RU2831084C2 - CONSTRUCTION OF OBLIGATE VECTOR BASED ON ONCOLYTIC HERPES SIMPLEX VIRUSES (oHSV) AND CONSTRUCT FOR CANCER THERAPY - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a recombinant oncolytic herpes simplex virus type 1 (HSV-1), comprising a HSV-1 vector containing a deletion modification in the wild-type HSV-1 genome, wherein said vector contains (i) only one copy of all represented in two copies of genes of HSV-1 genome of wild type, (ii) only one copy of duplicated non-coding sequences of genome HSV-1 of wild type, and (iii) sequences required for the expression of all wild-type HSV-1 genome open reading frames (ORF) presented in one copy, containing the ORF themselves regulatory sequences required for the expression of each ORF, wherein said regulatory sequences include promoters; a first heterologous nucleic acid sequence encoding IL-12; and a second nucleic acid sequence coding an anti-PD-1 agent, wherein said first and second nucleic acid sequences are stably inserted into at least a deleted region of the HSV-1 vector for treating various types of cancer. Also disclosed is a pharmaceutical composition for treating cancer, containing an effective amount of recombinant oncolytic HSV-1 and a pharmaceutically acceptable carrier.

EFFECT: invention extends the range of cancer treatment.

7 cl, 5 dwg, 1 tbl

Description

AREA OF TECHNOLOGY

[0001] The present invention generally relates to the treatment of cancer using oncolytic herpes simplex viruses (oHSV). In particular, the present invention relates to the production of an obligate HSV vector capable of carrying and expressing a plurality of genes encoding immunostimulatory and/or immunotherapeutic agents (means). The present invention also relates to an innovatively designed genome that can function as a vector capable of carrying and expressing a plurality of therapeutic genes for effective cancer therapy.

LEVEL OF TECHNOLOGY

[0002] Oncolytic herpes simplex viruses (oHSV) are being intensively studied for the treatment of solid tumors. This group of viruses offers many advantages over traditional cancer therapies (Markert et al., 2000; Russell et al., 2012; Shen and Nemunaitis, 2006). In particular, oHSVs typically contain a mutation that makes them sensitive to inhibition of one component of the innate immune system. As a result, these viruses replicate in cancer cells in which one or more innate immune responses to infection are impaired, but not in normal cells in which innate immune responses are intact. oHSVs are typically delivered directly into the tumor mass, where the virus can replicate. Because the virus is delivered to the target tissue rather than systemically, the side effects associated with cancer drugs are absent. Viruses typically trigger adaptive immune responses that limit their ability to be administered multiple times. oHSV has been administered to tumors multiple times with no evidence of loss of activity or induction of adverse reactions such as inflammatory responses. HSVs are large DNA viruses that can incorporate foreign DNA into their genomes and regulate the expression of these genes after administration to tumors. Foreign genes suitable for use with oHSV are those that help elicit an adaptive immune response to the tumor.

[0003] The defect in overcoming the cellular innate immune response determines the range of tumors in which the virus exerts its oncolytic oHSV as an anticancer agent. The more extensive the deletions, the more limited the range of cancer cells in which oHSV is effective, depending on the function of the deleted viral gene. Most novel oHSVs contain at least one cellular gene to enhance their anticancer activity (Cheema et al., 2013; Goshima et al., 2014; Markert et al., 2012; Walker et al., 2011).

[0004] It is convenient to consider separately the structure of oHSV, referred to as the "backbone," and the foreign genes suitable for insertion into the backbone. As noted above, the backbone structure determines the range of susceptible cancer types. The foreign genes enable the host to perceive cancer cells as legitimate targets for the adaptive immune response.

[0005] The HSV-1 genome consists of two covalently linked components designated L and S. Each component consists of unique sequences (U _L for the L component, U _S for the S component) flanked by inverted repeats. The inverted repeats of the L component are designated ab and b'a'. The inverted repeats of the S component are designated a'c' and ca. The inverted repeats b'a' and a'c' constitute the internal region of the inverted repeats. The inverted repeat regions of the L and S components are known to contain two copies of five protein-coding genes designated ICP0, ICP4, ICP34.5, ORF P, and ORF O, respectively, and large regions of DNA that are transcribed but do not encode proteins.

[0006] Historically, viruses studied in patients with cancer have been divided into three different designs. The first was based on the demonstration that deletion of the ICP34.5 gene significantly attenuated the virus (Andreansky et al., 1997; Chou et al., 1995; Chou et al., 1990; Chou and Roizman, 1992). Then, G207, the first virus studied in patients, was attenuated by an additional mutation in the gene encoding viral ribonucleotide reductase to ensure safety for the treatment of malignant glioblastomas (Mineta et al., 1995). G207, which carries mutations in both the ICP34.5 and ribonucleotide reductase genes, was over-attenuated and did not function in cancer cells expressing wild-type protein kinase R (Smith et al., 2006).

[0007] The second design was based on the demonstration that when a viral protein designated U _S 11 is expressed early in infection, it partially compensates for the absence of ICP34.5 and restores the ability to grow in cells expressing wild-type protein kinase R (Cassady et al., 1998a). A later backbone design for this virus was published by Cassady et al. (1998b) in which the U _S 12 gene and the U _S 11 promoter were deleted. As a consequence, U _S 11 is expressed as an immediate-early gene rather than a late gene.

[0008] The third virus backbone, initially designated R7020 and later renamed NV1020, is the result of modifications to a spontaneous mutant that was initially investigated as a live attenuated viral vaccine (Meignier et al., 1988; Weichselbaum et al., 2012). This mutant lacked the internal inverted repeats (which consist of b'a' and a'c', encoding one copy of the ICP0, ICP4, ICP34.5, ORF P, and ORF O genes) and the genes encoding U _L 56 and U _L 24. In addition, this mutant contained bacterial sequences and, because it was intended for use as a vaccine, it also contained genes encoding several HSV-2 glycoproteins. R7020 has been extensively studied in patients with liver metastases from colon cancer. In addition, R7020 has been studied in head and neck epithelial squamous cell carcinoma and prostate adenocarcinoma xenografts in nude mice and in bladder tumor models (Cozzi et al., 2002; Cozzi et al., 2001; Currier et al., 2005; Fong et al., 2009; Geevarghese et al., 2010; Kelly et al., 2008; Kemeny et al., 2006; Wong et al., 2001).

[0009] The success of oHSV-based therapy depends on the degree of destruction of cancer cells. Early in the development of oHSV, it was recognized that HSV alone was not able to kill all cancer cells in a solid tumor, that it was unlikely that oHSV-based treatment could effectively eliminate all cancer cells, and that tumor destruction using oHSV in clinical trials must involve an adaptive immune response to the tumor. Subsequent studies have shown that the antitumor immune response induced by debris from infected tumor cells can be enhanced by the addition of cytokines. Comparison of oHSV lacking the cytokine gene with oHSV containing the immune-stimulatory cytokine confirmed this hypothesis (Andreanski et al.) and ultimately led to the incorporation of GM-CSF (granulocyte-macrophage colony-stimulating factor) into oHSV developed for the treatment of melanoma (Andtbacka et al., 2015).

[0010] The safety profile of oHSV depends on gene deletions that disable one or more viral functions, blocking host innate immune responses to infection. Analysis of published data suggests that oHSV in clinical trials to date is over-attenuated and could be improved (Miest and Cattaneo, 2014).

[0011] Insertion of genes encoding immunostimulatory cytokines enhances the immune response to tumors but does not effectively enhance T cell-mediated cytotoxicity, which is important for antitumor effects. Tumors engage the PD-1 and CTLA-4 inhibitory pathways to silence the immune system. PD-1 is expressed on activated T cells and other hematopoietic cells, and CTLA-4 is expressed on activated T cells, including regulatory T cells (Fife and Pauken, 2011; Francisco et al., 2010; Keir et al., 2008; Krummel and Allison, 1995; Walunas et al., 1994). Tumors exploit the PD-1 and CTLA-4 inhibitory pathway to evade the host immune response. To maximize antitumor responses, it is important to activate cytotoxic T cells by neutralizing PD-1 with an anti-PD1 antibody and, in some cases, neutralizing CTLA4, which is present on the surface of T cells (Topalian et al., 2015). Although systemic administration of a single-chain antibody to PD-1 or CTLA4 is effective in enhancing the therapeutic effects of oHSV, such an antibody often causes side effects and can only be administered a limited number of times.

[0012] Thus, there is an urgent clinical need to formulate a strategy for developing a safe but more potent oHSV and for combining its administration with the administration of immunotherapeutic agents.

BRIEF DESCRIPTION

[0013] One aspect of the present invention relates to a modified herpes simplex virus type 1 (also referred to hereinafter as HSV-1, obligate vector or vector, HSV-1 virus(es)) comprising a modified HSV-1 genome. The modification comprises a deletion between the promoter of the U _L 56 gene and the promoter of the U _S 1 promoter of the wild-type HSV-1 genome such that (i) one copy of all genes present in two copies is missing, and (ii) the sequences necessary for the expression of all existing open reading frames (ORFs) in the viral DNA are intact after the deletion.

[0014] In another aspect of the present invention, there is provided an oncolytic herpes simplex virus type 1 (HSV-1) construct comprising (i) sequences necessary for the expression of all open reading frames (ORFs) present in a single copy in the viral genome; (ii) only one copy of each of all genes present in two copies in the viral genome; and (iii) one copy of duplicated DNA encoding non-coding RNAs.

[0015] A further aspect of the present invention relates to a recombinant oncolytic herpes simplex virus type 1 (HSV-1) comprising (a) a modified HSV-1 genome, wherein the modification comprises a deletion between the promoter of the U _L 56 gene and the promoter of the U _S 1 gene of the wild-type HSV-1 genome, as a result of which (i) one copy of all genes present in two copies is missing, and (ii) the sequences necessary for the expression of all existing open reading frames (ORFs) in the viral DNA are intact after the deletion; and (b) a heterologous nucleic acid sequence encoding an immunostimulatory and/or immunotherapeutic agent, wherein the heterologous nucleic acid sequence is stably inserted into at least the deleted region of the modified HSV-1 genome.

[0016] A further aspect of the present invention is that a viral vector comprising one copy of all open reading frames, namely from U _L 1 to U _L 56 and from U _S 1 to U _S 12, and comprising the "a" sequences at the ends of the genome, is an obligate vector, since it cannot replicate by itself in highly sensitive Vero cells. The vector can replicate after insertion of DNA comprising (a) coding or non-coding sequences of cellular DNA or (b) viral DNA consisting of non-coding sequences. The total amount of DNA that can be transferred by an obligate vector is at least 15 kb (thousand base pairs) or up to 22 kb.

[0017] A further aspect relates to a pharmaceutical composition comprising an effective amount of a recombinant oncolytic HSV-1 according to the present invention and a pharmaceutically acceptable carrier. The composition is in a form for, for example, intratumoral administration.

[0018] A further aspect relates to a method of treating cancer, said method comprising administering to a subject in need thereof an effective amount of a recombinant oncolytic HSV-1 or a pharmaceutical composition according to the present invention. Further, the present invention relates to the use of a recombinant oncolytic HSV-1 according to the present invention for use in a method of treating cancer.

[0019] Another aspect of the present invention relates to the use of a recombinant oncolytic HSV-1 or a pharmaceutical composition according to the present invention for the production of an anticancer medicament.

BRIEF DESCRIPTION OF DRAWINGS

[0020] These and other aspects and advantages of the present invention will be apparent from the following description, in which they are described in detail with reference to the accompanying drawings, in which:

[0021] Figure 1: Schematic representations of HSV-1 viruses. HSV-1, genome structure of wild-type HSV-1 showing the internal b'a'-a'c' inverted repeat region located between bp 117005 and bp 132096; IMMV201, genome structure of oHSV-1, also called the obligate vector; IMMV202, oHSV-1 expressing murine IL-12; IMMV203, oHSV-1 expressing human IL-12; IMMV303, oHSV-1 expressing an anti-human CTLA-4 scFv; IMMV403, oHSV-1 expressing an anti-human PD-1 scFv.

[0022] Figure 2: Schematic representations of HSV-1 oncolytic viruses based on obligate vectors that express immunostimulatory and/or immunotherapeutic agents. IMMV502, oHSV-1 expressing scFv to human PD-1 and murine IL-12; IMMV504, oHSV-1 expressing scFv to mouse CTLA-4 and murine IL-12; IMMV503, oHSV-1 expressing scFv to human PD-1 and human IL-12; IMMV505, oHSV-1 expressing scFv to human CTLA-4 and human IL-12; IMMV507, oHSV-1 expressing scFv to human CTLA-4 and scFv to human PD-1; IMMV603, oHSV-1 expressing scFv to human CTLA-4, scFv to human PD-1 and human IL-12.

[0023] Figure 3: Expression of anti-PD-1 scFv by secretion of test constructs. Expression of His-tagged anti-PD-1 scFv driven by the CMV promoter along with signal peptide coding regions from various environmental sources. Cell lysates and supernatant were collected and subjected to SDS-PAGE analysis and blotted with anti-His antibody. Lane 1, GM-CSF signal peptide; Lane 2, Gaussian luciferase signal peptide; Lane 3, Hidden Markov Model 38 (HMM38) signal peptide; Lane 4, antibody V gene signal peptide.

[0024] Figure 4: Binding affinity analysis of scFv to PD-1 to PD-1. ELISA analysis of His-tagged scFv to PD-1 driven by the CMV promoter together with the HMM38 signal peptide. The supernatant was collected and ELISA analysis was performed, detection was performed with an anti-His antibody.

[0025] Figure 5: Cell viability in an in vitro growth assay. (a): T24, human bladder carcinoma; (b) ECA109, human esophageal carcinoma; (c) CNE1, human nasopharyngeal carcinoma; (d) HCT116, human colon carcinoma; (e) Hep2, human laryngeal carcinoma; (f) MD-MB-231, human breast cancer; (g) Hela, human epithelial adenocarcinoma; (h) A549, human lung epithelial adenocarcinoma; (i) H460, human non-small cell lung carcinoma.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0026] It should be noted that a term denoting an entity in the singular means one or more such entities; for example, "recombinant oncolytic HSV-1" should be understood as referring to one or more recombinant oncolytic HSV-1 viruses. In this regard, the terms denoting an entity in the singular, "one or more" entities, and "at least one" entity may be used interchangeably herein.

[0027] "Homology" or "identity" or "similarity" means the similarity (relevance) of a sequence between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for comparison purposes. If a position in a reference sequence is occupied by the same base or the same amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions common to the sequences. "Unrelated" or "non-homologous" sequences are characterized by an identity of less than 40%, but preferably an identity of less than 25%, with one of the sequences of the present invention.

[0028] A polynucleotide or region of a polynucleotide (or a polypeptide or region of a polypeptide) has a specified percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of "sequence identity" with another sequence; this means that, when aligned, the specified percentage of bases (or amino acids) are the same when the two sequences are compared. This alignment and the percentage of homology or sequence identity can be determined using software known in the art.

[0029] As used herein, an "antibody" or "antigen-binding polypeptide" refers to a polypeptide or complex of polypeptides that specifically recognizes and binds one or more antigens. An antibody can be a whole antibody and any antigen-binding fragment or one chain of an antibody. Thus, the term "antibody" includes any protein or peptide comprising a molecule that comprises at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples thereof include, but are not limited to, the complementarity determining region (CDR) of a heavy or light chain or the ligand-binding portion of said chains, the variable region of a heavy chain or light chain, the constant region of a heavy chain or light chain, a framework region (FR) or any portion thereof, or at least one portion of a binding protein. The term "antibody" also includes polypeptides or polypeptide complexes that, upon activation, have antigen-binding properties.

[0030] The terms "antibody fragment" or "antigen-binding fragment" as used herein represent a portion of an antibody, such as F(ab') ₂ , F(ab) ₂ , Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered proteins that function similarly to an antibody by binding to a specific antigen to form a complex.

[0031] The antibodies, antigen-binding polypeptides, variants or derivatives thereof of the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab') ₂ , Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments containing a VK or VH domain, fragments produced in a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, the anti-Id antibodies to the LIGHT antibodies disclosed herein). The immunoglobulin molecules or antibodies of the present invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of an immunoglobulin molecule.

[0032] By "specifically binds" or "has specificity for" is generally meant that the antibody binds to an epitope with its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more readily than it would bind to an arbitrary unrelated epitope. The term "specificity" is used herein to refer to the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be said to have a higher specificity for a given epitope than antibody "B", or antibody "A" may be said to bind to epitope "C" with a higher specificity than it has for the related epitope "D".

[0033] As used herein, the terms "cancer" or "tumor" are used interchangeably to refer to a group of diseases that can be treated according to the present invention and that are accompanied by abnormal cell growth with the potential to invade or spread to other parts of the body. Not all tumors are cancerous; benign tumors do not spread to other parts of the body. Possible signs and symptoms include, but are not limited to: a new lump, abnormal bleeding, a persistent cough, unexplained weight loss, and a change in bowel movements. There are over 100 different known types of cancer that affect humans. The present invention relates primarily to solid tumors. Non-limiting examples of cancer or tumor include bladder cancer, basal cell carcinoma, cholangiocarcinoma, colon cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma, prostate cancer, gastric cancer, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, laryngeal carcinoma, liver cancer, lung cancer, melanoma, mesothelioma, pancreatic cancer, rectal cancer, kidney cancer, thyroid cancer, malignant peripheral nerve cell tumor, malignant peripheral nerve sheath tumor (MPNST), cutaneous and ramus neurofibromas, leiomyoadenomatoid tumor, fibroids, uterine fibroids, leiomyosarcoma, papillary thyroid cancer, anaplastic thyroid cancer, medullary thyroid cancer, follicular thyroid cancer, cell carcinoma Hürthle, thyroid cancer, ascites, malignant ascites, mesothelioma, salivary gland tumors, mucoepidermoid carcinoma of the salivary glands, acinar cell carcinoma of the salivary glands, gastrointestinal stromal tumor (GIST), tumors that cause effusions into potential spaces of the body, pleural exudates, pericardial exudates, peritoneal exudates, also called ascites, giant cell tumors (GCT), GCT of bone, pigmented villonodular synovitis (PVNS), tenosynovial giant cell tumor (TGCT), TGCT of the tendon sheath (TGCT-TS) and other sarcomas. According to a preferred embodiment, the present invention is used to treat esophageal cancer, lung cancer, prostate cancer or bladder cancer.

[0034] As used herein, the terms "treat" or "treatment" refer to both therapeutic treatment and prophylactic or preventative measures that aim to prevent or slow down (reduce) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease, delay or slowing of the progression of the disease, improvement or temporary alleviation of the disease, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean an increase in survival time compared to the expected survival without receiving the treatment. Subjects in need of treatment include subjects already suffering from the condition or disorder, as well as subjects susceptible to developing such a condition or disorder, or subjects in whom the condition or disorder is to be prevented.

[0035] By "subject" or "individual" or "animal" or "patient" or "mammal" is meant any subject, in particular a subject that is a mammal, for whom diagnosis, prognosis or therapy is desired. Mammalian subjects include humans, domesticated animals, farm animals and animals kept in zoos, used for sport or pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0036] As used herein, phrases such as "a patient in need of treatment" or "a subject in need of treatment" include subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present invention, when used, for example, for detection, for a diagnostic procedure, and/or for treatment.

[0037} One of ordinary skill in the art also understands that the modified genomes disclosed herein may be modified such that their nucleotide sequence differs from the modified polynucleotides from which they were derived. For example, a polynucleotide or nucleotide sequence derived from the disclosed DNA sequence may be similar to, e.g. , may have a certain percentage of identity, the original sequence, e.g. , the sequence may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to, the original sequence.

[0038] Additionally, nucleotide or amino acid substitutions, deletions, or insertions may be made that result in conservative substitutions or changes in "non-essential" regions of amino acids. For example, a polypeptide or amino acid sequence that is derived from a designated protein may be identical to the parent sequence except for one or more individual amino acid substitutions, insertions, or deletions, such as one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or more individual amino acid substitutions, insertions, or deletions. In some embodiments, a polypeptide or amino acid sequence that is derived from a designated protein comprises one to five, one to ten, one to fifteen, or one to twenty individual amino acid substitutions, insertions, or deletions compared to the parent sequence.

[0039] Antibodies can be labeled with a detectable label by attaching them to a chemiluminescent compound. The presence of the chemiluminescent compound-labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that occurs during a chemical reaction. Examples of particularly suitable chemiluminescent label compounds include luminol, isoluminol, a theromatic acridinium ester, imidazole, acridinium salt, and an oxalate ester.

Modified obligate HSV-1 vector

[0040] According to one aspect of the present disclosure, there is provided an HSV-1 virus comprising a modified HSV-1 genome, also referred to as an obligate HSV-1 vector. The HSV-1 genome consists of two covalently linked components designated L and S. Each component consists of unique sequences (U _L for the L component, U _S for the S component) flanked by inverted repeats. The inverted repeats of the L component are designated ab and b'a'. The inverted repeats of the S component are designated a'c' and ca. The inverted repeat regions contain two copies of transcription units. There are at least five open reading frames known in the art that are present in two copies, the proteins of which are designated ICP0, ICP4, ICP34.5, ORF P, and ORF O, respectively. The inverted repeats b'a' and a'c'(b'a'-a'c') are combined to form an internal inverted repeat region. In contrast, the inverted repeats ab and ca are referred to herein as external repeat regions.

[0041] According to one embodiment of the present invention, the modification comprises a deletion between the promoter of the U _L 56 gene and the promoter of the U _S 1 gene of the wild-type HSV-1 genome. In fact, the deleted sequences include the following:

(a) Single copies of transcription units encoding at least 5 proteins (ICP0, ICP4, ICP34.5, ORF-O and ORF-P), all other open reading frames are conserved.

(b) Transcription units that are contained entirely within the sequence b'a'-a'c'.

(c) Transcription units that start in the unique region but continue into the deleted region.

[0042] In the present invention, the deletion is carried out in a precise manner to ensure that the sequences necessary for the expression of all existing open reading frames (ORFs) in the viral DNA remain intact after the deletion. In this context, "sequences necessary for the expression of all existing ORFs" include the ORFs themselves and the regulatory sequences necessary for the expression of each ORF, such as promoters and enhancers, to ensure efficient expression of the ORF and the functionality of the proteins translated in this manner. By "intact" is meant sequences so defined that are at least functional, but this does not mean that the sequences must be 100% identical to naturally occurring sequences. The sequences may differ slightly in nucleotide sequence from naturally occurring sequences due to the content of, for example, conservative substitutions or changes in "non-essential" regions. In this context, sequences may be 90%, 95%, 98%, or 99% identical to natural sequences.

[0043] As will be appreciated by those skilled in the art, the precise first and last nucleotide positions to be deleted according to the present invention depend on the strains and isomers of the HSV-1 virus genome, and these positions can be readily determined using techniques known in the art. It will be appreciated that the present invention is not intended to be limited to any particular HSV-1 virus genome isomers or strains. In one embodiment, the deletion results in the removal of nucleotides 117005 to 132096 in the genome. It will also be apparent to those skilled in the art that other strains are also possible, provided that the genomic DNA is sequenced. Sequencing technologies are readily available in the literature and commercially. For example, in another embodiment, the deletion can be performed in HSV-1 strain 17, the genome of which is available under GenBank Accession No. NC_001806.2. According to another embodiment, the deletion can be performed in the KOS 1.1 strain, the genome of which is available under GenBank accession number KT899744. According to yet another embodiment, the deletion can be performed in the F strain, the genome of which is available under GenBank accession number GU734771.1.

[0044] In some embodiments, the deletion is performed at precisely predetermined positions, resulting in the removal of a DNA fragment starting from the promoter of the last known gene in the L component (such as U _L 56) to the promoter sequence of the first known gene in the S component (such as U _S 1). Thus, all ORFs of genes U _L 1 through U _L 56 in the U _L component and U _S 1 through U _S 12 in the U _S component, as well as the sequence necessary for expression of the ORFs, remain intact. The precise removal and preservation of sequences necessary for expression of all existing open reading frames (ORFs) in the viral DNA after the deletion has many advantages. By "retention" is meant that the modified vector contains all genes in unique sequences ( _UL and _US ) and only one copy of all genes present in two copies, for example, genes for ICP0, ICP4, ICP34.5, ORF P and ORF O. It should be noted that most of the deleted sequences do not encode proteins, but represent duplicated non-coding sequences occupying the spaces between the deleted regions, for example, ICP0 introns, the LAT domain, "a" sequences, etc. It is assumed that the obligate vector according to the present invention also contains only one copy of the duplicated non-coding sequences.

[0045] Retention of all ORFs provides a more potent virus before or after incorporation of the inserted foreign genes, which is maximally resistant to environmental factors such as temperature, pressure, UV light, etc. This approach also allows maximization of the range of cancer cells in which oncolytic HSV-1 is effective.

[0046] Various genetic manipulation techniques known in the art can be used to produce the modified HSV-1 vector described herein. For example, bacterial artificial chromosomes (BAC) technology can be used. See, for example, Horsburgh BC, Hubinette MM, Qiang D, et al. Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genome. Gene Ther , 1999, 6(5):922-30. As another example, a COS plasmid can be used with the present disclosure. See, for example, van Zijl M, Quint W, Briaire J, et al. Regeneration of herpes viruses from molecularly cloned subgenomic fragments. J Virol , 1988, 62(6):2191-5.

[0047] A key property of the construct described herein is that it functions as an obligate vector. The definition applicable to such constructs is that the constructs do not replicate in susceptible cells, but replicate after insertion of viral or cellular DNA sequences, and therefore function as vectors for the expression of genes inserted into the vector sequences.

Recombinant oncolytic virus HSV-1

[0048] The number of foreign DNA sequences that can be inserted into a wild-type virus is limited because they interfere with the packaging of DNA into virions. A precise deletion in a designated region provides an ideal space for insertion of foreign DNA sequences. In one embodiment of the present invention, the deletion removes at least 15 kb of the oncolytic viral vector, allowing a similar number of foreign DNA sequences to be accommodated. Other studies have shown that wild-type genomes carry an additional 7 kb of DNA.

[0049] Therefore, according to another aspect of the present disclosure, there is provided a recombinant oncolytic herpes simplex virus type 1 (HSV-1), comprising (a) a modified HSV-1 genome, wherein the modification comprises a deletion between the promoter of the U _L 56 gene and the promoter of the U _S 1 gene of the wild-type HSV-1 genome, whereby (i) one copy of all genes present in two copies is missing, and (ii) the sequences necessary for the expression of all existing open reading frames (ORFs) in the viral DNA are intact after the deletion; and (b) a heterologous nucleic acid sequence encoding an immunostimulatory and/or immunotherapeutic agent, wherein the heterologous nucleic acid sequence is stably inserted into at least the deleted region of the modified HSV-1 genome.

[0050] In one embodiment, the recombinant oncolytic HSV-1 comprises a heterologous nucleic acid sequence encoding an immunostimulatory agent. In some embodiments, the immunostimulatory agent is selected from the group consisting of GM-CSF, IL-2, IL-5, IL-12, IL-15, IL-24, and IL-27. In one embodiment, the immunostimulatory agent is IL-12. In one embodiment, the immunostimulatory agent is human IL-12 or humanized IL-12. In one embodiment, the immunostimulatory agent is murine IL-12. In another embodiment, the immunostimulatory agent is IL-15.

[0051] In one embodiment, the recombinant oncolytic HSV-1 comprises a heterologous nucleic acid sequence encoding an immunotherapeutic agent. In some embodiments, the immunotherapeutic agent is selected from an anti-PD-1 agent and an anti-CTLA-4 agent. In one embodiment, the immunotherapeutic agent is an anti-PD-1 agent. In another embodiment, the immunotherapeutic agent is an anti-CTLA-4 agent.

[0052] When only one heterologous nucleic acid sequence encoding an immunostimulatory or immunotherapeutic agent is inserted, the heterologous nucleic acid sequence is preferably inserted into the deleted region of the genome. In one embodiment, the heterologous nucleic acid sequence has a length similar to that of the deleted region. In one embodiment, the heterologous nucleic acid sequence has a length that is 20% greater or less than that of the deleted region. According to another embodiment, the heterologous nucleic acid sequence has a length that is 15%, 10%, 5%, 4%, 3%, 2%, or 1% greater or less than that of the deleted region.

[0053] In one embodiment, the heterologous nucleic acid sequence is less than about 18 kb, about 17 kb, or about 16 kb in length. In one embodiment, the heterologous nucleic acid sequence is greater than about 10 kb, 11 kb, 12 kb, 13 kb, or 14 kb in length. In one embodiment, the heterologous nucleic acid sequence is between about 14 kb and about 16 kb in length. In one embodiment, the heterologous nucleic acid sequence is about 15 kb in length.

[0054] In some embodiments, the recombinant oncolytic HSV-1 comprises at least two heterologous nucleic acid sequences encoding immunostimulatory and/or immunotherapeutic agents. In some embodiments, the recombinant oncolytic HSV-1 comprises heterologous nucleic acid sequences encoding two different immunostimulatory agents. For example, in one embodiment, the recombinant oncolytic HSV-1 comprises heterologous nucleic acid sequences encoding both IL-12 and GM-CSF. In another embodiment, the recombinant oncolytic HSV-1 comprises heterologous nucleic acid sequences encoding both IL-15 and GM-CSF. According to a further embodiment, the recombinant oncolytic HSV-1 comprises heterologous nucleic acid sequences encoding both IL-12 and IL-15.

[0055] In some embodiments, the recombinant oncolytic HSV-1 comprises heterologous nucleic acid sequences encoding two different immunotherapeutic agents. In one embodiment, for example, the recombinant oncolytic HSV-1 comprises heterologous nucleic acid sequences encoding both an anti-PD-1 agent and an anti-CTLA-4 agent.

[0056] In some embodiments, the recombinant oncolytic HSV-1 comprises heterologous nucleic acid sequences encoding three different immunostimulatory and/or immunotherapeutic agents. For example, in one embodiment, the recombinant oncolytic HSV-1 comprises heterologous nucleic acid sequences encoding IL-12, an anti-CTLA-4 agent, and an anti-PD-1 agent.

[0057] If more than one heterologous nucleic acid sequence encoding immunostimulatory and/or immunotherapeutic agents is inserted, the first heterologous nucleic acid sequence is preferably inserted into the deleted region of the genome. The second or subsequent heterologous nucleic acid sequence can be inserted into the L component of the genome. In one embodiment, the second heterologous nucleic acid sequence is inserted between the U _L 3 and U _L 4 genes of the L component. In one embodiment, the second heterologous nucleic acid sequence is inserted between the U _L 37 and U _L 38 genes of the L component.

[0058] In one embodiment, a first heterologous nucleic acid sequence is inserted into a deleted region of the genome, and a second heterologous nucleic acid sequence is inserted between the U _L 3 and U _L 4 genes. In one embodiment, a first heterologous nucleic acid sequence is inserted into a deleted region of an internal inverted repeat of the genome, and a second heterologous nucleic acid sequence is inserted between the U _L 37 and U _L 38 genes of the L component. In one embodiment, a first heterologous nucleic acid sequence is inserted into a deleted region of an internal inverted repeat of the genome, a second heterologous nucleic acid sequence is inserted between the U _L 3 and U _L 4 genes, and a third heterologous nucleic acid sequence is inserted between the U _L 37 and U _L 38 genes of the L component.

[0059] In one embodiment, the first heterologous nucleic acid sequence encodes IL-12. In one embodiment, the second heterologous nucleic acid sequence encodes an anti-CTLA-4 agent or an anti-PD-1 agent. In one embodiment, the third heterologous nucleic acid sequence encodes an anti-PD-1 agent or an anti-CTLA-4 agent.

[0060] It should be understood that insertions of one or more heterologous nucleic acid sequences into the oncolytic HSV-1 genome do not interfere with the expression of native HSV-1 genes, and the heterologous nucleic acid sequences are stably incorporated into the modified HSV-1 genome, such that functional expression of the heterologous nucleic acid sequences can be expected.

[0061] A recombinant gene encoding an immunostimulatory and/or immunotherapeutic agent comprises a nucleic acid encoding a protein together with regulatory elements for expression of the protein. Typically, the regulatory elements that are present in a recombinant gene, and which are selected depending on the host cells used for expression, operably linked to the nucleic acid sequence to be expressed include a transcriptional promoter, a ribosome binding site, and a terminator. Within a recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory sequence or sequences in a manner that allows expression of the nucleotide sequence ( e.g. , in an in vitro transcription/translation system or in a host cell when a virus is introduced into a host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence in multiple host cell types and sequences that direct expression of a nucleotide sequence exclusively in certain host cells (e.g., tissue-specific regulatory sequences).

[0062] Recently, regulatory sequences called insulators have been discovered, which comprise a class of DNA elements found on cellular chromosomes that protect genes in one region of the chromosome from the regulatory influence of another region. Amelio et al. discovered a 1.5 kb region containing a cluster of CTCF motifs in the LAT region that has insulator activities, namely, enhancer blocking and silencing (Amelio et al.. A Chromatin Insulator-Like Element in the Herpes Simplex Virus Type 1 Latency-Associated Transcript Region Binds CCCTC-Binding Factor and Displays Enhancer-Blocking and Silencing Activities. Journal of Virology , Vol. 80, No. 5, Mar. 2006, p. 2358-2368).

[0063] A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one that stimulates initiation of mRNA synthesis at a high frequency. A suitable element for processing in eukaryotic cells is a polyadenylation signal. Antibody-linked introns may also be present. Examples of expression cassettes for producing an antibody or antibody fragment are well known in the art (e.g., Persic et al ., 1997, Gene 187:9-18; Boel et al ., 2000, J Immunol. Methods 239:153-166; Liang et al ., 2001, J. Immunol. Methods 247:1 19-130; Tsurushita et al ., 2005, Methods 36:69-83).

[0064] Those skilled in the art can select appropriate regulatory elements based on, for example, the desired tissue specificity and level of expression. For example, a tissue type-specific or tumor-specific promoter can be used to restrict expression of a gene product to a particular cell type. In addition to the use of tissue-specific promoters, local administration of viruses can result in localized expression and action. Examples of non-tissue-specific promoters that can be used include the cytomegalovirus (CMV) early promoter (U.S. Patent No. 4,168,062) and the Rous sarcoma virus promoter. HSV promoters, such as the HSV-1 IE promoters, can also be used. In some embodiments, the promoter is selected from the promoter in the following table.

Promoter Tumor or target tissue B-myb Glioma metastases in the liver Nestine Glioma CEA (carcinoembryonic antigen) Colon cancer Albumen Hepatoma DF3/MUC1 (Mucin 1) Pancreatic cancer Saponin Leiomyosarcoma

[0065] Examples of tissue-specific promoters that can be used in this technique include, for example, the prostate-specific antigen (PSA) promoter, specific for prostate cells; the desmin promoter, specific for muscle cells; the enolase promoter, specific for neurons; the beta-globin promoter, specific for erythroid cells; the tau-globin promoter, also specific for erythroid cells; the growth hormone promoter, specific for pituic cells; the insulin promoter, specific for pancreatic beta cells; the glyofibrillar acidic protein promoter, specific for astrocytes; the tyrosine hydroxylase promoter, specific for catecholaminergic neurons; the amyloid precursor protein promoter, specific for neurons; the dopamine beta-hydroxylase promoter, specific for noradrenergic and adrenergic neurons; The tryptophan hydroxylase promoter is specific for serotonin/pineal cells; the choline acetyltransferase promoter is specific for cholinergic neurons; the aromatic L-amino acid decarboxylase (AADC) promoter is specific for catecholaminergic/5-HT/D-type cells; the proenkephalin promoter is specific for neuronal/spermatogenic epididymal cells; the reg (lipostatin) promoter is specific for colorectal tumors and pancreatic and kidney cells; and the parathyroid hormone-related peptide (PTHrP) promoter is specific for liver and cecum tumors and for neurilemomas of the kidney, pancreas, and adrenal glands.

[0066] Examples of promoters that function specifically in tumor cells include the stromelysin 3 promoter, which is specific for breast cancer cells; the surfactant protein A promoter, which is specific for non-small cell lung cancer cells; the secretory leukocyte protease inhibitor (SLPI) promoter, which is specific for SLPI-expressing carcinomas; the tyrosinase promoter, which is specific for melanoma cells; the stress-inducible grp78/BiP promoter, which is specific for fibrosarcoma/tumor-promoting cells; the adipose enhancer AP2, which is specific for adipocytes; the transthyretin a-1 antitrypsin promoter, which is specific for hepatocytes; the interleukin-10 promoter, which is specific for glioblastoma multiform cells; c-erbB-2 promoter, specific for pancreatic, breast, gastric, ovarian, and non-small cell lung cells; a-B-crystallin/heat shock protein 27 promoter, specific for brain tumor cells; basic fibroblast growth factor promoter, specific for glioma and meningioma cells; epidermal growth factor receptor promoter, specific for squamous cell carcinoma, glioma, and breast tumor cells; mucin-like glycoprotein (DF3, MUC1) promoter, specific for breast carcinoma cells; mtsl promoter, specific for metastatic tumor; NSE promoter, specific for small cell lung cancer cells; somatostatin receptor promoter, specific for small cell lung cancer cells; c-erbB-3 and c-erbB-2 promoters, specific for breast cancer cells; a c-erbB4 promoter specific for breast and gastric cancer; a thyroglobulin promoter specific for thyroid carcinoma cells; an afferent fetal protein (AFP) promoter specific for hepatoma cells; a villin promoter specific for gastric cancer cells; and an albumin promoter specific for hepatoma cells. According to another embodiment, a TERT promoter or a survivin promoter is used.

[0067] For example, in some embodiments, the heterologous nucleic acid sequences are operably linked to a promoter, such as a CMV promoter or an Egr promoter. In one embodiment, a nucleotide sequence encoding mIL12 (murine IL-12) is operably linked to an Egr promoter. In another embodiment, a nucleotide sequence encoding an anti-hPD1 (human PD1) scFv is operably linked to a CMV promoter.

Immunostimulating or immunotherapeutic agents

[0068] In certain embodiments, oHSV-1 of the present invention encodes one or more immunostimulatory agents (also referred to as immunostimulatory molecules), including cytokines such as IL-2, IL-4, IL-12, GM-CSF, IFNγ, chemokines such as MIP-1, MCP-1, IL-8, and growth factors such as FLT3 ligand.

[0069] Alternatively or additionally, the oHSV-1 of the present invention encodes one or more immunotherapeutic agents, such as a PD-1 binding agent (or anti-PD-1 agent) or a CTLA-4 binding agent (or anti-CTLA-4 agent), including antibodies or fragments thereof, such as an anti-PD1 antibody that specifically binds to PD-1 or an anti-CTLA-4 antibody that specifically binds to CTLA-4. The anti-PD-1 antibody can be a single chain antibody that antagonizes PD-1 activity. In other embodiments, the oncolytic virus expresses an agent that antagonizes the binding of PD-1 ligands to the receptor, such as anti-PD-L1 and/or anti-PD-L2, anti-PD-L1 and/or anti-PD-L2 antibodies, or a soluble PD-1 receptor.

[0070] The PD-1 signaling pathway plays an important role in tumor-associated immune dysfunction. Infection and lysis of tumor cells can induce a highly specific antitumor immune response that kills inoculated tumor cells as well as distant, established, uninoculated tumor cells. Tumors and their microenvironments have evolved mechanisms to evade, suppress, and inactivate the innate antitumor immune response. For example, tumors can down-regulate target receptors, enclose themselves in fibrous extracellular stromal matrix, or up-regulate host receptors or ligands involved in the activation or recruitment of regulatory immune cells. Innate and/or adaptive T-regulatory cells (Tregs) are involved in tumor-mediated immune suppression. Without intending to be limited by theoretical justification, it is believed that PD-1 blockade may inhibit Treg activity and improve the efficacy of tumor-reactive CTLs (cytotoxic lymphocytes). Other aspects of the technique will be described in more detail below. PD-1 blockade may also stimulate antitumor immune response by blocking the inactivation of T cells (CTLs and helpers) and B cells.

[0071] According to one aspect, the disclosed method provides an oncolytic virus that carries a gene encoding a PD-1 binding agent. Programmed Cell Death 1 (PD-1) is a 50-55 kDa type I transmembrane receptor originally identified by subtractive hybridization of a mouse T cell line that undergoes apoptosis (Ishida et al., 1992, Embo J. 11:3887-95). A member of the CD28 gene family, PD-1 is expressed on activated T, B, and myeloid cells (Greenwald et al. , 2005, Annu. Rev. Immunol. 23:515-48; Sharpe et al. , 2007, Nat. Immunol. 8:239-45). Human and mouse PD-1 share approximately 60% amino acid identity, with conservation of four potential N-glycosylation sites and residues that define the Ig-V domain. Two PD-1 ligands have been identified: PD ligand 1 (PD-L1) and PD ligand 2 (PD-L2); both belong to the B7 superfamily. PD-L1 is expressed on many cell types, including T, B, endothelial, and epithelial cells, as well as antigen-presenting cells. In contrast, PD-L2 is narrowly expressed on professional antigen-presenting cells such as dendritic cells and macrophages.

[0072] PD-1 negatively modulates T cell activation, and this inhibitory function is linked to the immunoreceptor tyrosine-based inhibitory motif (ITIM) of its cytoplasmic domain (Parry et al. , 2005, Mol. Cell. Biol. 25:9543-53). Disruption of this inhibitory function of PD-1 can lead to autoimmunity. The reverse scenario can also be detrimental. Persistent negative signals from PD-1 are implicated in the impairment of T cell function in many pathological situations, such as tumor immune evasion and chronic viral infections.

[0073] Host antitumor immunity is primarily dependent on tumor-infiltrating lymphocytes (TILs) (Galore et al. , 2006, Science 313:1960-4). Multiple lines of evidence have established that TILs are under the inhibitory regulation of PD-1. First, PD-L1 expression has been confirmed in multiple human and murine tumor lines, and expression can be further upregulated by IFN-γ in vitro (Dong et al. , 2002, Nat. Med. 8:793-800). Second, PD-L1 expression by tumor cells has been directly linked to their resistance to lysis by antitumor T cells in vitro (Blank et al. , 2004, Cancer Res. 64:1 140-5). Third, PD-1 knockout mice are resistant to tumor challenge (Iwai et al ., 2005, Int. Immunol. 17:133–44), and T cells from PD-1 knockout mice are highly effective at tumor rejection when adoptively transferred into tumor-bearing mice (Blank et al., cited above). Fourth, blocking PD-1 inhibitory signaling with a monoclonal antibody can enhance host antitumor immunity in mice (Iwai et al. , cited above; Hirano et al. , 2005, Cancer Res . 65:1089–96). Fifth, high levels of PD-L1 expression in tumors (detected by immunohistochemical staining) are associated with poor prognosis in many types of human cancer (Hamanishi et al. , 2007, Proc. Natl. Acad. Sci. USA 104:3360-5).

[0074] Oncolytic virotherapy is an effective way to shape the host immune system by expanding T or B cell populations specific for tumor-specific antigens released following oncolysis. The immunogenicity of tumor-specific antigens is largely dependent on the affinity of host immune receptors (B cell receptors or T cell receptors) for antigen epitopes and the host tolerance threshold. High-affinity interactions will direct host immune cells through multiple cycles of proliferation and differentiation to become long-lasting memory cells. Host tolerance mechanisms will balance such proliferation and expansion with minimization of potential tissue damage that results from local immune activation. PD-1 inhibitory signaling is part of such host tolerance mechanisms, as supported by the following set of data. First, PD-1 expression is increased in actively proliferating T cells, particularly in cells with terminally differentiated phenotypes, i.e., effector phenotypes. Effector cells are often associated with potent cytotoxic function and cytokine production. Second, PD-L1 is important for maintaining peripheral tolerance and for limiting overactive T cells locally. Therefore, inhibition of PD-1 using a PD-1 binding agent expressed in the tumor microenvironment may be an effective strategy to enhance IOL activity and stimulate an effective and durable antitumor immune response.

[0075] Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a member of the immunoglobulin (Ig) superfamily of proteins. The Ig superfamily is a group of proteins that are characterized by key structural properties of the variable (V) or constant (C) domain of the Ig molecule. Members of the Ig superfamily include, but are not limited to, immunoglobulins themselves, major histocompatibility complex (MHC) class molecules (i.e., MHC classes I and II), and TCR (T cell receptor) molecules. T cells require two types of signals from the antigen-presenting cell (APC) for activation and subsequent differentiation into effector function. First, there is an antigen-specific signal generated by interactions between TCRs on the T cell with MHC molecules presenting peptides on APCs. Second, there is an antigen-independent signal mediated by the interaction of CD28 with members of the B7 family (B7-1 (CD80) or B7-2 (CD86)). Where CTLA-4 fits into the milieu of immunologic reactivity, evasion was initially observed. Murine CTLA-4 was first identified and cloned (Brunet et al. Nature 328:267-270 (1987)) as part of a search for molecules potentially expressed on cytotoxic T lymphocytes. Human CTLA-4 was identified and cloned shortly thereafter (Dariavach et al. Eur. J. Immunol. 18:1901-1905 (1988)). Mouse and human CTLA-4 molecules share approximately 76% overall sequence homology and approach complete sequence identity in their cytoplasmic domains (Dariavach et al. Eur. J. Immunol . 18:1901–1905 (1988)).

[0076] Beginning in 1993, with a peak in 1995, researchers began to further elucidate the role of CTLA-4 in T cell stimulation. First, through the use of monoclonal antibodies to CTLA-4 (Walunas et al. Immunity 1:405-13 (1994)) evidence was obtained that CTLA-4 may function as a negative regulator of T cell activation.

[0077] In the cancer context, Kwon et al. PNAS USA 94:8099–103 (1997) established a syngeneic mouse model of prostate cancer and investigated two different manipulations to elicit an anti-prostate cancer response through enhanced T cell costimulation: (i) providing direct costimulation by transducing prostate cancer cells to express B7.1 ligand and (ii) antibody-mediated blockade of T cell CTLA-4 in vivo , which prevents T cell downregulation. It was demonstrated that antibody-mediated blockade of CTLA-4 in vivo enhanced immune responses against prostate cancer. Also, Yang et al. Cancer Res 57:4036–41 (1997) reported a study of whether blockade of CTLA-4 function enhances antitumor T cell responses at different stages of tumor growth. Based on in vitro and in vivo data, these investigators found that blockade of CTLA-4 in tumor-bearing individuals enhanced the ability to elicit antitumor T cell responses, but that this enhancing effect in this model was limited to early stages of tumor growth. Subsequently, Hurwitz et al. Proc Natl Acad Sci USA 95:10067–71 (1998) reported a study of the generation of T cell-mediated antitumor responses depending on T cell receptor major histocompatibility complex/antigen binding and CD28 binding by B7 family members. Some tumors, such as SM1 breast carcinoma, have been resistant to anti-CTLA-4 immunotherapy. For example, a combination of CTLA-4 blockade and a vaccine consisting of SM1 cells expressing granulocyte-monocyte colony-stimulating factor (SM1) resulted in regression of the parent tumor despite failure of either treatment alone. This combination therapy provided durable immunity to SM1 and was dependent on both CD4(+) and CD8(+) T cells. These results indicate that CTLA-4 blockade acts at the level of host antigen-presenting cells.

Anti-PD-1 agents and anti-CTLA-4 agents

[0078] According to one aspect, the present technique provides an oncolytic virus comprising a heterologous nucleic acid encoding an anti-PD-1 agent and/or an anti-CTLA-4 agent. In some embodiments, the anti-PD-1 agent or anti-CTLA-4 agent comprises an antibody variable region that provides specific binding to an epitope of PD-1 or CTLA-4. The antibody variable region can be present in, for example, a whole antibody, an antibody fragment, and a recombinant derivative of an antibody or antibody fragment. The term "antibody" describes an immunoglobulin, whether naturally occurring or partially or completely produced synthetically. Thus, the anti-PD-1 agent or anti-CTLA-4 agent of the present technology includes any polypeptide or protein comprising a binding domain specific for binding to an epitope of PD-1 or CTLA-4.

[0079] Different classes of antibodies are characterized by different structures. The different regions of an antibody can be illustrated by IgG. The IgG molecule contains four polypeptide chains: two longer heavy chains and two shorter light chains, which are linked together by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. The heavy chain contains the heavy chain variable region (V _H ) and the heavy chain constant region (CH1, CH2, and CH3). The light chain contains the light chain variable region (V _L ) and the light chain constant region ( _CL ). Within the variable regions, there are three hypervariable regions that are responsible for antigen specificity. (See, for example, Breitling et al. , Recombinant Antibodies , John Wiley & Sons, Inc. and Spektrum Akademischer Verlag, 1999; and Lewin, Genes IV , Oxford University Press and Cell Press, 1990).

[0080] The hypervariable regions, commonly referred to as complementarity determining regions (CDRs), are located between more conserved flanking regions called framework regions (FWs). There are four (4) FWs and three (3) CDRs, which are arranged from NH2-terminus to COOH-terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The amino acids associated with the framework regions and CDRs can be numbered and aligned using the approaches described in Kabat et al. , Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991; C. Chothia and A. M. Lesk, J Mol Biol 196(4):901 (1987); or B. Al-Lazikani, et al. , J Mol Biol 273(4): 27, 1997. For example, framework regions and CDRs can be identified based on both the Cabot and Chothia definitions. The variable regions of the heavy and light chains contain the binding domain that interacts with antigen. The two carboxyl regions of the heavy chain are constant regions joined by disulfide bonds to form the Fc region. The Fc region is important for effector functions. (Presta, Advanced Drug Delivery Reviews 58:640-656, 2006.) Each of the two heavy chains that form the Fc region continues into distinct Fab regions via the hinge region.

[0081] Anti-PD-1 agents or anti-CTLA-4 agents typically comprise the variable region of an antibody. Such antibody fragments include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the V _H , V _L , _{CH ,} and _{CL domains;} (ii) a Fab ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, consisting of the V _H and _CH1 domains; (iv) an Fv fragment, consisting of the V _H and V _L domains of one arm of an antibody; (v) a dAb fragment, which comprises a V _H or V _L domain; (vi) scAb, an antibody fragment comprising V _H and V _L , as well as C ₁ or _CH1 , and (vii) artificial antibodies based on protein backbones, including, but not limited to, fibronectin type III polypeptide antibodies (e.g., see U.S. Patent No. 6,703,199). Moreover, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be combined using recombinant methods through a synthetic linker, which allows them to be produced as a single protein chain in which the V _L and V _H regions are paired to form a monovalent molecule known as a single chain Fv (scFv). Thus, the variable region of the antibody can be present in the recombinant derivative. Examples of recombinant derivatives include single-chain antibodies, diabodies, triabodies, tetrabodies, and minibodies. An anti-PD-1 agent or an anti-CTLA-4 agent may also contain one or more variable regions that recognize the same or different epitopes.

[0082] In some embodiments, the anti-PD-1 agents or anti-CTLA-4 agents are encoded by an oncolytic virus produced using recombinant nucleic acid techniques. Various anti-PD-1 agents can be produced using a variety of techniques, including, for example, a single-chain protein comprising a V _H region and a V _L region connected by a linker sequence such as an scFv, and antibodies or fragments thereof; and a multichain protein comprising a V _H and V _L region on separate polypeptides. Recombinant nucleic acid techniques include engineering a nucleic acid template for protein synthesis. Suitable recombinant nucleic acid techniques are well known in the art. (See, for example, Ausubel, Current Protocols in Molecular Biology, John Wiley, 2005; Harlow et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Recombinant nucleic acid encoding an anti-PD-1 antibody or an anti-CTLA-4 antibody can be expressed in a cell infected with an oncolytic virus and released into the tumor microenvironment after viral lysis. In essence, the cell acts as a factory for the encoded protein.

[0083] A nucleic acid comprising one or more recombinant genes encoding one or both of the V _H region or the V _L region of an anti-PD-1 agent or CTLA-4 can be used to produce a complete PD-1/CTLA-4 binding protein/polypeptide. A complete binding agent can be produced, for example, by using a single gene to encode a single chain protein comprising a V _H region and a V _L region connected by a linker such as an scFv, or by using multiple recombinant regions, for example, to produce both V _H and V _L regions.

[0084] Examples of anti-PD-1 antibodies or anti-CTLA-4 antibodies, or fragments or derivatives thereof, suitable for the present invention are available in the art. See, for example, WO 2006/121168, WO 2014/055648, WO 2008/156712, US 2014/0234296, or U.S. Patent No. 6,984,720.

[0085] The recombinant oHSV-1 of the present invention delivers the immunostimulatory protein to the tumor exactly where the protein is needed, rather than systemically. Moreover, by reducing the production and very likely also the uptake of proteins in the tumor mass, cytotoxic effects are likely to be greatly reduced or absent.

Example of PD-1 scFv and CTLA-4 scFv sequences

1. scFv to mPD-1 (mouse PD-1) - nucleic acid (SEQ ID NO:1) ATGGGATGGT CATGTATCAT CCTTTTTCTA GTAGCAACTG CAACCGGCGC GCACTCCGAG
GTGCAGCTGG TGCAGTCTGG GGGAGGCGTG GTTCAGCCTG GGAGGTCCCT GAGACTCTCC
TGTGGCAGCGT CTGGATTCAC CTTTAGTAGC TATTGGATGA GCTGGGTCCG CCAGGCTCCA
GGGAAGGGGC TGGAGTGGGT CTCAGCTATTT AGTGGTAGTG GTGGTAGCAC ATACTACGCA
GACTCCGTGA AGGGCCGGTT CACCATCTCC AGAGACAATT CCAAGAACAC GCTGTATCTG
CAAATGAACA GCCTAAGAGC CGAGGACACG GCCGTATATT ACTGTGCGAA AGAGAACTGG
GGATCGTACT TCGATCTCTG GGGGCAAGGG ACCACGGTCA CCGTCTCCTC AGGTGGCGGA
GGGTCAGGTG GCGGAGGGTC AGGTGGCGGA GGGTCAGGCG TGCACTCCGA CATCGTGATG
ACCCAGTCTC CTTCCACCCT GTCTGCATCT GTAGGAGACA GAGTCACCAT CACTTGCCGG
GCCAGTCAGG GTATTAGTAG CTGGTTGGCC TGGTATCAGC AGAAACCAGG GAGAGCCCCT
AAGGTCTTGA TCTATAAGGC ATCTACTTTA GAAAGTGGGG TCCCATCAAG GTTCAGCGGC
AGTGGATCTG GGACAGATTT CACTCTCACC ATCAGCAGTC TGCAACCTGA AGATTTTGCA
ACTTACTACT GTCAACAGAG TTACAGTACC CCGTGGACGT TCGGCCAGGG GACCAAGCTG
GAAATCAAGA GATGATAA 2. scFv to mPD-1 - protein (SEQ ID NO:2) MGWSCIILFL VATATGAHSE VQLVQSGGGV VQPGRSLRLS CAASGFTFSS YWMSWVRQAP
GKGLEWVSAI SSGSGGSTYYA DSVKGRFTIS RDNSKNTLYL QMNSLRAEDT AVYYCAKENW
GSYFDLWGQG TTVTVSSGGG GSGGGGSGGG GSGVHSDIVM TQSPSTLSAS VGDRVTITCR
ASQGISSWLA WYQQKPGRAP KVLIYKASTL ESGGVPSRFSG SGSGTDFTLT ISSLQPEDFA
TYYCQQSYST PWTFGQGTKL EIKR 3. scFv to mCTLA-4 (mouse CTLA-4) - nucleic acid (SEQ ID NO:5) ATGGGATGGT CATGTATCAT CCTTTTTCTA GTAGCAACTG CAACCCAGAT CCAGCTTCAG
GAGTCAGGAC CTGGCCTGGT GAACCCCTCA CAATCACTGT CCCTCTCTTG CTCTGTCACT
GGTTACTCCA TCACCAGTGG TTATGGATGG AACTGGATCA GGCAGTTCCC AGGGCAGAAG
GTGGAGTGGA TGGGATTCAT ATATTATGAG GGTAGCACCT ACTACAACCC TTCCATCAAG
AGCCGCATCT CCATCACCAG AGACACATCG AAGAACCAGT TCTTCCTGCA GGTGAATTCT
GTGACCACTG AGGACACAGC CACATATTAC TGTGCGAGAC AAACTGGGTA CTTTGATTAC
TGGGGCCAAG GAACCATGGT CACCGTCTCC TCAGGTGGTG GTGGATCAGG TGGAGGCGGA
AGTGGAGGTG GCGGTTCCGA CATCATGATG ACCCAGTCTC CTTCATCCCT GAGTGTGTCA
GCGGGAGAGA AAGCCACTAT CAGCTGCAAG TCCAGTCAGA GTCTTTTCAA CAGTAACGCC
AAAACGAACT ACTTGAACTG GTATTTGCAG AAACCAGGGC AGTCTCCTAA ACTGCTGATC
TATTATGCAT CCACTAGGCA TACTGGGGTC CCTGATCGCT TCAGAGGCAG TGGATCTGGG
ACGGATTTCA CTCTCACCAT CAGCAGTGTC CAGGATGAAG ACCTGGCATT TTATTACTGT
CAGCAGTGGT ATGACTACCC ATACACGTTC GGAGCTGGGA CCAAGGTGGA AATCAAATGA
TAA 4. scFv to mCTLA-4 protein (SEQ ID NO:6) MGWSCIILFL VATATQIQLQ ESGPGLVNPS QSLLSLSCSVT GYSITSGYGW NWIRQFPGQK
VEWMGFIYYE GSTYYNPSIK SRISITRDTS KNQFFLQVNS VTTEDTATYY CARQTGYFDY
WGQGTMVTVS SGGGGSGGGG SGGGGSDIMM TQSPSSLSVS AGEKATISCK SSQSLFNSNA
KTNYLNWYLQ KPGQSPKLLI YYASTRHTGV PDRFRGSGSG TDFTLTISSV QDEDLAFYYC
QQWYDYPYTF GAGTKVEIK

Compositions

[0086] The oncolytic virus may be formulated in a suitable pharmaceutically acceptable carrier or excipient. Under ordinary conditions of storage and use, such preparations contain a preservative to prevent the growth of microorganisms. Dosage forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent No. 5,466,468). In all cases, the form must be sterile and must be fluid to the extent that it can be easily syringable. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0087] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyhydric alcohol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures of these components and/or vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic substances in the composition, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be ensured by including in the composition substances that delay absorption, for example, aluminum monostearate and gelatin.

[0088] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with a sufficient amount of saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art in light of the present disclosure. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of hypodermoclisis fluid or injected into the proposed infusion site (see, for example, Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variability in dosage will necessarily occur depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dosage for the individual subject. Moreover, for administration to humans, preparations must meet standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biologies standards.

[0089] Sterile injectable solutions are prepared by incorporating the active components in the required amount in an appropriate solvent with various other components enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating various sterilized active components in a sterile vehicle which contains the basic dispersion medium and the required other components from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield the active component in powder form in combination with any additional desired component from a previously filtered sterilized solution thereof.

[0090] The compositions disclosed herein may be presented in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein) which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be prepared from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and from organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. Once prepared, the solutions will be administered in a manner compatible with the dosage form and in an amount that is therapeutically effective. The compositions are easy to administer in various dosage forms, such as injection solutions, drug-releasing capsules, etc.

[0091] As used herein, "carrier" includes any and all solvents, dispersion media, fillers, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and vehicles for active pharmaceutical ingredients is well known in the art. Except where any conventional medium or vehicle is incompatible with the active component, their use in therapeutic compositions is contemplated. Additional active components can also be incorporated into the compositions.

[0092] The term "pharmaceutically acceptable" means chemical compounds and compositions that do not cause an allergic or similar undesirable reaction when administered to humans. The preparation of aqueous compositions that contain a protein as an active component is well known in the art. Typically, such compositions are prepared in the form of injection solutions, aqueous solutions or suspensions; solid forms suitable for solution or suspension in liquid prior to injection can also be prepared.

Treatment options

[0093] In a further aspect of the present disclosure, there is provided a method of treating or ameliorating cancer, said method comprising administering to a subject in need thereof an effective amount of a recombinant oncolytic HSV-1 virus or a pharmaceutical composition comprising a recombinant oncolytic HSV-1 virus as described above. Equally, the present invention provides an oncolytic HSV-1 virus as described above for use in a method of treating or ameliorating cancer.

[0094] According to some embodiments, the recombinant oncolytic HSV-1 virus or pharmaceutical composition is administered intratumorally. In one embodiment, the HSV-1 virus or pharmaceutical composition is injected directly into the tumor mass in the form of an injectable solution.

[0095] In some embodiments, it may be desirable to combine an oncolytic virus carrying a gene encoding an immunostimulatory and/or immunotherapeutic agent with other agents (agents) effective in treating cancer. For example, cancer can be treated using an oncolytic virus and other anti-cancer therapies, such as anti-cancer agents or surgery. In the context of the technology of the present invention, it is envisaged that oncolytic virus therapy can be used in combination with chemotherapeutic, radiotherapeutic, immunotherapeutic or other biological intervention.

[0096] An "anti-cancer" agent is capable of adversely affecting cancer in a subject, such as by killing cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing the size of a tumor, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, stimulating an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the survival of a subject suffering from cancer. Anti-cancer agents include biological agents (biotherapy), chemotherapeutic agents, and radiotherapeutic agents. More generally, these other compositions will be provided in a combined amount effective to kill or inhibit proliferation of a cell. This process may include contacting cells with an expression construct and the agent or agents or multiple factors simultaneously. This can be achieved by contacting the cell with a single composition or formulation that contains both agents, or by contacting the cell with two separate compositions or formulations simultaneously, one composition containing the expression construct and the other containing the second agent or agents.

[0097] In some embodiments, an oncolytic virus carrying a gene that encodes an immunostimulatory and/or immunotherapeutic agent is combined with an adjuvant. In one embodiment, the adjuvant is an oligonucleotide containing an unmethylated CpG motif. Unmethylated CpG dinucleotide motifs in bacterial deoxyribonucleic acid (DNA) have the advantage of stimulating certain immune cells to secrete cytokines to enhance innate and adaptive immunity.

[0098] Virotherapy may precede or follow treatment with other agents at intervals ranging from minutes to weeks. In embodiments in which the other agent and the oncolytic virus are administered to a cell separately, it should generally be ensured that no significant period of time has elapsed between the time of each delivery, so that the agent and virus are still able to exert their advantageous combined effect on the cell. In such cases, it is envisaged that the cell may be contacted with both agents within an interval of about 12 to 24 hours. However, if the interval between the respective administrations is from several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8), it may be desirable in some cases to significantly increase the time period for treatment.

oHSV constructs expressing immunostimulatory or immunotherapeutic genes

Construction of a modified obligate oHSV-1 vector (IMMV201)

[0099] In the production of the obligate vector IMMV201 using bacterial artificial chromosome (BAC) technology, the CMV promoter cassette, ATGCAGGTGCAGTAATAGTAA, which forms 3 stop codons, was inserted into the T-Easy vector. HSV-1 in the prototype (P) arrangement was used. The cassettes flanked upstream of nucleotide 117005 and downstream of nucleotide 132096 with respect to the wild-type genome were PCR amplified from the HSV-1 viral genome using two sets of primers, respectively (GAAGATCTAATATTTTTATTGCAACTCCCTG, CTAGCTAGCTTATAAAAGGCGCGTCCCGTGG) and (GCTCTAGATTGCGACGCCCCGGCTC, CCTTAATTAAGGTTACCACCCTGTAGCCCCGATGT), and inserted into a plasmid containing CMV and the three stop codons described above, then constructed into the gene replacement plasmid pKO5 to obtain pKO-CMV-STOP. IMMV201 was constructed by inserting pKO-CMV-STOP into E. coli RecA+ carrying the HSV BAC.

BAC-IMMV201 was unable to support viral growth in mammalian cells

[00100] Vero cells were transfected with 2-3 micrograms of BAC-IMMV201 constructed as described above at 70% confluence using OptiMEM medium (Life Technologies, Inc.) according to the manufacturer's instructions. Cells were incubated in an incubator at 37°C with 5% _CO2 for 4 hours. After incubation, the medium was replaced with 4 ml of fresh complete growth medium (5% newborn calf serum/DMEM). After 3-4 days, no viral plaques had formed. The experiment was repeated three times and no viral plaques were formed.

oHSV-1 constructs expressing a single immunostimulatory or immunotherapeutic gene (IMMV202, 203, 303, 403)

[00101] CMV promoter cassettes driving immunostimulatory genes (mouse IL-12, human IL-12) or immunotherapeutic genes (scFv to human PD-1 (SEQ ID No. 1 or 3), scFV to human CTLA-4 (SEQ ID No. 5)) were constructed by electroporation of pKO cassettes into E. coli RecA+ carrying IMMV201 (shown in Figure 1).

oHSV-1 constructs expressing two immunostimulatory or immunotherapeutic genes (IMMV502, 503, 504, 505, 507)

[00102] Then, CMV promoter cassettes driving immunostimulatory genes (IL-12) and immunotherapeutic genes (scFv to PD-1, scFV to CTLA-4) were inserted between the UL3 and UL4 genes into the IMMV202, 203, 303, 403 vector to generate recombinant oHSVs that expressed combinations of immunostimulatory genes (IL-12) and immunotherapeutic genes and which are shown in Figure 2.

oHSV-1 construct expressing one immunostimulatory gene and two immunotherapeutic genes (IMMV603)

[00103] IMMV603 expressed all three immunostimulatory and immunotherapeutic cDNAs encoding IL-12, anti-PD-1 scFv and anti-CTLA-4 scFV, by inserting anti-CTLA-4 scFV between the UL37 and UL38 genes in the IMMV503 vector (shown in Figure 2).

In vitro assays

Expression of scFv to PD-1

[00104] In the series of experiments described herein, 2 x 10 ⁶ H293T cells were mock transfected or transfected with plasmids containing cDNA encoding a His-tagged anti-PD-1 scFv driven by the CMV promoter, together with signal peptide coding regions from various natural sources. Cell lysates and supernatant were collected 46 hours after transfection, then analyzed by SDS-PAGE and blotted with an anti-His antibody. Forty microliters of 2 ml of supernatant, 30 μl of 200 μl of cell lysates were loaded onto a 12% PAA gel. The amounts of anti-PD-1 scFv accumulated in the cell culture supernatant (Figure 3) reflected the potency of the various signal peptides.

Binding affinity of scFv to PD-1 with PD-1

[00105] 2 x 10 ⁶ H293T cells were mock transfected or transfected with plasmids containing cDNA encoding a His-tagged anti-PD-1 scFv driven by the CMV promoter along with the HMM38 signal peptide. Supernatants were collected 46 hours post-transfection and analyzed by ELISA, detected with an anti-His antibody (Figure 4). Secreted anti-PD-1 scFv bound to PD-1 in a dose-dependent manner.

Cell viability in in vitro growth assay

[00106] 5 x 10 ³ human tumor cells, as indicated, seeded in 96-well plates were mock infected (negative control) or infected with IMMV507 (oHSV-1 expressing antibodies to PD-1 and CTLA-4) at a multiplicity of infection of 0.01, 0.1, 1, 10, and 100 pfu (plaque forming units) per cell, respectively. Cell viability and growth were measured by applying the CCK-8 kit every 24 hours for 96 hours (Figure 5). Optical absorbance was determined at 450 nm using a microplate reader (BiotekEpoch).

[00107] Tumor cell lines used in these studies: T24, human bladder carcinoma; ECA109, human esophageal carcinoma; CNE1, human nasopharyngeal carcinoma; HCT116, human colon carcinoma; Hep2, human laryngeal carcinoma; MD-MB-231, human breast cancer; Hela, human epithelial adenocarcinoma; A549, human lung epithelial adenocarcinoma; H460, human non-small cell lung carcinoma.

[00108] IMMV507 killed tumor cells in a dose-dependent manner. Tumor cells decreased over time after exposure to oHSV-1 viruses.

[00109] It should be understood that although the present invention has been described in detail by means of preferred embodiments and optional characteristics, one skilled in the art will be able to make changes, improvements and variations of the present concepts embodied in the present invention and disclosed herein, and such modifications, improvements and variations are considered to be included within the scope of the present invention. The materials, methods and examples presented herein characterize preferred embodiments, are illustrative and are not intended to limit the scope of the present invention.

[00110] The present invention has been described herein in a broad and general sense. Each of the narrower species and subgeneric groups falling within the general disclosure also forms part of the present disclosure. This includes the general description of the present invention subject to a condition or negative limitation excluding any inventive subject matter from the genus, whether or not the material to be excluded is specifically stated herein. In addition, where features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will understand that the present invention is thereby also described with respect to any individual member or subgroup of members of the Markush group.

[00111] All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entireties to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control. The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "including," etc. are to be construed expansively and without limitation. Additionally, the terms and expressions used herein have been used as terms of description and not limitation, and there is no intention by the use of such terms and expressions to exclude any equivalents of the features shown or described, or portions thereof, but it is to be understood that various modifications are possible within the claimed scope of the present invention.

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Claims (9)

1. A recombinant oncolytic herpes simplex virus type 1 (HSV-1) comprising an HSV-1 vector comprising a deletion modification in the wild-type HSV-1 genome, wherein said vector comprises (i) only one copy of all of the wild-type HSV-1 genome genes present in two copies, (ii) only one copy of the duplicated non-coding sequences of the wild-type HSV-1 genome, and (iii) sequences necessary for the expression of all of the wild-type HSV-1 genome open reading frames (ORFs) present in one copy, the ORFs themselves containing regulatory sequences necessary for the expression of each ORF, wherein said regulatory sequences include promoters; a first heterologous nucleic acid sequence encoding IL-12; and a second nucleic acid sequence encoding an anti-PD-1 agent, wherein said first and second nucleic acid sequences are stably integrated into at least the deleted region of the HSV-1 vector. 2. The recombinant oncolytic HSV-1 according to claim 1, characterized in that the genes, presented in two copies, contain the genes ICP0, ICP4, ICP34.5, ORF P and ORF O. 3. The recombinant oncolytic HSV-1 according to claim 1, characterized in that HSV-1 is selected from the group consisting of strains F, KOS and 17. 4. The recombinant oncolytic HSV-1 according to claim 3, characterized in that the said deletion results in the removal of nucleotide positions 117005 to 132096 in the genome of strain F. 5. The recombinant oncolytic HSV-1 according to claim 1, characterized in that said recombinant oncolytic HSV-1 additionally contains a promoter sequence operably linked to the corresponding heterologous nucleic acid sequence. 6. A pharmaceutical composition for treating cancer, comprising an effective amount of a recombinant oncolytic HSV-1 according to any one of claims 1-5 and a pharmaceutically acceptable carrier. 7. A pharmaceutical composition according to item 6, characterized in that said composition is made in a form for intratumoral administration.