some other immune cells during viral infection.

To determine the ability of the incorporated mCD47ECD to enable systemic delivery of the virus, CD26 murine colon cancer cells were implanted into the right flank of immunocompetent Balb/c mice. Once the tumor reached a size of about 8mm in diameter, we injected 2X 106 pfu of FusOn-CD47-Luc or FusOn-Luc systemically into each mouse. Mice were monitored for luciferase activity by the IVIS spectroscopic system starting on day 2 and then daily until the signal was completely lost. The results in FIG. 4 show that on day 2 post-viral administration, the imaging signal from FusOn-CD47-Luc was about 1.5 log stronger than the imaging signal from FusOn-Luc. This suggests that the former is more efficiently delivered to the tumor site by the systemic route than the latter. Furthermore, fusOn-CD47-Luc appeared to have a significantly longer residence time in tumor tissue than FusOn-Luc. On day 5, little imaging signal was detected in tumors from mice receiving FusOn-Luc, while signals from tumors from FusOn-CD47-Luc remained detectable until day 7. However, since this HSV-2 based oncolytic virus grew poorly in CT26 tumor cells, neither virus showed any significant amplification in tumor tissue. Interestingly, in one case where several mice were imaged on day 1 post viral delivery, a significant imaging signal was detected transiently in the liver. These signals completely disappeared on day 2.

To determine if systemic delivery of FusOn-CD47-Luc was also superior to FusOn-Luc in other tumor models, the above experiment was repeated, but in mice bearing tumors established by implantation of 4T1 murine mammary tumor cells in the right flank, fusOn-H2 was found to be able to grow moderately. In addition, 4T1 tumor cells secrete macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF), which enhance macrophage infiltration and phagocytosis. This would allow for a more powerful test of CD 47-mediated escape strategies. The IVIS imaging results in fig. 5A do show that the signal in the 4T1 tumor is generally lower than the signal detected in the CT26 tumor (as shown in fig. 4). However, the results again indicate that FusOn-CD47-Luc can be delivered more efficiently to local tumors by the systemic route than FusOn-Luc. The difference in image signal intensity between the two viruses was about 1.5 log on day 2. The maximum difference was recorded on day 4, at which time the image signal from FusOn-Luc was reduced to near background level, while it reached the highest level of FusOn-CD 47-Luc. These data again demonstrate that CD47 modification allows the virus to be delivered more efficiently by the systemic route and once reaching tumor tissue, it remains in tumor tissue longer than the control virus.

To generate a FusOn-H2 product that can withstand all three major limiting factors in the blood stream (neutralizing antibodies, complement, and phagocytes), we performed the FusOn-CD47 anti-HSV-2 serum selection as described above. It was subjected to 7 consecutive selections with anti-HSV rat serum, followed by 23 consecutive passages in the presence of rat serum plus one of the human serum with extremely high anti-HSV-2 antibody levels as described above. Again, several tests were performed during selection to determine if the virus gained the ability to resist immune serum neutralization effects. One of the results of such a test is shown in fig. 5, which was performed after 7 rounds of selection with rat serum first and 17 rounds of selection with rat serum plus human serum. The results clearly show that, similar to FusOn-SS9, trained FusOn-CD47 (designated FusOn-CD47-SS 24) acquired substantial capacity in maintaining infectivity in the presence of immune anti-HSV-2 serum.

To ensure more significant resistance to neutralizing antibodies in humans, we performed three additional rounds of selection of viruses with a mixture of HSV-2 positive sera obtained from 12 individuals, 4 were mixed together at a time. The selection rounds are as follows: batch 1 was 8 runs, batch 2 was 6 runs, and batch 3 was 4 runs. After these extensive selections, the obtained virus plaques were purified and 6 plaque pickups were amplified for further analysis. They are designated HR49-N2-5, HR49-N7 and HR49-N8. First, they were tested for their ability to resist neutralization by the eight human serum mixtures used in the selection, which is the most stringent neutralization test we performed. FusOn-SS9 and FusOn-CD47-SS24 were included in this experiment for comparison. Different dilutions of anti-HSV serum mixtures were used to incubate with the indicated viruses, and viral infectivity was then determined. The results in fig. 6 show that under such stringent neutralization conditions (containing a mixture of antiviral sera from multiple individuals), the selected viruses can still produce a large amount of plaque, while the unselected viruses do not produce any plaque until the serum mixture is diluted 80-fold. The data also show that the subsequent three rounds of selection in human serum mixtures further improves the ability of the selected viruses to resist neutralization. Furthermore, the data show that some of the picked viruses have better resistance to neutralization by mixed human antiviral serum than others.

Example 3

The oncolytic virus FusOn-H2 was evaluated for its ability to escape anti-HSV antibody neutralization with and without insertion of the extracellular domain of CD47 into the N-terminus of gC. In this in vitro experiment, 500 plaque forming units (pfu) of FusOn-CD47 or FusOn-Luc were mixed with or without diluted anti-HSV-2 serum (at a dilution of 1:40 or 1:160) and incubated at 37℃before they were applied to Vero cell monolayers to determine infectivity and quantitate plaque formation. The results in FIG. 7 show that serum can almost completely neutralize the viral infectivity of FusOn-Luc when diluted 1:40. Even at 1:160 dilution, serum can significantly reduce infection by FusOn-Luc (and thus reduce the number of plaque formations). In contrast, fusOn-CD47 was much less neutralized in the presence of the same dilution of anti-HSV serum. FusOn-CD47 produced a significant number of plaques (about 15% of wells without anti-HSV serum added) in the presence of 1:40 dilution of serum, whereas FusOn-Luc showed almost no viral plaques at all.

FusOn-CD47, fusOn-Luc and FusOn-SD delivered in vivo by the systemic route were evaluated in Balb/c mice vaccinated with HSV-2 and implanted with CT26 murine colon cancer cells in the right flank. 1X 10 by tail vein injection ⁷ Individual FusOn-CD47, fusOn-Luc or FusOn-SD were delivered systemically to mice in different groups. Mice were imaged with an IVIS imager on the days shown in fig. 8. The results showed that FusOn-Luc was found to be resistant to HSV in the presence of immunityCannot be delivered to tumors by systemic route. In contrast, fusOn-CD47 is readily detected in tumor tissue by the same delivery route and in the presence of anti-HSV immunity. Furthermore, fusOn-CD47 remained in tumor tissue for nearly eight days after reaching the tumor site despite the presence of antiviral immunity. CD47 unexpectedly helps the oncolytic virus escape the neutralizing effect of anti-HSV antibodies, helping FusOn-SD to escape the overall ability of neutralizing antibodies for systemic delivery. Thus, fusOn-SD performs best in systemic delivery to tumor sites. Thus, fusOn-SD may be used in patients with HSV-1 and/or HSV-2 antibodies, such as patients vaccinated against HSV-1 and/or HSV-2 or with HSV-1 and/or HSV-2.

Unexpectedly, it was found that gE was not present in FusOn-SD virus particles. This is illustrated by fig. 9A to 9C.

FIG. 9A is a bar graph showing absorbance of FusOn-H2, fusOn-SD and PBS (negative control) after treatment with mouse anti-HSV-2 gE (1:1000 dilution) and HPR conjugated rabbit anti-mouse IgG (1:10,000 dilution). More specifically, in FIG. 9A, 1X 10 will be ⁶ The pfu viruses (parental FusOn-H2 and FusOn-SD) were coated onto 96-well plates for ELISA assays. Wells coated with PBS served as negative controls. The primary antibody used was mouse anti-HSV-2 gE (1:1000 dilution) and the secondary antibody was HPR conjugated rabbit anti-mouse IgG (1:10,000 dilution). Significant readings were detected in FusOn-H2, but not in FusOn-SD, indicating that gE was present in FusOn-H2 virus particles, but not in FusOn-SD.

FIG. 9B is a Western blot showing gE detection of FusOn-SD and FusOn-H2. From 1X 10 ⁶ Proteins were subtracted from the pfu viruses (FusOn-H2 and FusOn-SD) and electrophoresed on an acrylamide gel for Western blot analysis. The same antibodies to the mouse anti-HSV-2 gE and HPR conjugated rabbit anti-mouse IgG used in FIG. 9A were used for Western blotting. The results showed that the gE band for FusOn-H2 was clear, whereas the lanes loaded with FusOn-SD protein had no gE band. This result confirms the findings in fig. 9A.

Fig. 9C is a schematic diagram of NK cell mechanisms that recognize HSV or HSV infected cells via gE. The figure shows that gE binds IgG (virus specific or non-virus specific). cD16a is one of the most important NK cell activating receptors, which binds to the Fc region of IgG, resulting in NK cell activation and clearance of virus particles or virus infected cells. This results in reduced oncolytic viral delivery and replication, thus minimizing the therapeutic effects of viral therapy. Since FusOn-SD cannot be recognized by this mechanism, NK cells do not clear them and the therapeutic efficacy of FusOn-SD remains unchanged.

Reference to the literature

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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.

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.

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Eing，B.R.，Kühn，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.

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

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.

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

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.

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

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.

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

Kinchen，J.M.and Ravichandran，K.S.，2008.Phagocytic signaling：you can touch，but you can′t eat.Curr Biol 18，R521-4.

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.

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.

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.Sciance 339，971-5.

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.

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

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.

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.

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

All publications, patents, and patent applications cited herein are hereby incorporated by reference as if set forth in their entirety herein. While this invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of the illustrative embodiments, as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to the description. Accordingly, the appended claims are intended to cover such modifications and enhancements.

Claims (34)

1. A composition comprising an oncolytic herpes simplex virus type 1 (HSV-1) or 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 an immune serum having elevated levels of anti-HSV antibodies.

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

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

4. The composition of claim 3, wherein the glycoprotein is glycoprotein C.

5. The composition of any one of the preceding claims, wherein said passage in said immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of said oncolytic HSV-1 or HSV-2.

6. The composition of any one of claims 2-5, wherein the extracellular CD47 domain comprises amino acids 19-141 of CD 47.

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

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

9. The composition of any of the preceding claims, wherein the composition comprises an oncolytic HSV-2, the oncolytic HSV-2 having been prepared by passaging oncolytic HSV-2 seven times in the presence of a rat serum having elevated levels of anti-HSV antibodies, then passaging seventeen times in the presence of a mixture of a rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

10. The composition of any of the preceding claims, wherein the composition comprises an oncolytic HSV-2, the oncolytic HSV-2 having been prepared by passaging oncolytic HSV-2 seven times in the presence of a rat serum having elevated levels of anti-HSV antibodies, then passaging twenty-three times in the presence of a mixture of a rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

11. The composition of any one of the preceding claims, wherein the oncolytic HSV-1 or oncolytic HSV-2 comprises a modified ICP10 coding region that lacks nucleotides 1 to 1204 of an endogenous ICP10 coding region, wherein the oncolytic HSV-1 or HSV-2 comprises a 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 wherein the oncolytic HSV-1 or HSV-2 is capable of selectively killing cancer cells.

12. The composition of any one of the preceding claims, wherein the composition comprises oncolytic HSV-2 and the oncolytic HSV-2 passaged is a FusOn-H2 oncolytic virus.

13. The composition of any one of the preceding claims, wherein the composition comprises oncolytic HSV-2 and the oncolytic HSV-2 passaged is a FusOn-CD47 oncolytic virus.

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

15. The method of claim 14, wherein the oncolytic HSV1 or HSV-2 has an envelope comprising glycoproteins, wherein at least one glycoprotein comprises an extracellular CD47 domain inserted into the N-terminus of the glycoprotein.

16. The method of claim 15, wherein the glycoprotein is selected from the group consisting of glycoprotein C, glycoprotein B, glycoprotein D, glycoprotein H, and glycoprotein L.

17. The method of claim 16, wherein the glycoprotein is glycoprotein C.

18. The method of any one of claims 14 to 17, wherein said passaging in said immune serum mutates neutralizing epitopes on glycoprotein B and glycoprotein D of said oncolytic HSV-1 or HSV-2.

19. The method of any one of claims 15 to 18, wherein the extracellular domain comprises amino acids 19-141 of CD 47.

20. The method of any one of claims 14-19, wherein the immune serum comprises mammalian anti-HSV antibodies.

21. The method of any one of claims 14 to 20, wherein the method comprises passaging the oncolytic HSV-2 at least twice in the presence of rat serum having elevated levels of anti-HSV antibodies, then passaging at least once in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

22. The method of claim 21, wherein the method comprises passaging the oncolytic HSV-2 seven times in the presence of rat serum having elevated levels of anti-HSV antibodies, then passaging seventeen times in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

23. The method of claim 21, wherein the method comprises passaging the oncolytic HSV-2 seven times in the presence of rat serum having elevated levels of anti-HSV antibodies, then passaging twenty-three times in the presence of a mixture of rat serum having elevated levels of anti-HSV antibodies and at least one human serum.

24. The method of any one of claims 14 to 23, wherein the composition comprises oncolytic HSV-2 and the oncolytic HSV-2 passaged is a FusOn-H2 oncolytic virus.

25. A method of treating cancer in a patient in need thereof with an HSV-based oncolytic viral therapy, the method comprising administering the composition of any one of claims 1-14.

26. The method of claim 25, wherein the composition is administered systemically.

27. The method of claim 25 or 26, wherein the cancer is a metastatic cancer.

28. A method of treating cancer in a patient in need thereof with an HSV-based oncolytic viral therapy, the method comprising administering a composition prepared by the method of any one of claims 15-24.

29. The method of claim 28, wherein the composition is administered systemically.

30. The method of claim 28, wherein the composition is administered by intratumoral injection or by intraperitoneal injection.

31. The method of claim 28 or 29, wherein the cancer is a metastatic cancer.

32. The method of any one of claims 25 to 31, wherein the patient has been vaccinated against HSV-1 and/or HSV-2 or has HSV-1 and/or HSV-2.

33. The method of any one of claims 25-32, wherein the method further comprises treating the patient with checkpoint blocking immunotherapy.

34. The method of claim 33, wherein the checkpoint blocking immunotherapy is selected from (a) administering a PD-L1 inhibitor, a PD-1 inhibitor, or a CTLA-4 inhibitor to the patient, or (b) treating the patient by adoptive T cell transfer.