TWI868410B - Oncolytic herpes simplex virus type 1 for brain tumor treatment - Google Patents

Abstract

The present invention discloses a genetically engineered oncolytic HSV-1 virus for brain tumor treatment, which lacks two copies of the γ34.5 gene and the internal inverted repeat region and optionally incorporates an immunostimulatory and/or immunotherapeutic gene. The oncolytic HSV-1 virus exhibits excellent anti-tumor activity, especially in brain tumors. The present invention also discloses a pharmaceutical composition comprising an oncolytic HSV-1 virus and a pharmaceutically acceptable carrier, and a method of using the pharmaceutical composition for brain tumor treatment.

Description

Oncolytic herpes simplex virus type 1 for brain tumor treatment

The present invention relates to an oncolytic virus for tumor treatment, and in particular to a genetically engineered oncolytic herpes simplex virus type I (oHSV-1) for brain tumor treatment. The present invention also relates to a method for treating brain tumors using the recombinant oncolytic virus disclosed herein, and its pharmaceutical composition and application.

Primary tumors of the brain arise from different types of cells in the central nervous system. Medulloblastomas are derived from precursors of neuronal cells, while astrocytomas are derived from the astrocyte subpopulation of neuroglia, and oligodendrogliomas are derived from the oligodendroglial precursor subpopulation of neuroglia. Other types of primary tumors are derived from cells that form the lining and outer lining of the brain, such as ependymomas, which are derived from ependymal cells, and meningiomas, which are derived from cells that comprise the meninges, respectively. Glioblastoma multiforme (GBM), derived from astrocytes, is the most common and lethal primary brain tumor and is therefore classified as a WHO grade IV astrocytoma.

Current treatment for malignant glioblastoma multiforme (GBM) is tumor resection followed by chemotherapy and radiation. Although oncolytic herpes simplex virus (oHSV) has been shown to be safe in clinical trials for GBM, its efficacy has been suboptimal, primarily due to insufficient viral spread after tumor resection. Glioblastoma multiforme (GBM) is the most common brain tumor in adults and remains one of the most difficult malignancies to treat despite tremendous advances in molecular understanding. Although GBM tumor resection is an important therapeutic intervention, standard treatment of tumor resection followed by radiation and temozolomide chemotherapy provides only modest clinical benefit. Therefore, there is an urgent need to develop novel local therapies that can be administered directly into the GBM tumor resection cavity after tumor debulking.

Previous studies have attempted local treatment in the cavity of resected GBM using the clinically approved Gliadel wafer (polyanhydride wafer containing the chemotherapeutic agent BCNU), which showed limited therapeutic effect. In the ongoing search for therapies that can eliminate such tumor remnants after tumor resection, oncolytic viruses have shown great potential in preclinical studies. These viruses are usually genetically engineered to replicate only in and kill tumor cells, an approach that is ideal for actively proliferating tumor cells in the brain among non-proliferating or slowly proliferating normal cells. Among therapeutic viruses, oHSV is one of the most promising candidates for GBM treatment because it is an inherently neurotropic virus and its oncolytic activity is less dependent on specific host cell receptors, mutations, or intracellular pathways. In addition, oHSV has a well-studied genome and significant transgenic capacity for the insertion of additional therapeutic genes to further enhance its oncolytic potency. Although phase I and Ib oHSV clinical trials conducted to date in GBM have shown signs of antitumor activity, clinical response rates have been suboptimal.

The inventors surprisingly found that compared with the existing oHSV-1 virus, oHSV-1 lacking two copies of the γ34.5 gene and the inverted internal repeat region has unexpectedly superior anti-tumor activity against brain tumors compared to non-brain tumors.

On the one hand, the present invention provides an oncolytic herpes simplex virus type 1 (oHSV-1) comprising a modified genome, wherein the modification comprises: (a) an alteration of the copy of the γ34.5 gene in the terminal repeat sequence of the genome, such that the copy of the γ34.5 gene cannot express a functional ICP34.5 protein, and (b) a deletion of the internal inverted repeat region of the genome, such that one copy of each of the double copy (multicopy) genes and one copy of the repeated non-coding sequence within the internal inverted repeat region are deleted, wherein the double copy gene comprises genes encoding ICP0, ICP4, ICP34.5, ORF P and ORF O, and wherein all single copy genes in the _UL and _US components of the genome are intact, such that all single copy genes can express their respective functional proteins.

In some embodiments, the alteration comprises a deletion of all or part of the coding region or regulatory region of a copy of the γ34.5 gene.

In some embodiments, the repeated non-coding sequences include introns, LAT domains, and "a" sequences of ICP0.

In some embodiments, all single copy genes in the _UL and _US components include _UL1 to _UL56 genes in the _UL component and _US1 to _US12 genes in the _US component.

In some embodiments, oHSV-1 is selected from the F strain, the KOS strain, and the 17 strain. In some embodiments, the deletion of the internal inverted repeat region results in the excision of nucleotides 117005 to 132096 in the genome of the F strain.

In some embodiments, oHSV-1 has a prototype (P) genomic isoform and lacks the internal inverted repeat region starting from the stop codon of the last gene in the _UL component (e.g., _UL56 ) to the promoter of the first gene in the _US component (e.g., _US1 ).

In some embodiments, heterologous nucleic acid sequences encoding immunostimulants and/or immunotherapeutics are incorporated into oHSV-1, wherein the incorporation does not interfere with the expression of native genes of the HSV-1 genome. In some embodiments, heterologous nucleic acid sequences encoding immunostimulants and immunotherapeutics are incorporated into oHSV-1.

In some embodiments, the immunostimulant is selected from GM-CSF, IL-2, IL-12, IL-15, IL-24, and IL-27. In some embodiments, the immunostimulant is IL-12.

In some embodiments, the immunotherapeutic agent is an anti-PD-1 agent, an anti-CTLA-4 agent, or both. In some embodiments, the immunotherapeutic agent is an anti-PD-1 agent. In some embodiments, the anti-PD-1 agent includes an anti-PD-1 antibody or an antigen-binding fragment thereof, such as Fab, scFv, (scFv)2, Fab', or F(ab')2. In some embodiments, the anti-CTLA-4 agent includes an anti-CTLA-4 antibody or an antigen-binding fragment thereof, such as Fab, scFv, (scFv)2, Fab', or F(ab')2. In some embodiments, the anti-PD-1 antibody or anti-CDLA-4 antibody includes a modified antibody form, including an antibody-drug conjugate (ADC), a bispecific antibody, and a nanobody (or VHH).

In some embodiments, the heterologous nucleic acid sequence is incorporated into the internal inverted repeat region and/or between the _UL3 and _UL4 genes in the _UL element.

In some embodiments, heterologous nucleic acid sequences encoding IL-12 and anti-PD-1 agents are incorporated into oHSV-1. In some embodiments, the heterologous nucleic acid sequence encoding IL-12 is incorporated into the internal inverted repeat region, and the heterologous nucleic acid sequence encoding the anti-PD-1 agent is incorporated between the _UL3 and _UL4 genes in the _UL component.

In another aspect, a pharmaceutical composition for the treatment of a brain tumor is provided, comprising an effective amount of any oHSV-1 disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the brain tumor is selected from neuroglioma, glioblastoma, oligodendroglioma, astrocytoma, ependymoma, primitive neuroectodermal tumor, atypical meningioma, malignant meningioma, and neuroblastoma. In some embodiments, the brain tumor is glioblastoma multiforme.

In another aspect, a use of any oHSV-1 disclosed herein in the preparation of a medicament for the treatment of a brain tumor is provided. In some embodiments, the brain tumor is selected from neuroglioma, glioblastoma, oligodendroglioma, astrocytoma, ependymoma, primitive neuroectodermal tumor, atypical meningioma, malignant meningioma, and neuroblastoma. In some embodiments, the brain tumor is glioblastoma multiforme.

In another aspect, there is provided a use of any oHSV-1 disclosed herein for the treatment of a brain tumor. In some embodiments, the brain tumor is selected from neuroglioma, glioblastoma, oligodendroglioma, astrocytoma, ependymoma, primitive neuroectodermal tumor, atypical meningioma, malignant meningioma, and neuroblastoma. In some embodiments, the brain tumor is glioblastoma multiforme.

In another aspect, a method for treating a brain tumor in an individual is provided, comprising administering to the individual a therapeutically effective amount of any oHSV-1 disclosed herein or any pharmaceutical composition disclosed herein.

In some embodiments, a second treatment is administered to a subject before, simultaneously with, or after administration of oHSV-1 disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the second treatment is chemotherapy, radiation therapy, immunotherapy, and/or surgical intervention. In some embodiments, the individual is a human. In some embodiments, the brain tumor is selected from neuroglioma, glioblastoma, oligodendroglioma, astrocytoma, ependymoma, primitive neuroectodermal tumor, atypical meningioma, malignant meningioma, and neuroblastoma. In some embodiments, the brain tumor is glioblastoma multiforme.

These and other aspects and advantages of the present invention will be apparent from the following detailed description taken in conjunction with the accompanying drawings, in which:

Figure 1 shows the genomic structures of oHSV-1 constructs T3011, C5252, C8282, C1212, and R3616.

Figure 2 shows the results of the lack of ICP34.5 protein expression in C5252, C8282, and C1212. Six-well plates of Vero cells were mock infected or infected with 1 PFU of HSV-1 (F), R3616, C5252, C8282, and C1212 per cell. Cells were harvested at 6, 12, and 24 hours post infection (H). ICP34.5 protein expression was detected by Western blot.

Figure 3 shows the results of in vitro inhibition evaluation of C5252 on proliferation of human malignant neuroglioma cells. Each sample had 6 replicate wells, and these results were confirmed by another independent experiment. U87-MG, U138-MG, U373-MG, D54-MG and U251-MG were inoculated into 96-well plates (5000 cells/well) and infected with a series of titers of C5252/C1212 (0.01, 0.1, 1, 10, 33.33, 100 PFU/unit). After 48 hours of infection, the inhibition rate of U87-MG, U373-MG, U138-MG, D54-MG and U251-MG was determined by Cell Titer-Glo Luminescent Cell Viability Assay, and IC ₅₀ was calculated.

Figure 4 shows the results of the inhibitory effect of C5252 on normal cells and tumor cells. U373-MG, ACHN, HA and HRGEC were inoculated into 96-well plates (5000 cells/well) and infected with a series of titers of C5252 (0.01-500 PFU/cell). 48 hours after infection, the relative cell activity of U373-MG, ACHN, HA and HRGEC was determined by Cell Titer-Glo Luminescent Cell Viability Assay, and IC ₅₀ was calculated.

Figure 5 shows the results of the efficacy study of C8282 in the treatment of the GL261 subcutaneous implant model in C57BL/6 mice. 32 female C57BL/6J mice were inoculated subcutaneously with GL261 tumor cells (1×10 ⁶ ) in the right flank. When the tumor volume reached ~70 mm ³ , the mice were randomly divided into 4 groups of 8 each. Mice were treated intratumorally with C8282 (green line: 5×10 ⁴ , blue line: 5×10 ⁵ or red line: 5×10 ⁶ PFU/animal, 3 times in total, Q3d). Tumor volume and body weight were measured twice a week. Tumor volume and body weight are expressed as mean ± SEM.

Figure 6 shows the results of the efficacy study of C5252 in the treatment of the orthotropic U87 human neuroglioma model in nude mice. 30 female Balb/c nude mice were inoculated with 5 μL of U87-Luc tumor cells in the left striatum. Two weeks after inoculation, the mice were randomly divided into 3 groups of 8 mice each based on the luminescence signal in the region of interest (ROI) acquired by the IVIS imaging system. Mice were treated with C5252 (blue line: 3×10 ⁴ PFU/mouse, 5 μL or red line: 3×10 ⁵ PFU/mouse, 5 μL) every 3 days for a total of 6 times (D1, 4, 7, 10, 13, 16). IVIS was performed once a week to monitor tumor growth.

It is to be noted that the term "a" or "an" or "an" entity refers to one or more or one or more of that entity; for example, "oncolytic HSV-1" is understood to mean one or more oncolytic HSV-1 viruses. Therefore, the terms "a" or "a", "one or more" or "one or more" and "at least one" or "at least one" can be used interchangeably herein.

"Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing alignable positions in each sequence. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is related to the number of matching or homologous positions shared by the sequences. "Unrelated" or "non-homologous" sequences share less than 40% identity, preferably less than 25% identity with one of the sequences herein.

A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a particular percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same when comparing the two sequences. The alignment and percentage homology or sequence identity can be determined using software programs known in the art.

As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds one or more antigens. The antibody can be a complete antibody and any antigen-binding fragment thereof or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide that is at least a portion of an immunoglobulin molecule that has antigen-binding biological activity. Such examples include, but are not limited to, a heavy chain or light chain complementary determining region (CDR) or a ligand-binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region or any portion thereof, or at least a portion of a binding protein. The term antibody also includes a polypeptide or polypeptide complex that has antigen-binding ability when activated.

As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror image isomers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

The antibodies, antigen-binding polypeptides, or variants or derivatives thereof herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, antigen-determining binding fragments (e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv)), fragments comprising a VK or VH domain, fragments generated by a Fab expression library, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecule herein may be any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). For example, an anti-PD-1 antibody may refer to a Fab fragment or its scFv.

"Specific binding" or "having specificity" generally refers to the binding of an antibody to an epitope via its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen binding domain more readily than to a random, unrelated epitope. The term "specificity" is used herein to quantify the relative affinity with which an antibody binds to an 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 described as binding to epitope "C" with a higher specificity than to a related epitope "D".

As used herein, "cancer" or "tumor" as used interchangeably herein refers to a group of diseases that can be treated according to the present disclosure and involve abnormal cell growth, which may 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: new lumps, abnormal bleeding, prolonged coughing, unexplained weight loss and bowel changes, etc. There are more than 100 different known cancers that affect humans. The present invention is preferably applicable to solid tumors, and more preferably to brain tumors.

As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and preventive measures intended to prevent or slow down (lessen) an undesirable physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, relief of symptoms, reduction in severity of disease, stabilization (i.e., non-worsening) of the disease state, slowing or slowing the progression of disease, improvement or relief of the disease state, and disappearance of symptoms (whether partial or complete), whether detectable or undetectable. "Treatment" also means prolonging survival as compared to that expected if not receiving treatment. Patients in need of treatment include those already suffering from the disease or condition, as well as those susceptible to the disease or condition, or those being prevented from the disease or condition.

"Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include humans, livestock, farm and zoo animals, competition animals or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

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, who would benefit from administration of oHSV-1 or compositions of the invention for, e.g., detection, diagnostic procedures and/or treatment.

It will also be understood by those of ordinary skill in the art that modified genomes as disclosed herein may be modified so that they differ in nucleotide sequence from the modified polynucleotides from which they are derived. For example, a polynucleotide or nucleotide sequence derived from a specified DNA sequence may be similar, such as having a certain percentage identity with the starting sequence, such as it may have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the starting sequence.

In addition, nucleotide or amino acid substitutions, deletions or insertions may be made to make conservative substitutions or changes in "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence derived from a specified protein may be identical to the starting sequence except for one or more individual amino acid substitutions, insertions or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more individual amino acid substitutions, insertions or deletions). In certain embodiments, a polypeptide or amino acid sequence derived from a specified protein has 1 to 5, 1 to 10, 1 to 15 or 1 to 20 individual amino acid substitutions, insertions or deletions relative to the starting sequence.

Oncolytic herpes simplex virus type 1

The HSV-1 genome consists of two covalently linked components, namely L and S. Each component consists of a unique sequence (UL for the _L component and US for the _S component), and the two sides of the unique sequence are reverse repeat sequences, namely terminal repeat sequences and internal repeat sequences. The reverse repeat sequences of the L component are called ab and b'a'. The reverse repeat sequences of the S component are called a'c' and ca. The reverse repeat sequences b'a' and a'c' constitute the internal reverse repeat region. It is known that the reverse repeat regions of the L and S components contain two copies of five genes and a large amount of transcribed but non-protein encoding DNA. These five genes encode proteins ICP0, ICP4, ICP34.5, ORF P and OFR O, respectively, and the DNA includes, for example, the intron of ICP0, the LAT domain and the "a" sequence.

Homologous recombination between the terminal repeat sequences results in the inversion of the L and S components of the HSV-1 genome, producing four linear isomers of equimolar concentration. The isomers are called P (prototype), IL (inversion of the L component), IS (inversion of the S component), and ISL (inversion of both the L and S components). The HSV-1 genome encodes approximately 90 unique transcription units (genes), of which approximately half are essential for viral replication in a permissive tissue culture environment. The remainder are non-essential for cell growth in culture. However, these so-called "non-essential" genes are most likely not required for replication in animal systems. They usually encode functions involved in virus-host interactions, such as inducing immune evasion and host cell shut-off.

Infected cell protein 34.5 (ICP34.5) is a protein encoded by the gamma 34.5 gene (also called gamma ₁ 34.5) that blocks the cellular stress response to viral infection. When a cell is infected with HSV, protein kinase R is activated by the double-stranded RNA of the virus. Protein kinase R then phosphorylates a protein called eukaryotic initiation factor 2A (eIF-2A), inactivating eIF-2A. EIF-2A is required for translation, so by shutting down eIF-2A, the cell prevents the virus from hijacking its own protein-making machinery. The virus in turn forms ICP34.5 to defeat the defense; it activates protein phosphatase 1A to dephosphorylate eIF-2A, allowing translation to occur again. HSV lacking the γ34.5 gene will not be able to replicate in normal cells because it cannot produce protein. The HSV-1 genome has two copies of the γ34.5 gene, located on either side of the _UL element, one in the terminal repeat sequence and the other in the internal repeat sequence.

In one aspect, the present invention provides an oncolytic herpes simplex virus type I (oHSV-1) that has been genetically modified so that both copies of the γ34.5 gene are unable to express functional ICP34.5 protein, and the oHSV-1 is further modified to delete the internal inverted repeat region of the genome. The deletion of the internal inverted repeat region results in the deletion of one copy of each of the double-copy genes including genes encoding ICP0, ICP4, ICP34.5, ORF P and ORF O and one copy of the repeated non-coding sequence in the internal inverted repeat region. However, all single-copy genes in the _UL and _US components of the genome (including _UL 1 to _UL 56 and _US 1 to _US 12) are intact, so they can express their respective functional proteins.

In some embodiments, the modification comprises an alteration of a copy of the γ34.5 gene in the terminal repeat sequence of the genome, so that the copy of the γ34.5 gene cannot express a functional ICP34.5 protein. "Cannot express a functional ICP34.5 protein" means that γ34.5 cannot be detected at the protein or mRNA level in the engineered virus, or the virus expresses the ICP34.5 protein but it is non-functional or partially functional. Measures to achieve the above purpose are readily available in the field of genetic engineering and are known to technicians. For example, the alteration may comprise the insertion, mutation or addition of one or more nucleotides in the coding region or regulatory region of the copy of the γ34.5 gene, or the deletion of all or part of the coding region or regulatory region of the copy of the γ34.5 gene. In some embodiments, the alteration comprises the deletion of all or part of the coding region or regulatory region of the copy of the γ34.5 gene.

The oHSV-1 disclosed herein lacks two copies of the γ34.5 gene. Another copy of the γ34.5 gene is located within the internal repeat sequence of the _UL component, which is deleted by the deletion of the internal inverted repeat region of the genome. As described above, the internal inverted repeat region is composed of the internal repeat sequence of the _UL component and the internal repeat sequence of the _US component. One copy of the double copy gene (including genes encoding ICP0, ICP4, ICP34.5, ORF P and ORF O) and one copy of the repeated non-coding sequence are located within the internal inverted repeat region. Therefore, the deletion of the internal inverted repeat region will result in the deletion of one copy of the double copy gene (including another copy of the γ34.5 gene) and one copy of the repeated non-coding sequence. In some embodiments, the repeated non-coding sequences include, for example, introns, LAT domains, and "a" sequences of ICP0. Thus, in some embodiments, the deletion of the internal inverted repeat region of the genome results in the deletion of one copy of each of ICP0, ICP4, ICP34.5, ORF P, and ORF O, and one copy of each of the introns, LAT domain, and "a" sequence of ICP0. Thus, another copy of each of ICP0, ICP4, ORF P, and ORF O, and another copy of each of the introns, LAT domain, and "a" sequence of ICP0 are retained in the engineered oHSV-1 genome.

In the present invention, the deletion of the internal inverted repeat region is carried out in a precise manner to ensure that all single copy genes in the _UL and _US components of the genome (including _UL1 to _UL56 and _US1 to _US12 ) are intact so that they can express their respective functional proteins. In this case, "all single copy genes in the _UL and _US components of the genome are intact" means that the ORF of each of these single copy genes and the regulatory sequences (such as promoters and enhancers) required for the expression of each ORF are intact to ensure that the expression of the ORF is successful and the protein translated from the ORF is functional. "Intact" means that the coding sequence of each of the single copy genes is at least functional, but does not mean that the sequence must be 100% identical to the naturally occurring sequence. By including, for example, conservative substitutions or changes in "non-essential" regions, the sequence may have slight variations in nucleotide sequence compared to the naturally occurring sequence. In this case, the sequence may have 90%, 95%, 98% or 99% identity with the naturally occurring sequence.

Given that the location of each of the single copy genes in the HSV-1 genome is known in the art and depends on the strain and genomic isoform of the HSV-1 virus, one skilled in the art will appreciate that the exact start and end positions of the deleted nucleotides in the internal inverted repeat region will vary from strain to strain and isoform to isoform, but can be readily determined by techniques known in the art. It should be understood that the present invention is not intended to be limited to any particular genomic isoform or strain of the HSV-1 virus. Rather, the present invention contemplates that all strains and isoforms of the HSV-1 virus are useful.

For example, in an embodiment using the HSV-1 F strain, whose genome is obtained by GenBank Accession No. GU734771.1, the deletion of the internal inverted repeat region results in the excision of nucleotides 117005 to 132096 in the genome. Those skilled in the art will also understand that other strains can also be used in the present invention as long as their genomic DNA is sequenced. Sequencing technology is easily available in the literature and on the market. For example, in another embodiment, the deletion can be made on the HSV-1 strain 17, whose genome can be obtained by GenBank Accession No. NC_001806.2. In another embodiment, the deletion can be made on the KOS1.1 strain, whose genome can be obtained by GenBank Accession No. KT899744.

In some embodiments, deletion is performed precisely at a predetermined position, thereby achieving excision of a DNA fragment from the last gene in the L component (e.g., _UL56 in the case of the P isomer) to the first gene in the S component (e.g., _US1 in the case of the P isomer). In view of the fact that there are four different isomers of HSV-1 (i.e., isomers P, IS, IL, and ISL), the names of the first gene and the last gene are different depending on the isomer. In the context of the present invention, the numbering of the genes in the _UL component (i.e., the first and the last) is defined as the direction from the terminal repeat sequence of the _UL component to the internal repeat sequence of the _UL component, and the numbering of the genes in the _US component is defined as the direction from the internal repeat sequence of the US component to the terminal repeat sequence of the _US component. Thus, in the case of the isomeric prototype (P), the first gene in the _UL component would be, for example, the _UL1 gene, and the last gene in the _UL component would be, for example, _UL56 , and the first gene in the _US component would be, for example, the _US1 gene, and the last gene in the _US component would be, for example, _US12 . In the case of the isomeric IS, the first gene in the _UL component would be, for example, the _UL1 gene, and the last gene in the _UL component would be, for example, _UL56 , and the first gene in the _US component would be, for example, the _US12 gene, and the last gene in the _US component would be, for example, _US1 . In the case of isoform IL, the first gene in the _UL component will be, for example, the _UL56 gene, and the last gene in the _UL component will be, for example, the _UL1 , and the first gene in the _US component will be, for example, the _US1 gene, and the last gene in the _US component will be, for example, the _{US12 gene} . In the case of isoform ISL, the first gene in the _UL component will be, for example, the _UL56 gene, and the last gene in the _UL component will be, for example, the _UL1 , and the first gene in the _US component will be, for example, the _US12 gene, and the last gene in the _US component will be, for example, the _US1 .

The deletion of the internal inverted repeat region does not cause damage to the single copy gene in the _US or _UL component, so that the coding sequence and regulatory sequence of the single copy gene (including the promoter sequence necessary for the expression of the single copy gene) are intact. For example, in the case of isoform P, the deletion results in the excision of a DNA fragment starting from the end of the stop codon of the _UL56 gene, such as to the beginning of the promoter sequence of the _US1 gene, such as. For example, in the case of isoform IL, the deletion results in the excision of a DNA fragment starting from the beginning of the promoter sequence of the _UL1 gene, such as to the beginning of the promoter sequence of the _US1 gene, such as.

The retention of all single copy genes in the engineered oHSV-1 genome as well as another copy of each of ICP0, ICP4, ORF P and ORF O and another copy of each of the introns, LAT domain and "a" sequence of ICP0 provides a more robust virus, both before and after the incorporation of the inserted foreign gene. Thus, oHSV-1 is resistant to environmental factors such as temperature, pressure, UV light, etc. to the greatest extent. It also maximizes the range of cancer cells in which oncolytic HSV-1 is effective.

Various gene manipulation methods known in the art can be used to obtain the modified HSV-1 vector as described in the present invention. For example, bacterial artificial chromosome (BAC) technology is used. As another embodiment, COS plasmid can be used in the present invention. WO 2017/181420 discloses an oHSV-1 vector constructed by BAC technology, the entire contents of which are incorporated herein by reference.

The amount of exogenous DNA sequences that can be inserted into wild-type viruses is limited because it interferes with DNA packaging into viral particles. Precise deletions in designated regions provide ideal space for the insertion of exogenous DNA sequences. According to one embodiment of the present invention, the deletion removes at least 15k bp of the oncolytic viral vector, allowing a similar amount of exogenous DNA sequences to be accommodated. Other studies have shown that the wild-type genome can tolerate an additional 7KB of DNA.

In some embodiments, a heterologous nucleic acid sequence encoding an immunostimulant and/or an immunotherapeutic agent is incorporated into the genetically engineered oHSV-1. In the present invention, the incorporation of the heterologous nucleic acid sequence does not interfere with the expression of the natural genes of the HSV-1 genome (such as any of the above-mentioned single copy genes or other double copy genes).

In some embodiments, the heterologous nucleic acid sequence is incorporated into an internal inverted repeat region. In some embodiments, the heterologous nucleic acid sequence is incorporated between adjacent single copy genes in the _UL or _US component. In some embodiments, the heterologous nucleic acid sequence is incorporated into an internal inverted repeat region and between adjacent single copy genes in the _UL or _US component. In some embodiments, the heterologous nucleic acid sequence is incorporated into an internal inverted repeat region and between _UL3 and _UL4 genes.

In some embodiments, oHSV-1 comprises a heterologous nucleic acid sequence encoding an immunostimulant. In some embodiments, the immunostimulant is selected from GM-CSF, IL-2, IL-12, IL-15, IL-24, and IL-27. In one embodiment, the immunostimulant is IL-12. In one embodiment, the immunostimulant is human IL-12 or humanized IL-12.

In some embodiments, oHSV-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, an anti-CTLA-4 agent, or both. In one embodiment, the immunotherapeutic agent is an anti-PD-1 agent.

In some embodiments, oHSV-1 comprises heterologous nucleic acid sequences encoding an immunostimulatory agent and an immunotherapeutic agent. In some embodiments, the immunostimulatory agent is selected from GM-CSF, IL-2, 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 some embodiments, the immunotherapeutic agent is selected from anti-PD-1 agent, anti-CTLA-4 agent, or both. In one embodiment, the immunotherapeutic agent is anti-PD-1 agent.

In an embodiment, when only one heterologous nucleic acid sequence encoding an immunostimulatory agent or encoding an immunotherapeutic agent is inserted, the heterologous nucleic acid sequence is preferably incorporated into the deleted internal inverted repeat region of the genome. In one embodiment, the heterologous nucleic acid sequence has a length similar to the deleted fragment. In one embodiment, the heterologous nucleic acid sequence has a length that is 20% longer or shorter than the length of the deleted fragment. In another embodiment, the heterologous nucleic acid sequence has a length that is 15%, 10%, 5%, 4%, 3%, 2% or 1% longer or shorter than the deleted fragment.

In one embodiment, the heterologous nucleic acid sequence has a length of less than about 18k bp, about 17k bp, or about 16k bp. In one embodiment, the heterologous nucleic acid sequence has a length of greater than about 10k bp, 11k bp, 12k bp, 13k bp, or 14k bp. In one embodiment, the heterologous nucleic acid sequence has a length between about 14k bp and about 16k bp. In one embodiment, the heterologous nucleic acid sequence has a length of about 15k bp.

In some embodiments, oHSV-1 comprises at least two heterologous nucleic acid sequences encoding immunostimulants and/or immunotherapeutics. In some embodiments, oHSV-1 comprises heterologous nucleic acid sequences encoding two different immunostimulants. For example, in one embodiment, oHSV-1 comprises heterologous nucleic acid sequences encoding IL-12 and GM-CSF. In another embodiment, oHSV-1 comprises heterologous nucleic acid sequences encoding IL-15 and GM-CSF. In another embodiment, oHSV-1 comprises heterologous nucleic acid sequences encoding IL-12 and IL-15.

In some embodiments, oHSV-1 comprises heterologous nucleic acid sequences encoding two different immunotherapeutic agents. In one embodiment, for example, oHSV-1 comprises heterologous nucleic acid sequences encoding both an anti-PD-1 agent and an anti-CTLA-4 agent.

In embodiments, when more than one heterologous nucleic acid sequence encoding an immunostimulant and/or immunotherapeutic agent is incorporated, the first heterologous nucleic acid sequence is preferably inserted into the internal repeat region of the genome deletion. The second or additional 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 _UL3 and _UL4 genes of the L component. In one embodiment, the second heterologous nucleic acid sequence is inserted between the _UL37 and _UL38 genes of the L component.

In one embodiment, the first heterologous nucleic acid sequence encodes IL-12 inserted into the deleted internal repeat region of the genome. In one embodiment, the second heterologous nucleic acid sequence encodes an anti-PD-1 agent inserted between the _UL3 and _UL4 genes of the L component.

It is understood that the insertion of one or more heterologous nucleic acid sequences into the oncolytic HSV-1 genome does not interfere with the expression of the native HSV-1 gene, and the heterologous nucleic acid sequence is stably incorporated into the modified HSV-1 genome, so that the functional expression of the heterologous nucleic acid sequence can be expected.

The heterologous nucleic acid sequence encoding the immunostimulatory agent and/or immunotherapeutic agent contains a nucleic acid encoding a peptide or protein and a regulatory element for expression. Typically, regulatory elements (including transcription promoters, ribosome binding sites, and terminators) present in the recombinant gene and selected based on the host cell to be used for expression are operably linked to the nucleic acid sequence to be expressed. Within the recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription/translation system, or in a host cell when a virus is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression regulatory elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of the nucleotide sequence in many types of host cells, as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).

One of ordinary skill in the art can select appropriate regulatory elements based on, for example, desired tissue specificity and expression levels. For example, cell type-specific or tumor-specific promoters can be used to restrict expression of a gene product to a specific cell type. In addition to the use of tissue-specific promoters, local administration of viruses can achieve local expression and effects. Examples of non-tissue-specific promoters that can be used include the early cytomegalovirus (CMV) promoter (U.S. Patent No. 4,168,062) and the Rous Sarcoma Virus promoter. Furthermore, HSV promoters, such as the HSV-1 IE promoter, can be used.

For example, examples of tissue-specific promoters that can be used in the present technology include the prostate-specific antigen (PSA) promoter, which is specific for prostate cells; the desmin promoter, which is specific for muscle cells; the enolase promoter, which is specific for neurons; the beta globin promoter, which is specific for erythroid cells; the tau-globin promoter, which is specific for prostate cells; , which is also specific for erythroid cells; the growth hormone promoter, which is specific for pituitary cells; the insulin promoter, which is specific for pancreatic beta cells; the neurofibromic acid protein promoter, which is specific for astrocytes; the tyrosine hydroxylase promoter, which is specific for catecholaminergic neurons; and the amyloid precursor protein promoter, which is specific for neurons. The promoter of dopamine β-hydroxylase is specific for norepinephrine and adrenergic neurons; the promoter of tryptophan hydroxylase is specific for 5-hydroxytryptamine/pineal cells; the promoter of choline acetyltransferase is specific for cholinergic neurons; the promoter of aromatic L-amino acid decarboxylase (AADC) is specific for catecholaminergic/5-HT/D-type cells; the proenkephalin promoter, which is specific for neurons/spermatogenic epididymal cells; the reg (pancreatic stone protein) promoter, which is specific for colon and rectal tumors as well as pancreatic and kidney cells; and the parathyroid hormone-related peptide (PTHrP) promoter, which is specific for liver and cecal tumors as well as neurilemmoma, kidney cells, pancreatic cells, and adrenal cells.

Examples of promoters that specifically function 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 cancers expressing SLPI; the tyrosinase promoter, which is specific for melanoma cells; the stress-induced grp78/BiP promoter, which is specific for fibroblasts; The AP2 lipoprotein enhancer is specific for adipocytes; the alpha-1 antitrypsin transthyretin promoter is specific for hepatocytes; the interleukin-10 promoter is specific for glioblastoma multiforme; the c-erbB-2 promoter is specific for pancreatic, breast, gastric, ovarian, and non-small cell lung cells; and the alpha-B-crystallin/heat shock protein 27 promoter is specific for brain tumor cells. The promoters are: the basic fibroblast growth factor promoter, which is specific for neuroglioma and meningioma cells; the epidermal growth factor receptor promoter, which is specific for squamous cell carcinoma, neuroglioma, and breast tumor cells; the mucin-like glycoprotein (DF3, MUC1) promoter, which is specific for breast cancer cells; the mts1 promoter, which is specific for metastatic tumors; the NSE promoter, which is specific for small cell lung cancer cells; the somatostatin receptor promoter, which is specific for The promoters are specific for small cell lung cancer cells; c-erbB-3 and c-erbB-2 promoters, which are specific for breast cancer cells; c-erbB4 promoter, which is specific for breast cancer and gastric cancer; thyroglobulin promoter, which is specific for thyroid cancer cells; alpha-fetoprotein (AFP) promoter, which is specific for liver cancer cells; villin promoter, which is specific for gastric cancer cells; and albumin promoter, which is specific for liver cancer cells. In another embodiment, the TERT promoter or survivin promoter is used.

For example, in some embodiments, the heterologous nucleic acid sequence is operably linked to a promoter, such as a CMV promoter or an Egr-1 promoter. In one embodiment, a nucleotide sequence encoding IL-12 is operably linked to an Egr-1 promoter. In another embodiment, a nucleotide sequence encoding scFv-anti-hPD1 is operably linked to a CMV promoter.

In certain embodiments, the oHSV-1 of the present invention encodes one or more immunostimulatory agents (also referred to as immunostimulatory molecules), including cytokines (such as IL-2, IL4, IL-12, GM-CSF, IFNγ), chemokines (such as MIP-1, MCP-1, IL-8), and growth factors.

Alternatively or in addition, the oHSV-1 of the present invention encodes one or more immunotherapeutic agents, such as PD-1 binding agents (or anti-PD-1 agents) or CTLA-4 binding agents (or anti-CTLA-4 agents), including antibodies or fragments thereof, such as anti-PD1 antibodies that specifically bind to PD-1 or anti-CTLA-4 antibodies that specifically bind 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 receptors, such as anti-PD-L1 and/or PD-L2 antibodies, PD-L1 and/or PD-L2 baits, or soluble PD-1 receptors.

The PD-1 signaling pathway plays an important role in tumor-associated immune dysfunction. Infection and lysis of tumor cells can trigger highly specific anti-tumor immune responses that kill cells of the inoculated tumor as well as cells of distant established non-inoculated tumors. Tumors and their microenvironment have developed mechanisms to evade, inhibit, and inactivate natural anti-tumor immune responses. For example, tumors may downregulate target receptors, encase themselves in fibrous extracellular matrix, or upregulate host receptors or ligands involved in the activation or recruitment of regulatory immune cells. Innate and/or adaptive regulatory T cells (Tregs) participate in tumor-mediated immunosuppression. Without wishing to be limited by theory, PD-1 blockade can suppress Treg activity and improve the efficacy of tumor-reactive CTLs. Other aspects of this technology are described in further detail below. PD-1 blockade can also stimulate anti-tumor immune responses by blocking the inactivation of T cells (CTLs and helper cells) and B cells.

In one aspect, the present technology provides an oncolytic virus carrying a gene encoding a PD-1 binder. Programmed cell death 1 (PD-1) is a 50-55 kDa type I transmembrane receptor originally identified by subtractive hybridization of mouse T cell lines undergoing apoptosis. PD-1, a member of the CD28 gene family, is expressed on activated T cells, B cells, and myeloid lineage cells. Human and mouse PD-1 have approximately 60% amino acid identity, with four potential N-glycosylation sites and conservation of residues defining the Ig-V domain. Two ligands for PD-1, PD ligand 1 (PD-L1) and ligand 2 (PD-L2), have been identified, both belonging to the B7 superfamily. PD-L1 is expressed on many cell types, including T cells, B cells, endothelial cells and epithelial cells, as well as antigen-presenting cells. In contrast, PD-L2 is only expressed on professional antigen-presenting cells, such as dendritic cells and macrophages.

PD-1 negatively regulates T cell activation, and this inhibitory function is associated with the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. Disruption of this inhibitory function of PD-1 can lead to autoimmunity. The opposite situation can also be harmful. In many pathological conditions, persistent negative signaling of PD-1 is involved in T cell dysfunction, such as tumor immune escape and chronic viral infection.

Host antitumor immunity is primarily influenced by tumor-infiltrating lymphocytes (TILs). Multiple lines of evidence indicate that TILs are inhibitoryly regulated by PD-1. First, expression of PD-L1 has been demonstrated in many human and mouse tumor lines and can be further upregulated in vitro by IFN-γ. Second, expression of PD-L1 in tumor cells is directly associated with their resistance to lytic activity by antitumor T cells in vitro. Third, PD-1 knockout mice are resistant to tumor attack, and T cells from PD-1 knockout mice are highly effective in tumor rejection when adopted into tumor-bearing mice. Fourth, blocking PD-1 inhibitory signals by monoclonal antibodies can enhance host antitumor immunity in mice. Fifth, high expression of PD-L1 in tumors (detected by immunohistochemical staining) is associated with poor prognosis in many human cancer types.

Oncolytic virotherapy is an effective method to shape the host immune system by expanding the T or B cell population specific for tumor-specific antigens (released after oncolysis). The immunogenicity of tumor-specific antigens depends largely on the affinity of the host immune receptors (B cell receptors or T cell receptors) for the antigenic determinants and the host tolerance threshold. High-affinity interactions will drive host immune cells through multiple rounds of proliferation and differentiation to become long-lasting memory cells. Host tolerance mechanisms will counteract this proliferation and expansion to minimize potential tissue damage caused by local immune activation. PD-1 inhibitory signaling is part of this host tolerance mechanism, which can be supported by the following evidence. First, PD-1 expression is elevated in actively proliferating T cells, especially in T cells with a terminally differentiated phenotype (effector phenotype). Effector cells are generally associated with potent cytotoxic effects and cytokine production. Second, PD-L1 is important for maintaining peripheral tolerance and locally limiting overactive T cells. Therefore, PD-1 inhibition using PD-1 binders expressed in the tumor microenvironment can be an effective strategy to increase the activity of TILs and stimulate effective and durable anti-tumor immune responses.

In one aspect, the present technology provides an oncolytic virus comprising a heterologous nucleic acid encoding an anti-PD-1 agent. In some embodiments, the anti-PD-1 agent contains an antibody variable region that provides specific binding to the PD-1 antigenic determinant. The antibody variable region can be present in, for example, a complete antibody, an antibody fragment, and a recombinant derivative of an antibody or an antibody fragment. The term "antibody" refers to an immunoglobulin that can be natural, partially synthetic, or fully synthetic. Therefore, the anti-PD-1 agent of the present technology includes any polypeptide or protein having a binding domain that specifically binds to the PD-1 antigenic determinant.

Different types of antibodies have different structures. IgG can be used to illustrate different antibody regions. IgG molecules contain four polypeptide chains, two long heavy chains and two shorter light chains connected to each other by disulfide bonds. The heavy chain and light chain each contain a constant region and a variable region. The heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH1, CH2, and CH3). The light chain consists of a light chain variable region (VL) and a light chain constant region (CL). There are three hypervariable regions in the variable region that are responsible for antigen specificity.

The hypervariable regions are often referred to as complementarity determining regions (CDRs) and are located between more conserved flanking regions called framework regions (FWs). There are four (4) FW regions and three (3) CDRs from the NH2-terminus to the COOH-terminus: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. For example, the framework regions and CDRs can be identified considering the Kabat and Chothia definitions. The variable regions of the heavy and light chains contain the binding domains that interact with the antigen. The two heavy chain carboxyl regions are constant regions that are linked by disulfide bonds to produce the Fc region. The Fc region is important for providing effector functions. Each of the two heavy chains that make up the Fc region extends to a different Fab region via a hinge region.

Anti-PD-1 agents or anti-CTLA-4 agents generally contain antibody variable regions. Such antibody fragments include, but are not limited to, (i) Fab fragments, monovalent fragments consisting of VH, VL, CH and CL domains; (ii) Fab2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments, consisting of VH and CH1 domains; (iv) Fv fragments, consisting of the VH domain and VL domain of a single antibody arm; (v) dAb fragments, which contain VH domains or VL domains; (vi) scAb, antibody fragments containing VH and VL and C1 or CH1, and (vii) artificial antibodies based on protein scaffolds, including but not limited to fibronectin type III polypeptide antibodies. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, allowing them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule, called a single-chain Fv (scFv). Therefore, the antibody variable region can be present in a recombinant derivative. Examples of recombinant derivatives include single-chain antibodies, biantibodies, triabodies, tetrabodies, and miniantibodies. Anti-PD-1 agents or anti-CTLA-4 agents may also contain one or more variable regions that recognize the same or different antigenic determinants.

In some embodiments, the anti-PD-1 agent or anti-CTLA-4 agent is encoded by an oncolytic virus produced using recombinant nucleic acid technology. Different anti-PD-1 agents can be produced by different technologies, including, for example, single-chain proteins (such as scFv) containing VH and VL regions connected by a linker sequence, and antibodies or fragments thereof; and multi-chain proteins containing VH and VL regions on separate polypeptides. Recombinant nucleic acid technology involves the construction of nucleic acid templates for protein synthesis. Suitable recombinant nucleic acid technology is well known in the art. Recombinant nucleic acids encoding anti-PD-1 antibodies or anti-CTLA-4 antibodies can be expressed in cells infected with oncolytic viruses and released into the tumor microenvironment after viral lysis. Cells actually act as factories for encoding proteins.

Nucleic acids containing one or more recombinant genes encoding either or both of the VH or VL regions of anti-PD-1 or anti-CTLA-4 agents can be used to produce complete proteins/polypeptides that bind to PD-1/CTLA-4. For example, a single gene is used to encode a single-chain protein (such as scFv) containing a VH region and a VL region connected by a linker, or, for example, multiple recombinant regions are used to produce VH and VL regions, thereby providing a complete binding agent.

Exemplary anti-PD-1 antibodies or anti-CTLA-4 antibodies or fragments or derivatives thereof that can be used in the present invention are available in the art. See, for example, WO 2006/121168, WO 2014/055648, WO 2008/156712, US2014/0234296 or U.S. Patent No. 6,984,720.

Drug Combinations

On the other hand, the present invention provides a pharmaceutical composition for tumor treatment, comprising an effective amount of the genetically engineered oHSV-1 as described herein and a pharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition for tumor treatment comprises an effective amount of a genetically engineered oHSV-1 and a pharmaceutically acceptable carrier, wherein the genetically engineered oHSV-1 comprises a modified genome, wherein the modification comprises: (a) a change in the copy of the γ34.5 gene in the terminal repeat sequence of the genome, so that the copy of the γ34.5 gene cannot express a functional ICP34.5 protein, and (b) a deletion of the internal inverted repeat region of the genome, so that one copy of each of the double copy genes and one copy of the repeated non-coding sequence within the internal inverted repeat region are deleted, wherein the double copy gene comprises genes encoding ICP0, ICP4, ICP34.5, ORF P and ORF O, and wherein all single copy genes in the _UL and _US components of the genome are intact, so that all single copy genes can express their respective functional proteins.

In some embodiments, the alteration comprises the deletion of all or part of the coding region or regulatory region of the copy of the γ34.5 gene. In some embodiments, the repeated non-coding sequence comprises introns, LAT domains and "a" sequences of ICPO. In some embodiments, all single copy genes in the _UL and _US components comprise _UL1 to _UL56 genes in the _UL component and _US1 to _US12 genes in the _US component.

In some embodiments, oHSV-1 is selected from the F strain, the KOS strain, and the 17 strain. In some embodiments, the deletion of the internal inverted repeat region results in the excision of nucleotides 117005 to 132096 in the genome of the F strain.

In some embodiments, oHSV-1 has a prototype (P) genomic isoform and lacks the internal inverted repeat region starting from the stop codon of the last gene in the _UL component (e.g., _UL56 ) to the promoter of the first gene in the _US component (e.g., _US1 ).

In some embodiments, heterologous nucleic acid sequences encoding immunostimulants and/or immunotherapeutics are incorporated into oHSV-1, wherein the incorporation does not interfere with the expression of native genes of the HSV-1 genome. In some embodiments, heterologous nucleic acid sequences encoding immunostimulants and immunotherapeutics are incorporated into oHSV-1.

In some embodiments, the immunostimulatory agent is selected from GM-CSF, IL-2, IL-12, IL-15, IL-24, and IL-27. In some embodiments, the immunostimulatory agent is IL-12. In some embodiments, the immunotherapeutic agent is an anti-PD-1 agent, an anti-CTLA-4 agent, or both. In some embodiments, the immunotherapeutic agent is an anti-PD-1 agent.

In some embodiments, the heterologous nucleic acid sequence is incorporated into an internal inverted repeat region and/or between _UL3 and _UL4 genes in the _UL component. In some embodiments, heterologous nucleic acid sequences encoding IL-12 and an anti-PD-1 agent are incorporated into oHSV-1. In some embodiments, the heterologous nucleic acid sequence encoding IL-12 is incorporated into an internal inverted repeat region, and the heterologous nucleic acid sequence encoding an anti-PD-1 agent is incorporated between _UL3 and _UL4 genes in the _UL component.

Oncolytic viruses can be prepared in suitable pharmaceutically acceptable carriers or formulations. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be easily injected. The form must be stable under the conditions of manufacture and storage and must be protected against contamination by microorganisms (e.g., bacteria and fungi).

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and/or vegetable oils. For example, by using a coating such as lecithin, by maintaining the required fineness in the case of dispersion and by using a surfactant to maintain appropriate fluidity. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents such as sugars or sodium chloride. The absorption of injectable compositions can be prolonged by using agents that delay absorption in the composition (such as aluminum monostearate and gelatin).

For parenteral administration in aqueous solution, for example, the solution should be appropriately buffered (if necessary) and the liquid diluent first made isotonic with sufficient 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 will be known to those skilled in the art in light of the present invention. For example, one dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous perfusion fluid or injected at the proposed infusion site. Depending on the condition of the subject being treated, some variation in dosage will necessarily occur. In any case, the person responsible for administration will determine the appropriate dosage for the individual subject. In addition, for human administration, the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by FDA standards for biologics.

Sterile injectable solutions are prepared by incorporating the active compound in the required amount with the various other ingredients listed above as required in a suitable solvent and then filtering and sterilizing. Dispersions are usually prepared by incorporating the various sterilized active ingredients into a sterile carrier containing a basic dispersion medium and the required other ingredients from the above list. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient and any additional required ingredients from a previously sterile filtered solution.

The compositions disclosed herein can be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), including salts formed with inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as sodium, potassium, ammonium, calcium or iron hydroxide) and organic bases (such as isopropylamine, trimethylamine, histidine, procaine, etc.). When formulated, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The preparation is easily administered in various dosage forms (such as injectable solutions, drug release capsules, etc.).

As used herein, "carrier" includes any and all solvents, dispersion media, carriers, 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 agents for pharmaceutical actives is well known in the art. In addition to any conventional media or reagents that are incompatible with the active ingredient, it is contemplated that other media or reagents may be used in the therapeutic compositions. Supplementary active ingredients may also be incorporated into the compositions.

The term "pharmaceutically acceptable" refers to molecular entities and ingredients that do not produce allergic or similar adverse reactions when administered to humans. The preparation of aqueous compositions containing proteins as active ingredients is well known in the art. Typically, such compositions are prepared as injections, either as liquid solutions or suspensions; they can also be prepared in solid form suitable for dissolution or suspension in liquid prior to injection.

In some embodiments, the compositions disclosed herein are used for tumor treatment. In some embodiments, the compositions disclosed herein are used for solid tumor treatment. In some embodiments, the compositions disclosed herein are used for brain tumor treatment. In some embodiments, the compositions disclosed herein are used for brain tumor treatment, wherein the brain tumor is selected from neuroglioma, glioblastoma, oligodendroglioma, astrocytoma, ependymoma, primitive neuroectodermal tumor, atypical meningioma, malignant meningioma and neuroblastoma. In some embodiments, the brain tumor is glioblastoma multiforme.

Applications and treatments

In another aspect, the present invention provides a genetically engineered oHSV-1 as described herein for use in the treatment of a tumor in a subject. In another aspect, the present invention provides a genetically engineered oHSV-1 as described herein for use in the treatment of a solid tumor in a subject. In another aspect, the present invention provides a genetically engineered oHSV-1 as described herein for use in the treatment of a brain tumor in a subject.

In some embodiments, the genetically engineered oHSV-1 comprises a modified genome, wherein the modification comprises: (a) an alteration of the copy of the γ34.5 gene in the terminal repeat sequence of the genome, such that the copy of the γ34.5 gene cannot express a functional ICP34.5 protein, and (b) a deletion of the internal inverted repeat region of the genome, such that one copy of each of the double copy genes and one copy of the repeated non-coding sequence within the internal inverted repeat region are deleted, wherein the double copy gene comprises genes encoding ICP0, ICP4, ICP34.5, ORF P and ORF O, and wherein all single copy genes in the _UL and _US components of the genome are intact, such that all single copy genes can express their respective functional proteins.

In some embodiments, the alteration comprises the deletion of all or part of the coding region or regulatory region of the copy of the γ34.5 gene. In some embodiments, the repeated non-coding sequence comprises introns, LAT domains and "a" sequences of ICPO. In some embodiments, all single copy genes in the _UL and _US components comprise _UL1 to _UL56 genes in the _UL component and _US1 to _US12 genes in the _US component.

In some embodiments, oHSV-1 is selected from the F strain, the KOS strain, and the 17 strain. In some embodiments, the deletion of the internal inverted repeat region results in the excision of nucleotides 117005 to 132096 in the genome of the F strain.

In some embodiments, oHSV-1 has a prototype (P) genomic isoform and lacks the internal inverted repeat region starting from the stop codon of the last gene in the _UL component (e.g., _UL56 ) to the promoter of the first gene in the _US component (e.g., _US1 ).

In some embodiments, heterologous nucleic acid sequences encoding immunostimulants and/or immunotherapeutics are incorporated into oHSV-1, wherein the incorporation does not interfere with the expression of native genes of the HSV-1 genome. In some embodiments, heterologous nucleic acid sequences encoding immunostimulants and immunotherapeutics are incorporated into oHSV-1.

In some embodiments, the immunostimulatory agent is selected from GM-CSF, IL-2, IL-12, IL-15, IL-24, and IL-27. In some embodiments, the immunostimulatory agent is IL-12. In some embodiments, the immunotherapeutic agent is an anti-PD-1 agent, an anti-CTLA-4 agent, or both. In some embodiments, the immunotherapeutic agent is an anti-PD-1 agent.

In some embodiments, the heterologous nucleic acid sequence is incorporated into an internal inverted repeat region and/or between _UL3 and _UL4 genes in the _UL component. In some embodiments, heterologous nucleic acid sequences encoding IL-12 and an anti-PD-1 agent are incorporated into oHSV-1. In some embodiments, the heterologous nucleic acid sequence encoding IL-12 is incorporated into an internal inverted repeat region, and the heterologous nucleic acid sequence encoding an anti-PD-1 agent is incorporated between _UL3 and _UL4 genes in the _UL component.

In another aspect, the present invention provides the use of a genetically engineered oHSV-1 as described herein in the preparation of a drug for the treatment of a tumor in a subject. In another aspect, the present invention provides the use of a genetically engineered oHSV-1 as described herein in the preparation of a drug for the treatment of a solid tumor in a subject. In another aspect, the present invention provides the use of a genetically engineered oHSV-1 as described herein in the preparation of a drug for the treatment of a brain tumor in a subject.

In some embodiments, the genetically engineered oHSV-1 comprises a modified genome, wherein the modification comprises: (a) an alteration of the copy of the γ34.5 gene in the terminal repeat sequence of the genome, such that the copy of the γ34.5 gene cannot express a functional ICP34.5 protein, and (b) a deletion of the internal inverted repeat region of the genome, such that one copy of each of the double copy genes and one copy of the repeated non-coding sequence within the internal inverted repeat region are deleted, wherein the double copy gene comprises genes encoding ICP0, ICP4, ICP34.5, ORF P and ORF O, and wherein all single copy genes in the _UL and _US components of the genome are intact, such that all single copy genes can express their respective functional proteins.

In some embodiments, the alteration comprises the deletion of all or part of the coding region or regulatory region of the copy of the γ34.5 gene. In some embodiments, the repeated non-coding sequence comprises introns, LAT domains and "a" sequences of ICPO. In some embodiments, all single copy genes in the _UL and _US components comprise _UL1 to _UL56 genes in the _UL component and _US1 to _US12 genes in the _US component.

In some embodiments, oHSV-1 is selected from the group consisting of F strain, KOS strain, and 17 strain. In some embodiments, the deletion of the internal inverted repeat region results in the excision of nucleotides 117005 to 132096 in the genome of the F strain.

In some embodiments, oHSV-1 has a prototype (P) genomic isoform and lacks the internal inverted repeat region starting from the stop codon of the last gene in the _UL component (e.g., _UL56 ) to the promoter of the first gene in the _US component (e.g., _US1 ).

In some embodiments, heterologous nucleic acid sequences encoding immunostimulants and/or immunotherapeutics are incorporated into oHSV-1, wherein the incorporation does not interfere with the expression of native genes of the HSV-1 genome. In some embodiments, heterologous nucleic acid sequences encoding immunostimulants and immunotherapeutics are incorporated into oHSV-1.

In some embodiments, the immunostimulatory agent is selected from GM-CSF, IL-2, IL-12, IL-15, IL-24, and IL-27. In some embodiments, the immunostimulatory agent is IL-12. In some embodiments, the immunotherapeutic agent is an anti-PD-1 agent, an anti-CTLA-4 agent, or both. In some embodiments, the immunotherapeutic agent is an anti-PD-1 agent.

In some embodiments, the heterologous nucleic acid sequence is incorporated into an internal inverted repeat region and/or between _UL3 and _UL4 genes in the _UL component. In some embodiments, heterologous nucleic acid sequences encoding IL-12 and an anti-PD-1 agent are incorporated into oHSV-1. In some embodiments, the heterologous nucleic acid sequence encoding IL-12 is incorporated into an internal inverted repeat region, and the heterologous nucleic acid sequence encoding an anti-PD-1 agent is incorporated between _UL3 and _UL4 genes in the _UL component.

In another aspect, the present invention provides a method for treating or alleviating a tumor in a subject, comprising administering to the subject in need thereof an effective amount of an oHSV-1 virus or a pharmaceutical composition comprising the oHSV-1 virus as described above. In certain embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a brain tumor. In certain embodiments, the brain tumor is selected from neuroglioma, glioblastoma, oligodendroglioma, astrocytoma, ependymoma, primitive neuroectodermal tumor, atypical meningioma, malignant meningioma, and neuroblastoma. In certain embodiments, the brain tumor is glioblastoma multiforme.

In some embodiments, the oHSV-1 virus or drug composition is administered intratumorally. In one embodiment, the HSV-1 virus or drug composition is injected directly into the tumor mass in the form of an injectable solution.

The method of the invention can be used to treat brain tumors. This includes all tumors in the human skull (head) or in the central spinal canal. The tumors can originate from the brain itself, or from lymphatic tissue, blood vessels, cranial nerves, brain capsule (meninges), skull, pituitary gland or pineal body. Within the brain itself, the cells involved can be neurons or neuroglia (including astrocytes, oligodendrocytes and ependymal cells). Brain tumors can also be spread from cancers that are primarily located in other organs (metastatic tumors).

In some embodiments, the brain tumor is a neuroglioma, such as an ependymoma, an astrocytoma, an oligoastrocytoma, an oligodendroglioma, a ganglioneuroma, a glioblastoma (also known as glioblastoma multiforme), or a mixed neuroglioma. Neurogliomas are primary brain tumors that are divided into four grades (I, II, III, and IV) based on their appearance under a microscope, particularly the presence of atypical cells, mitosis, endothelial proliferation, and necrosis. Grade I and II tumors, called "low-grade neurogliomas," have none or one of these features, and include diffuse astrocytoma, pilocytic astrocytoma, low-grade astrocytoma, low-grade oligoastrocytoma, low-grade oligodendroglioma, ganglioglioma, dysembryoplastic neuroepithelial tumor, pleomorphic xanthoastrocytoma, and mixed neuroglioma. Grade III and IV tumors, referred to as "high-grade neurogliomas," have two or more of these features, and include anaplastic astrocytomas, anaplastic oligodendrogliomas, anaplastic oligoastrocytomas, anaplastic ependymomas, and glioblastomas (including giant cell glioblastomas and gliosarcomas). In one aspect of these embodiments, the neuroglioma is a low-grade neuroglioma. In another aspect of these embodiments, the neuroglioma is a high-grade neuroglioma. In another aspect of these embodiments, the neuroglioma is a glioblastoma.

In some embodiments, oHSV-1 can be combined with other agents that are effective in treating cancer. For example, treatment of cancer can be implemented with oncolytic viruses and other anticancer therapies (e.g., anticancer agents or surgery). In the present technology, it is expected that oncolytic virus therapy can be used in combination with chemotherapy, radiotherapy, immunotherapy, or other biological interventions.

An "anti-cancer" agent is capable of negatively affecting the cancer in a subject, for example, by killing cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the life expectancy of a cancer patient. Anti-cancer agents include biologics (biotherapy), chemotherapeutic agents, and radiotherapeutic agents. More generally, these other compositions will be provided in a combined amount effective to kill or inhibit cell proliferation. The process may involve contacting the cells with the expression construct and the reagent or factors simultaneously. This can be accomplished by contacting the cells with a single composition or pharmacological formulation that includes both agents, or by contacting the cells simultaneously with two different compositions or formulations, one of which includes the expression construct and the other of which contains the second agent.

In some embodiments, oHSV-1 disclosed herein is combined with an adjuvant. In one embodiment, the adjuvant is an oligonucleotide comprising an unmethylated CpG motif. Unmethylated dinucleotide CpG motifs in bacterial deoxyribonucleic acid (DNA) have the advantage of stimulating several immune cells to secrete cytokines to enhance innate and adaptive immunity.

Viral therapy can be administered within a time interval of several minutes to several weeks before or after other drug treatment. In embodiments where the other agent and the oncolytic virus are administered to the cells separately, it will generally be ensured that there is not a considerable period of time between each delivery time so that the agent and the virus can still exert a beneficial combined effect on the cells. In such a case, it is expected that the cells can be exposed to the two therapies within about 12-24 hours of each other. However, in some cases, when the respective administrations are separated by a period of several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks), it may be necessary to significantly extend the duration of treatment.

In some embodiments, a second treatment is administered to a subject prior to, concurrently with, or after administration of oHSV-1 disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the second treatment is chemotherapy, radiation therapy, immunotherapy, and/or surgical intervention. In some embodiments, the subject is a human.

The sequences used in the present invention are summarized as follows.

Embodiment

As demonstrated in the following examples, a genetically engineered oHSV-1 virus that lacks two copies of the γ34.5 gene and further lacks an internal inverted repeat region exhibits higher anti-tumor activity against various brain tumor cells compared to non-brain tumor cells or normal cells. oHSV-1 viruses with similar genomic structures known in the art (e.g., T3011, R3616, WT strain F) are less efficient in killing brain tumor cells than the oHSV-1 disclosed herein (i.e., C1212, C5252, C8282), and these results are surprising.

oConstruction of HSV-1C5252, C8282, and C1212

Construction of oHSV-1 C5252

The genome structures of oHSV-1T3011, C5252, C8282 and C12122 are shown in Figure 1.

In detail, C5252 contains a deletion of the γ34.5 gene, an insertion of an anti-human PD-1 antibody expression cassette between _UL 3 and _UL 4, and a modified internal repeat (IR) region replaced by an IL-12 expression cassette. Recombinant viruses were constructed in several steps using the instructions of the bacterial artificial chromosome (BAC) system. Details of the viral constructs are described below.

Using an HSV-1 BAC with two copies of the γ34.5 gene deletion arrangement (BAC-Δ34.5), the following IL-12 expression gene cassette was PCR-amplified from the HSV-1 viral genome with nucleotides 117005 on the upstream side and 132096 on the downstream side in the wild-type genome background by two sets of primers (GAAGATCTAATATTTTTATTGCAACTCCCTG (SEQ ID NO: 5), CTAGCTAGCTTATAAAAGGCGCGTCCCGTGG (SEQ ID NO: 6)) and (GCTCTAGATTGCGACGCCCCGGCTC (SEQ ID NO: 7), CCTTAATTAAGGTTACCACCCTGTAGCCCCGATGT (SEQ ID NO: 8)), respectively, and inserted into the gene replacement plasmid pKO5 to generate pKO1407. pKO1407 was then electroporated into E. coli carrying BAC-Δ34.5 to produce BAC-Δ34.5-IL12. Then, in the background of wild-type genome, the gene cassette of the CMV promoter driving the PD-1 Fab gene was amplified from the HSV-1 viral genome by two sets of primers (TCCCATGGATTTAACAAACGGGGGGGTGTCG (SEQ ID NO: 9), GGCCCCCGAGGCCAGCATGACGTTATCT (SEQ ID NO: 10)) and (GAGTAACCGCCCCCCCCCCATGCCACCCTCAC (SEQ ID NO: 11), GTGTTTTACTGCCACTACACCCCCGGGGAAC (SEQ ID NO: 12)) respectively: upstream flanked by nucleotide 11658, downstream linked to nucleotide 11659, and ligated to pKO5 at the BglII and PacI sites to generate the pKOE1002 plasmid. The pKOE1002 plasmid was then electroporated into E. coli carrying BAC-Δ34.5-IL12 to produce BAC-5252. The C5252 virus was obtained by transfection of the BAC-5252 plasmid, followed by several steps of plaque purification and expansion in Vero cells, and then by detecting the γ34.5 gene encoding protein ICP34.5 for secretion of IL-12 and PD-1 Fab (Table 1) and expression of the protein ICP34.5 (Figure 2).

C8282 is a functionally identical mouse version of C5252, except that C8282 carries the mouse version of IL-12 and a mouse anti-PD-1 antibody (single chain antibody fragment, scFv, containing a heavy chain variable region and a light chain variable region having sequences as shown in SEQ ID NO: 13 and 14, respectively) at the same location on the viral genome, while C5252 carries human IL-12 and an anti-human PD-1 antibody.

C1212 is a functionally identical version of C5252, except that C1212 carries the CMV promoter followed by three repeated stop codons and green fluorescent protein (GFP) at the same position on the viral genome, while C5252 carries human IL-12 and anti-human PD-1 antibody (PD-1 Fab, containing heavy chain variable and constant regions and light chain variable and constant regions having sequences shown in SEQ ID NOs: 1-4, respectively).

Confirmation of IL-12 and anti-PD-1 antibody expression and ICP34.5 protein expression in C5252, C8282, and C1212 viruses

Vero cells were seeded into 6-well plates at a density of 4×10 ⁵ cells per well. After overnight incubation, cells were mock infected or infected with 1 PFU of HSV-1 (F), R3616, C5252, C8282, C1212 per cell. Cells were harvested at 6, 12, and 24 hours (H) after infection. Proteins were separated by electrophoresis in 10% denaturing gel and reacted with antibodies ICP34.5 or GAPDH. GAPDH was used as a loading control (Figure 2). Cell supernatants collected 24 hours (H) after infection with C5252, C8282, and C1212 were used for ELISA assays to detect the expression levels of IL-12 and anti-PD-1 antibodies. The results are shown in Table 1.

Table 1. Detection of IL-12 and anti-PD-1 antibody (PD-1 Ab) expression in C5252, C8282, and C1212

As shown in Table 1, C5252 and C8282 viruses expressed comparable levels of IL-12 and anti-PD-1 antibodies. C1212 is a backbone virus, and no IL-12 or anti-PD-1 antibody expression was detected by ELISA.

The expression of ICP34.5 protein detected by immunoblotting is shown in Figure 2, indicating that ICP34.5 protein is absent in C5252, C8282, C1212, and R3616 infected samples, but is expressed in wild type (WT) F infected samples.

The above results all indicate that the recombinant viruses C5252, C8282, and C1212 were confirmed by the expression of IL-12, anti-PD-1 antibodies, and the absence of ICP34.5 protein expression.

In vitro cytotoxic activity - brain tumor cell lines

A172, D54-MG, U87-MG, U138-MG, and D458 cells were seeded onto 96-well plates (4000 cells/well) and infected with F, R3616, T3011, and C5252 (0.1 and 1.0 PFU/cell). Cell viability was determined by CCK8-Kit 48 hours after infection (48Hp.i.). Inhibition rate = (OD of uninfected wells - OD of wells infected with oHSV) / (OD of uninfected wells - OD of blank wells) × 100%. Blank wells contained only culture medium. All values in the experiment are expressed as mean ± SEM. The results are shown in Table 2.

Table 2. In vitro cytotoxic activity of HSV-1 WTF, R3616, T3011 and C5252 against brain tumor cell lines

As shown in Table 2, oHSV-1 C5252 was an effective cytocidal agent in all tumor brain cells tested at 1.0 PFU/cell. In cell lines A172, D54-MG, and U138-MG, C5252 showed the highest cytocidal ability among the oHSV-1 viruses tested. For cell lines U87-MG and D458, the antitumor effect of C5252 was comparable to that of T3011. Compared with R3616, an oHSV-1 also null for the γ34.5 gene, C5252 was almost 2 to 3 times more effective in most cell lines tested.

In vitro cytotoxic activity - non-brain tumor cell lines

Cells were inoculated into 96-well plates (4000 cells/well) and infected with F, T3011, and C5252 (0.1 and 1.0 PFU/cell). Cell activity was determined by CCK8-Kit 48 hours after infection (48H p.i.). Inhibition rate = (OD of uninfected wells-OD of wells infected with oHSV)/(OD of uninfected wells-OD of blank wells) × 100%. Blank wells contained only culture medium. All values in the experiment are expressed as mean ± SEM. The results are shown in Table 3.

Table 3. In vitro cytotoxic activity of HSV-1 WTF, R3616, T3011 and C5252 against non-brain tumor cell lines

As shown in Table 3, when tested in non-brain tumor cell lines, T3011 and C5252 were both effective tumor killers against various non-brain tumor cells. Note that in all eight cell lines tested in this example, the anti-tumor activity of C5252 was essentially equivalent to that of T3011 at both low and high multiplicity of infection (MOI). This was unexpected because C5252 is a further attenuated version of T3011, lacking the second copy of the γ34.5 gene. However, the deletion of the second copy of the γ34.5 gene showed no adverse effect on the anti-tumor activity of the oHSV-1 virus against non-brain tumor cells, but significantly improved its tumoricidal effect against brain tumor cells (as shown in Table 2). Therefore, the oHSV-1 virus disclosed in this article is generally more effective in tumoricidal than the oHSV-1 from which it is derived.

In vitro evaluation of the inhibitory effect of C5252 on the proliferation of human malignant neuroglioma cells

As shown in Figure 3, the sensitivity of C5252 to human neuroglioma cell lines U87-MG, U138-MG, U373-MG, D54-MG, and U251-MG was basically the same, and the _IC50 values of C5252 and C1212 against these neuroglioma cells were less than 10 MOI. The inhibitory effect of C5252 was comparable to that of the backbone C1212. Incorporation of heterologous genes into the viral genome did not significantly affect the replication and inhibitory capacity of oHSV, but when administered in vivo, due to the immunostimulatory (IL-12) and immunotherapeutic (anti-PD-1 antibody) properties expressed by the oHSV-1 virus, it will significantly indicate that the individual's immune system kills tumor cells.

Inhibitory effects of C5252 on normal cells and tumor cells

As shown in Figure 4, the IC ₅₀ values of C5252 against tumor cells U373-MG and ACHN were 6.890 and 9.102 MOI, respectively, and the IC ₅₀ values of C5252 against normal cells HA and HRGEC were both greater than 500 MOI. Under the conditions of this experiment, C5252 had no significant inhibitory effect on normal cells, but had a significant inhibitory effect on tumor cells. Compared with normal cells, the inhibitory effect of C5252 has a higher targeting effect on human tumor cells. The results show that C5252 selectively kills tumor cells while leaving normal cells unharmed.

Efficacy of C8282 in the treatment of GL261 subcutaneous implantation model in C57BL/6 mice

5×10⁶ PFU/動物的C8282治療時，動物耐受良好。5×10⁵ PFU/動物的中等劑量水準在測試的劑量範圍中表現出最高的功效。 C8282 is a mouse replacement for C5252, in which mouse IL-12 (m-IL-12) and anti-mouse PD-1 (m-PD-1) antibodies are introduced into the viral genome to replace the corresponding human counterparts. As shown in Figure 5, intratumoral injection of C8282 showed significant efficacy in the GL261 subcutaneous tumor model.

C8282 treatment was well tolerated by animals at 5×10 ⁶ PFU/animal. The mid-dose level of 5×10 ⁵ PFU/animal showed the highest efficacy within the dose range tested.

Efficacy study of C5252 in the treatment of the orthotopic U87 human neuroglioma model in nude mice

As shown in Figure 6, intracerebral injection of C5252 showed significant efficacy against U87-MG cells in nude mice. There was no significant difference among different dose levels.

It should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, modifications, improvements and variations of the contents presented herein are easily recognized by those skilled in the art, and these modifications, improvements and variations are considered to be within the scope of the present invention. The materials, methods and embodiments provided herein are representative of preferred embodiments and are exemplary, and are not intended to limit the scope of the present invention.

<110> Shenzhen IMMVIRA Pharmaceutical Technology Co., Ltd. (IMMVIRA CO., LIMITED)

<120> Oncolytic herpes simplex virus type 1 for brain tumor treatment

<130>

<140> TW110145204

<141> 2021-12-03

<150> PCT/CN2020/133943

<151> 2020-12-04

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<170> PatentIn version 3.5

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Claims (21)

A use of an oncolytic herpes simplex virus type 1 (oHSV-1) for preparing a drug for treating brain tumors, wherein the oncolytic herpes simplex virus type 1 comprises a modified genome, wherein the modification comprises: a) a change in one copy of the γ34.5 gene in the terminal repeat sequence of the genome, so that the copy of the γ34.5 gene cannot express functional ICP34.5 protein; and b) a deletion of the internal inverted repeat region of the genome, so that one copy of each of the double copy genes and one copy of the repeated non-coding sequence in the internal inverted repeat region are deleted, wherein the double copy gene comprises genes encoding ICP0, ICP4, ICP34.5, ORF P and ORF O, wherein all single copy genes in the _UL and _US components of the genome are intact, so that all single copy genes can express their respective functional proteins. The use as described in claim 1, wherein the change comprises the deletion of all or part of the coding region or regulatory region of the copy of the γ34.5 gene. The use as described in claim 1, wherein the repeated non-coding sequence includes the intron, LAT domain and "a" sequence of ICP0. The use as described in claim 1, wherein all single copy genes in the _UL and _US components include _UL1 to _UL56 genes in the _UL component and _US1 to _US12 genes in the _US component. The use as described in any one of claims 1 to 4, wherein HSV-1 is selected from F strain, KOS strain and 17 strain. The use as described in any one of claims 1 to 4, wherein HSV-1 has a genomic isoform of the prototype (P). The use as described in any one of claims 1 to 4, wherein the deletion of the internal inverted repeat region results in the excision of nucleotides 117005 to 132096 in the genome of the F strain. The use as described in claim 6, wherein the deletion of the internal inverted repeat region starts from the stop codon of the last gene in the _UL component to the promoter of the first gene in the _US component. The use as described in claim 8, wherein the last gene in the _UL component is the _UL56 gene. The use as described in claim 8 or 9, wherein the first gene in the _US component is the _US 1 gene. The use as described in claim 1, further comprising incorporating a heterologous nucleic acid sequence encoding an immunostimulant and/or an immunotherapeutic into the oHSV-1, and the incorporation does not interfere with the expression of the natural genes of the HSV-1 genome. The use as described in claim 11, further comprising incorporating the heterologous nucleic acid sequence encoding the immunostimulant and the heterologous nucleic acid sequence encoding the immunotherapeutic into the oHSV-1. The use as described in claim 11, wherein the immunostimulant is selected from GM-CSF, IL-2, IL-12, IL-15, IL-24 and IL-27. The use as described in claim 13, wherein the immunostimulant is IL-12. The use as described in any one of claims 11 to 14, wherein the immunotherapy agent is an anti-PD-1 agent, an anti-CTLA-4 agent, or both. The use as described in claim 15, wherein the immunotherapy agent is an anti-PD-1 agent. The use according to any one of claims 11 to 14, wherein the heterologous nucleic acid sequence is incorporated into the internal inverted repeat region and/or between _UL3 and _UL4 genes in the _UL element. The use as described in any one of claims 11 to 14, wherein the immunostimulant is IL-12 and the immunotherapeutic agent is an anti-PD-1 agent. The use as described in claim 18, wherein the heterologous nucleic acid sequence encoding IL-12 is incorporated into the internal inverted repeat region, and the heterologous nucleic acid sequence encoding the anti-PD-1 agent is incorporated between the _UL3 and _UL4 genes in the _UL component. The use as described in claim 1, wherein the brain tumor is selected from neuroglioma, glioblastoma, oligodendroglioma, astrocytoma, ependymoma, primitive neuroectodermal tumor, atypical meningioma, malignant meningioma and neuroblastoma. The use as described in claim 1, wherein the brain tumor is glioblastoma multiforme.