Use of oncolytic viruses to treat cancer

Abstract

The present invention relates to the use of oncolytic viruses (e.g., modified HSV-1 viruses) to treat various types of cancer. Additionally, the present invention relates to compositions and kits related to such uses of oncolytic viruses.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority and benefit to U.S. Provisional Patent Application No. 62 / 813,961, filed on March 5, 2019, which is hereby incorporated by reference in its entirety.

[0002] Reference to Sequence Listing This application contains a Sequence Listing in computer - readable form. The Sequence Listing is provided as a text file named A - 2353 - WO - PCT_SeqListing_ST25.txt, created on January 10, 2020, and having a size of 37,667 bytes. The information in the electronic format of the Sequence Listing is hereby incorporated by reference in its entirety.

Background Art

[0003] Recent advances in the treatment of many forms of cancer have significantly improved the survival rates for both men and women with the most common types of cancer, such as lung cancer, colon cancer, breast cancer, and prostate cancer. The emergence of checkpoint inhibitors, which have successfully directed a patient's immune system to attack a given form of cancer, has significantly improved patient survival rates for a given cancer. For example, checkpoint inhibitors such as ipilimumab (anti - CTLA - 4 antibody), pembrolizumab, and nivolumab (anti - PD - 1 antibodies), and atezolizumab (anti - PD - L1 antibody) have demonstrated efficacy in various types of tumors. See (Non - Patent Document 1); (Non - Patent Document 2); and (Non - Patent Document 3).

[0004] Oncolytic viruses have also demonstrated clinical efficacy in the treatment of certain forms of cancer. Oncolytic viruses are typically genetically engineered to preferentially replicate within cancer cells (hyperactive cells) and to contain a "payload" that can be used to enhance the anti-tumor response. Such genetic engineering initially focused on the use of replication-deficient viruses with the aim of preventing virus-induced damage to non-tumor cells. More recently, the genetic engineering of oncolytic viruses has focused on the generation of "replication-restricted" viruses that can spread to other tumor cells while avoiding systemic infection.

[0005] Currently, the only approved oncolytic virus-based agent in the United States and Europe is talimogene laherparepvec (IMLYGIC®). Talimogene laherparepvec is HSV-1 derived from the clinical strain JS1 (deposited with the European collection of cell cultures (ECAAC) under accession number 01010209). In talimogene laherparepvec, the HSV-1 viral genes encoding ICP34.5 and ICP47 are functionally deleted. The functional deletion of ICP47 results in the early expression of US11, a gene that promotes virus growth within tumor cells without reducing tumor selectivity. In addition, the coding sequence for human GM-CSF has been inserted into the former ICP34.5 gene site in the viral genome. See (Non-Patent Document 4).

[0006] The therapeutic combination of oncolytic viruses and checkpoint inhibitors is being investigated. For example, the combination of talimogene laherparepvec and immunotherapy (e.g., ipilimumab and pembrolizumab) is currently being investigated in clinical trials for melanoma (NCT01740297 and NCT02263508) and head and neck squamous cell carcinoma (NCT02626000).

Prior Art Documents

Non-Patent Literature

[0007]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] Although oncolytic viruses have shown great promise in cancer treatment, there is still a need to develop oncolytic viruses that can not only limit their replication and lytic damage to cancer cells, but also help enhance and maintain a more robust systemic anti-tumor immune response.

[0009] The present invention addresses these and other needs.

Means for Solving the Problems

[0010] The present invention relates to an oncolytic virus comprising a nucleic acid encoding a heterologous dendritic cell growth factor and a nucleic acid encoding a first heterologous cytokine. The heterologous dendritic cell growth factor and the first heterologous cytokine may be linked by a polycistronic linker element. In some embodiments, the polycistronic linker element is porcine teschovirus 2a (P2A) or an internal ribosome entry site (IRES). This oncolytic virus can be a herpes simplex virus, such as herpes simplex virus type 1. In certain embodiments, the oncolytic virus is derived from the HSV-1 strain JS1.

[0011] The oncolytic virus can also be modified so that it does not contain a functional ICP34.5 gene and does not contain a functional ICP47 gene.

[0012] Furthermore, the oncolytic virus may further comprise a promoter under the control of the same promoter to which both the dendritic cell growth factor and the nucleic acid sequence encoding the first cytokine are under the control of the same promoter. In other embodiments, the oncolytic virus may comprise a first promoter in which the nucleic acid sequence encoding the dendritic cell growth factor is under the control of the first promoter; and a second promoter in which the nucleic acid sequence encoding the first cytokine is under the control of the second promoter.

[0013] The first heterologous cytokine can be an interleukin, such as interleukin-12 (IL12). The heterologous dendritic cell growth factor can be a second cytokine, such as Fms-related tyrosine kinase 3 ligand (FLT3L).

[0014] In certain embodiments, the oncolytic virus of the present invention comprises HSV-1 lacking a functional ICP34.5 coding gene and lacking a functional ICP47 coding gene, comprises a nucleic acid encoding FLT3L, and further comprises a nucleic acid encoding IL12. In some embodiments, the nucleic acid encoding IL12 and the nucleic acid encoding FLT3L are Original present at the site of the ICP34.5 coding gene. In one embodiment, the nucleic acid encoding IL12 and the nucleic acid encoding FLT3L are linked by P2A.

[0015] The nucleic acids encoding IL12, FLT3L and P2A can be present as [Flt3L]-[P2A]-[IL12], where the [Flt3L]-[P2A]-[IL12] construct is under the control of a single promoter, and this construct is the OriginalIt is present in the part. Suitable promoters include cytomegalovirus (CMV), Rous sarcoma virus (RSV), human elongation factor 1α promoter (EF1a), simian virus 40 early promoter (SV40), phosphoglycerate kinase 1 promoter (PGK), ubiquitin C promoter (UBC), and murine stem cell virus (MSCV). In certain embodiments, the promoter is CMV.

[0016] The oncolytic virus of the present invention may contain a bovine growth hormone polyadenylation signal sequence (BGHpA). The oncolytic virus of the present invention may further contain a nucleic acid that enhances mammalian translation. In some embodiments, the nucleic acid that enhances mammalian translation is a Kozak sequence or a consensus Kozak sequence. In certain embodiments, the Kozak sequence is set forth in SEQ ID NO: 20.

[0017] In one embodiment, the oncolytic virus contains one nucleic acid or a plurality of nucleic acids (also referred to as a construct or expression cassette) encoding [CMV]-[Kozak]-[Flt3L]-[P2A]-[IL12]-[BGHpA]. In another embodiment, IL12 exists as [P40 subunit]-[GGGGS]-[P35 subunit]. In another embodiment, the signal peptide within the IL12 P35 subunit is absent. In another embodiment, the oncolytic virus contains one nucleic acid or a plurality of nucleic acids encoding [CMV]-[Kozak]-[Flt3L]-[P2A]-[IL12(p40-GGGGS-SP None -p3 5) -[BGHpA]. In yet another embodiment, the construct is at the Original site of the ICP34.5 coding gene. HSV-1 / ICP34.5 - / ICP47 - The geometric arrangement of the construct within the ICP34.5 coding gene used to generate / FLT3L / IL12 is shown in FIG. 9, but the Original site of the ICP34.5 coding gene OriginalMultiple geometric arrangements of the expression cassette within the site could be generated / utilized.

[0018] In some embodiments, the oncolytic virus comprises an FLT3L sequence comprising SEQ ID NO: 1 and an IL12 sequence comprising SEQ ID NO: 7.

[0019] In some embodiments, the oncolytic virus comprises a CMV promoter comprising SEQ ID NO: 24, a Kozak sequence comprising SEQ ID NO: 20, an FLT3L sequence comprising SEQ ID NO: 1, a P2A sequence (GSG-P2A) comprising SEQ ID NO: 17, an IL12 sequence comprising SEQ ID NO: 7, and a BGHpA sequence comprising SEQ ID NO: 21.

[0020] The present invention further includes a method of treating cancer using the oncolytic virus of the present invention. Further, the present invention includes a therapeutically effective amount of an oncolytic virus for use in the treatment of cancer.

[0021] The present invention further includes a pharmaceutical composition for use in treating cancer. The pharmaceutical composition may further include a checkpoint inhibitor.

[0022] In some embodiments, the present invention includes a kit comprising the oncolytic virus of the present invention.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0024] The headings of the sections used in this specification are for organizational purposes only and are not to be construed as limiting the subject matter described. All references cited in the text of this specification are hereby incorporated by reference in their entirety.

[0025] In this specification, unless otherwise defined, scientific and technical terms used in connection with this application have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context requires a different interpretation, singular terms shall include the plural, and plural terms shall include the singular.

[0026] In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of the proteins and nucleic acids described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and procedures of the present application are generally carried out according to conventional methods well known in the art and as described in various general and specific references cited and discussed throughout this specification. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992) and Harlow and Lane, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990) (incorporated herein by reference). Enzyme reactions and purification procedures are performed according to the manufacturer's instructions, as commonly accomplished in the art, or as described herein. The terminology used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as their laboratory procedures and techniques, are well known and commonly employed in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0027] The present invention is not limited to the specific methodologies, protocols, reagents, etc. described herein, and thus should be understood to be capable of variation. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the disclosure, which scope is defined only by the claims.

[0028] Unless otherwise indicated in an Example or otherwise described, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about." The term "about," when used in connection with percentages, can mean ±1%.

[0029] All embodiments within a narrower range than that defined by a particular paragraph herein should be considered to be included in this disclosure. For example, a given aspect is described by way of genus, and it should be understood that all components of the genus can be, individually, embodiments. Further, aspects of selecting an aspect or component of a genus described as such should be understood to include combinations of two or more components of the genus. Further, while the language "comprising" is presented in various circumstances of the various embodiments herein, related embodiments can also be described using the language "consisting of" or "consisting essentially of."

[0030] Definitions The term "functionally deleted," when referring to a gene, means that the gene has been modified (e.g., by partially or completely deleting, substituting, rearranging, or otherwise altering the gene) such that a functional protein can no longer be expressed from that gene. In the context of herpes simplex virus (such as an oncolytic virus), a gene is "functionally deleted" when the viral gene has been modified within the herpes simplex virus genome such that a functional viral protein can no longer be expressed from that gene by herpes simplex virus.

[0031] The term "heterologous" when referring to a nucleic acid present within a viral genome (or a protein encoded by such nucleic acid) refers to a nucleic acid that is not naturally present in that virus (or a protein that is not naturally produced by that virus). For example, a nucleic acid encoding human IL12 or a nucleic acid encoding human FLT3L would be "heterologous" with respect to HSV-1.

[0032] The term "oncolytic virus" refers to a virus that preferentially infects and kills cancer cells over non-cancer cells, either naturally or as a result of modification.

[0033] As used herein, the terms "patient" or "subject" are used interchangeably and mean a mammal including, but not limited to, a human or non-human mammal such as a bovine, equine, canine, ovine or feline. Preferably, the patient is a human.

[0034] The term "HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12" refers to a modified HSV-1 derived from strain JS1, where HSV-1 lacks a functional ICP34.5 coding gene, lacks a functional ICP47 coding gene, and has inserted at the site of the ICP34.5 gene the following: [CMV]-[Kozak]-[Flt3L]-[P2A]-[IL12(p40-GGGGS-SP Original -p3 None -p3 5) -[BGHpA].

[0035] Oncolytic virus Any virus can be used to generate the oncolytic virus of the present invention. Generally, the virus can be modified, for example, to regulate its replication (e.g., to preferentially replicate within tumor cells over healthy cells) and to regulate its ability to contain exogenous nucleic acids, such as those detected by the host immune system.

[0036] In some embodiments, the oncolytic virus is herpes simplex virus (HSV). In other embodiments, the oncolytic virus is herpes simplex virus type 1 (HSV-1). In still other embodiments, the oncolytic virus is derived from JS1 (HSV-1). JS1 has been deposited with the European Collection of Authenticated Cell Cultures (ECACC) under accession number 01010209.

[0037] In some embodiments, the oncolytic virus is HSV-1 in which the viral gene encoding ICP34.5 is functionally deleted. The functional deletion of ICP34.5, which acts as a pathogenicity factor during HSV infection, restricts replication in non-dividing cells and renders the virus non-pathogenic. The safety of HSV with a functionally deleted ICP34.5 has been demonstrated in multiple clinical trials (MacKie et al, Lancet 357:525-526, 2001; Markert et al, Gene Ther 7:867-874, 2000; Rampling et al, Gene Ther 7:859-866, 2000; Sundaresan et al, J.Virol 74:3822-3841, 2000; Hunter et al, J Virol Aug;73(8):6319-6326, 1999).

[0038] In other embodiments, the oncolytic virus is HSV-1 in which the viral gene encoding ICP47 (which blocks viral antigen presentation to major histocompatibility complex class I and II molecules) is functionally deleted. The functional deletion of ICP47 also results in early expression of US11, a gene that promotes viral growth in tumor cells without reducing tumor selectivity.

[0039] In some embodiments, the viral gene encoding ICP34.5 is deleted. In some embodiments, the viral gene encoding ICP47 is deleted. In some embodiments, both the viral gene encoding ICP34.5 and the viral gene encoding ICP47 are deleted. In some embodiments, both the viral gene encoding ICP34.5 and the viral gene encoding ICP47 are deleted, and the deletion of ICP47 results in early expression of US11.

[0040] Herpes virus strains and methods for making such virus strains are described in U.S. Patent Nos. 5,824,318; 6,764,675; 6,770,274; 7,063,835; 7,223,593; 7,749,745; 7,744,899; 8,273,568; 8,420,071; 8,470,577; WIPO Publication Nos. WO 96 / 00007; WO 96 / 39841; WO 99 / 07394; WO 00 / 54795; WO 06 / 002394; WO 13 / 06795; Chinese Patent Nos. 128,303; 10,230,334; and 10,230,335; Varghese and Rabkin, (2002) Cancer Gene Therapy 9:967-97; and Cassady and Ness Parker, (2010) The Open Virology Journal 4:103-108, each of which is incorporated herein by reference.

[0041] The oncolytic viruses of the present invention are further modified such that they contain proteins encoding exogenous nucleic acids. Such proteins are rationally selected to enhance the immunostimulatory ability of the virus. Increasing the immunostimulatory ability allows the oncolytic virus to elicit a more robust anti-tumor response. Thus, in one aspect, the oncolytic virus comprises a nucleic acid encoding a heterologous dendritic cell growth factor, a first heterologous cytokine, or both. FLT3L enhances the proliferation and survival of dendritic cells, particularly the cDC1 subset, which is crucial for cross-presentation of tumor antigens to T cells. Furthermore, IL12 enhances T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) functions, resulting in maximal tumor killing activity. Without being bound by theory, it is believed that the combination of these two sets of attributes will produce an oncolytic virus that can surprisingly induce a systemic immune response against cancer cells.

[0042] In certain embodiments, the oncolytic virus comprises a nucleic acid encoding a heterologous dendritic cell growth factor and a nucleic acid encoding a first heterologous cytokine (sometimes referred to as the "payload"). Examples of the first heterologous cytokine include interleukin-2 (IL2), IL7, IL12, IL15, IL21, TNF, and other members of the interleukin family of cytokines and proteins that can bind to receptors on immune cells and / or enhance T cell function or memory phenotype. In certain embodiments, the first heterologous cytokine is IL12 (mouse or human). The nucleic acid sequences encoding muIL12a and muIL12b are set forth in SEQ ID NOs: 11 and 13, respectively. The nucleic acid sequences encoding huIL12a and huIL12b are set forth in SEQ ID NOs: 3 and 5, respectively. The amino acid sequences of muIL12a and muIL12b are set forth in SEQ ID NOs: 12 and 14, respectively. The amino acid sequences of huIL12a and huIlL2b are set forth in SEQ ID NOs: 4 and 6, respectively.

[0043] In its native form, IL12 is a heterodimeric cytokine comprising IL12A (p35 subunit) and IL12B (p40 subunit), with each subunit encoded by a separate gene. Thus, in some embodiments, the oncolytic virus of the invention comprises two heterologous nucleic acids, one encoding the IL12 p35 subunit and the other encoding the IL12 p40 subunit. In other embodiments, the oncolytic virus of the invention comprises a single-stranded IL12 variant. In such single-stranded IL12 variants, the p35 and p40 subunits can be fused directly to each other (i.e., without a linker) or can be joined to each other via a (synthetic or peptide-based) linker. Examples of suitable linkers include elastin-based linkers (VPGVGVPGVGGS; nucleic acid sequence shown in SEQ ID NO: 22; amino acid sequence shown in SEQ ID NO: 23), G 4 S, 2×(G 4 S), 3×(G 4 S), 4×(G 4 S), 5×(G 4 S), 6×(G 4 S), 7×(G 4 S), 8×(G 4 S), 9×(G 4 S) and 10×(G 4 S). In some embodiments, the linker is VPGVGVPGVGGS, G 4 S, 2×(G 4 S) or 3×(G 4 S). In certain embodiments, the linker is G 4 S.

[0044] The IL12 variant may or may not contain the signal peptides (one for each subunit) present in the native IL12 protein. In some embodiments, the IL12 variant contains no signal peptides, one signal peptide, or both signal peptides. In certain embodiments, the IL12 variant has a single signal peptide - for example, [IL12(p40-GGGGS-SP None -p3 5)(The nucleic acid sequence present in SEQ ID NO: 7; the amino acid sequence present in SEQ ID NO: 8), wherein the p40 signal peptide is maintained and the p35 signal peptide is removed. See Figure 3.

[0045] Examples of heterologous dendritic cell growth factors include cytokines, C-type lectins, and CD40L. In some embodiments, the heterologous dendritic cell growth factor is a cytokine (i.e., a second cytokine) selected from the list comprising Fms-related tyrosine kinase 3 ligand (FLT3L), GMCSF, TNFα, IL36γ, and IFN. In certain embodiments, the heterologous dendritic cell growth factor is FLT3L. The nucleic acid sequence encoding muFLT3L is described in SEQ ID NO: 9. The nucleic acid sequence encoding huFLT3L is described in SEQ ID NO: 1. The amino acid sequence of muFLT3L is described in SEQ ID NO: 10. The amino acid sequence of huFLT3L is described in SEQ ID NO: 2.

[0046] In some embodiments, the oncolytic virus comprises nucleic acids encoding FLT3L and IL12. In other embodiments, the oncolytic virus is HSV-1 lacking the viral genes encoding ICP34.5 and ICP47, and this oncolytic virus comprises nucleic acids encoding FLT3L and IL12.

[0047] The exogenous nucleic acids can be under the control of the same promoter or different promoters. In certain embodiments, the nucleic acid encoding the heterologous dendritic cell growth factor and the nucleic acid encoding the first heterologous cytokine are under the control of the same promoter. Using a single promoter (e.g., the CMV promoter) provides the benefit of producing both the heterologous dendritic cell growth factor and the first heterologous cytokine at the same rate and simultaneously in the same infected cells.

[0048] Examples of suitable promoters include cytomegalovirus (CMV), Rous sarcoma virus (RSV), human elongation factor 1α promoter (EF1a), simian virus 40 early promoter (SV40), phosphoglycerate kinase 1 promoter (PGK), ubiquitin C promoter (UBC), and murine stem cell virus (MSCV). In certain embodiments, the promoter is CMV (the nucleic acid sequence shown in SEQ ID NO: 24).

[0049] When under the control of the same promoter, the nucleic acids encoding the payloads can be linked by additional nucleic acids that, for example, permit polycistronic translation (polycistronic linker elements). Examples of suitable polycistronic linker elements include ribosome entry sites (e.g., internal ribosome entry site (IRES) (SEQ ID NO: 19)), 2A sequences (e.g., porcine teschovirus 2a (GSG-P2A; the nucleic acid sequence set forth in SEQ ID NO: 17; the amino acid sequence set forth in SEQ ID NO: 18), Thosea asigna virus 2A (T2A), foot-and-mouth disease virus 2A (F2A), and equine rhinitis A virus (E2A)). Such sequences can be used to link two nucleic acids in any geometric arrangement. For example, the nucleic acids within the viral genome can be arranged as [heterologous dendritic cell growth factor]-[P2A]-[first heterologous cytokine] or [first heterologous cytokine]-[P2A]-[heterologous dendritic cell growth factor].

[0050] It has been observed that the use of an IRES results in a decrease in the production of the second nucleic acid 3' of the IRES in the construct. For example, the production of FL53L in the [IL12]-[IRES]-[FLT3L] construct decreased, while the production of IL12 in [FLT3L]-[IRES]-[IL12] decreased. See Example 4. Accordingly, in one embodiment, the polycistronic linker element is 2A. In certain embodiments, the polycistronic linker element is P2A.

[0051] The oncolytic virus of the present invention may further contain a sequence that enhances the translation of exogenous nucleic acids (e.g., mammalian translation). For example, the Kozak sequence is known to enhance mammalian translation. Thus, in some embodiments, the oncolytic virus contains a Kozak sequence. In one embodiment, the Kozak sequence is the consensus Kozak sequence (SEQ ID NO: 20).

[0052] The oncolytic virus of the present invention may further contain a sequence that enhances the stability of the mRNA expressed by the virus. Examples of such sequences include the bovine growth hormone polyadenylation signal sequence (BGHpA) and the rabbit β-globin (RBGpA), SV40 polyA and hGH polyA. In certain embodiments, the sequence is BGHpA (SEQ ID NO: 21).

[0053] Examples of other oncolytic viruses that can be modified as described herein include RP1 (HSV-1 / ICP34.5 - / ICP47 - / GM-CSF / GALV-GP R(-); RP2 (HSV-1 / ICP34.5 - / ICP47 - / GM-CSF / GALV-GP R(-) / anti-CTLA-4 binder; and RP3 (HSV-1 / ICP34.5 - / ICP47 -Examples include / GM-CSF / GALV-GP R(-) / anti-CTLA-4 binder / costimulatory ligands (e.g., CD40L, 4-1BBL, GITRL, OX40L, ICOSL). In such oncolytic viruses, GALV (gibbon ape leukemia virus) is modified by specific deletion of the R-peptide to obtain GALV-GP R(-). Such oncolytic viruses are described in International Publication Nos. WO 2017 / 118864, WO 2017 / 118865, WO 2017 / 118866, WO 2017 / 118867 and WO 2018 / 127713A1, each of which is incorporated herein by reference in its entirety. Further examples of oncolytic viruses that can be modified as described herein include NSC-733972, HF-10, BV-2711, JX-594, Myb34.5, AE-618, Brainwel™ and Heapwel™, Cavatak® (coxsackievirus, CVA21), HF-10, Seprehvir®, Reolysin®, enadenotucirev, ONCR-177 and those described in U.S. Patent No. 10,105,404, International Publication Nos. WO 2018 / 006005, WO 2018 / 026872A1 and WO 2017 / 181420, each of which is incorporated herein by reference in its entirety.

[0054] Further examples of oncolytic viruses that can be modified as described herein include the following: G207, an oncolytic HSV-1 derived from the wild-type HSV-1 F strain, having deletions in both copies of the ICP34.5 gene, a major determinant of HSV neurotoxicity, and an inactivating insertion of the E. coli lacZ gene into UL39, which encodes protein 6 of infected cells (ICP6) (see Mineta et al. (1995) Nat Med. 1:938-943). OrienX010, a herpes simplex virus having deletions of both copies of the γ34.5 and ICP47 genes, an insertion of the ICP6 gene, and an insertion of the human GM-CSF gene (see Liu et al., (2013) World Journal of Gastroenterology 19(31):5138-5143). NV1020, a herpes simplex virus having a deletion in the junction region of the long (L) and short (S) regions, containing one copy of ICP34.5, UL24, and UL56.34,35. The deleted region was replaced with a fragment of HSV-2 US DNA (US2, US3(PK), gJ, and gG) (see Todo, et al. (2001) Proc Natl Acad Sci USA. 98:6396-6401). M032, a herpes simplex virus having deletions of both copies of the ICP34.5 gene and an insertion of interleukin 12 (see Cassady and Ness Parker, (2010) The Open Virology Journal 4:103-108). ImmunoVEX HSV2, a herpes simplex virus (HSV-2) having a functional loss of the genes encoding vhs, ICP47, ICP34.5, UL43, and US5. OncoVEX GALV / CD which is similarly derived from the JS1 strain of HSV-1, has functionally deleted genes encoding ICP34.5 and ICP47, and has genes encoding cytosine deaminase and the feline leukemia fusion glycoprotein inserted into the viral genome in place of the ICP34.5 gene. GALV / CD .

[0055] In certain embodiments, the oncolytic virus of the invention is HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12. In another embodiment, the oncolytic virus of the present invention is HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12, where the virus is derived from the HSV-1 strain JS1 deposited with the European Collection of Authenticated Cell Cultures (ECACC) under accession number 01010209.

[0056] Combination with other agents The oncolytic virus of the present invention can be used as a single agent for treating diseases such as cancer. Oncolytic viruses have generally been found to be safe with a favorable safety profile. Thus, the oncolytic virus of the present invention can be used in combination with other agents without a significant negative contribution to the safety profile.

[0057] The oncolytic virus of the present invention (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) can be used in combination with immune checkpoint inhibitors, immune cytokines, agonists of costimulatory molecules, targeted therapies, and standard agents of supportive care. For example, the oncolytic virus of the present invention (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) can be used in combination with targeted cancer therapies (e.g., MEK inhibitors such as cobimetinib, trametinib, and binimetinib) and / or cytokines (e.g., pegylated IL2 (e.g., bempegaldesleukin) or pegylated IL10 (e.g., pegilodecakin)).

[0058] Checkpoint inhibitors Immune checkpoints are proteins that regulate certain types of immune system cells, such as T cells, which play a central role in cell-mediated immunity. Immune checkpoints assist in suppressing the immune response, but they can also prevent T cells from killing cancer cells. Immune checkpoint inhibitors (or simply "checkpoint inhibitors") can block immune checkpoint protein activity, release the "brakes" of the immune system, and enable T cells to better kill cancer cells.

[0059] As used herein, the term "immune checkpoint inhibitor" or "checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. Checkpoint proteins control the activation or function of T cells. For example, numerous checkpoint proteins are known, such as CTLA-4 and its ligands CD80 and CD86; and PD-1, including its ligands PD-L1 and PD-L2 (Pardoll, Nature Reviews Cancer 12:252-264, 2012). These proteins are responsible for co-stimulatory or inhibitory interactions of T cell responses. Immune checkpoint proteins control and maintain self-tolerance as well as the duration and amplitude of physiological immune responses. Immune checkpoint inhibitors can include antibodies or can be derived from antibodies.

[0060] Checkpoint inhibitors can include small molecule inhibitors or antibodies or antigen-binding fragments thereof that bind to and block or inhibit an immune checkpoint receptor, or antibodies that bind to and block or inhibit an immune checkpoint receptor ligand. Exemplary checkpoint molecules that can be targeted for blocking or inhibition include CTLA-4, PD-L1, PD-L2, PD-1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (which belongs to the CD2 family of molecules and is present on all NK, γδ, and memory CD8 +(expressed on (αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK 1 and CHK2 kinases, A2aR and various B-7 family ligands, including but not limited to these. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and B7-H7. Checkpoint inhibitors include antibodies or antibody-binding fragments, other binding proteins, biological therapeutics or small molecules that bind to one or more of CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160 and CGEN-15049 and block or inhibit the activity of one or more of these.

[0061] Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) is an immune checkpoint molecule that downregulates the pathway of T cell activation. CTLA-4 is a negative regulator of T cell activation. Blockade of CTLA-4 has been shown to enhance T cell activation and proliferation. The combination of herpes simplex virus and anti-CTLA-4 antibody is intended to improve T cell activation through two different mechanisms to enhance the anti-tumor immune response against tumor antigens released after lytic replication of the virus in the tumor. Therefore, the combination of herpes simplex virus and anti-CTLA-4 antibody increases the destruction of injected and uninjected / distal tumors, improves the overall tumor response, and in particular, can extend the overall survival period compared to that obtained by using anti-CTLA-4 antibody alone.

[0062] Programmed cell death protein 1 (PD-1) is a cell surface protein molecule consisting of 288 amino acids that is expressed on T cells and pro-B cells and plays a role in their fate / differentiation. Two ligands of PD-1, PD-L1 and PD-L2, are members of the B7 family. PD-1 restricts the activity of T cells in peripheral tissues during the inflammatory response to infection, and the fact that PD-1 inhibition in vitro restricts autoimmune PD-1 inhibition improves T cell proliferation and cytokine production in response to challenge by specific antigen targets or allogeneic cells during the mixed lymphocyte reaction. The strong correlation between PD-1 expression and response has been demonstrated by inhibition of PD-1 (Pardoll, Nature Reviews Cancer, 12:252-264, 2012). PD-1 inhibition can be achieved by various mechanisms including antibodies that bind to PD-1 or PD-L1.

[0063] Programmed cell death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene. See Entrez Gene: CD274 CD274 molecule. PD-L1 is a 40 kDa type I transmembrane protein that plays a role in the suppression of the immune system and binds to its receptor (PD-1), which is found on activated T cells, B cells, and myeloid cells, to regulate cell activation or inhibition. See Chemnitz et al., Journal of Immunology, 173(2):945-54 (2004).

[0064] Other immune checkpoint inhibitors include lymphocyte activation gene 3 (LAG-3) inhibitors, such as IMP321, which is a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Also included are B7 inhibitors such as B7-H3 and B7-H4 inhibitors (e.g., anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15(18)3834)). Another checkpoint inhibitor is TIM3 (T cell immunoglobulin domain and mucin domain 3) (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al., 2010, J. Exp. Med. 207:2187-94).

[0065] As further described herein, in one aspect, the present invention relates to the use of a combination of an oncolytic virus and a checkpoint inhibitor for treating cancer. In another aspect, the present invention relates to a pharmaceutical composition comprising a combination of an oncolytic virus and a checkpoint inhibitor.

[0066] Accordingly, in one aspect of the present invention, the checkpoint inhibitor is a blocker or inhibitor of CTLA-4, PD-1, PD-L1 or PD-L2. In some embodiments, the checkpoint inhibitor is a blocker or inhibitor of CTLA-4 such as tremelimumab, ipilimumab (also known as 10D1, MDX-D010), BMS-986249, AGEN-1884, and anti-CTLA-4 antibodies described in U.S. Patent Nos. 5,811,097, 5,811,097, 5,855,887, 6,051,227, 6,207,157, 6,682,736, 6,984,720, and 7,605,238, each of which is incorporated herein by reference.In some embodiments, the checkpoint inhibitor is pembrolizumab (anti-PD-1 antibody), nivolumab (anti-PD-1 antibody), CT-011 (anti-PD-1 antibody), CX-072 (anti-PD-L1 antibody), IO-103 (anti-PD-L1), BGB-A333 (anti-PD-L1), WBP-3155 (anti-PD-L1), MDX-1105 (anti-PD-L1), LY-3300054 (anti-PD-L1), KN-035 (anti-PD-L1), FAZ-053 (anti-PD-L1), CK-301 (anti-PD-L1), AK-106 (anti-PD-L1), M-7824 (anti-PD-L1), CA-170 (anti-PD-L1), CS-1001 (anti-PD-L1 antibody); SHR-1316 (anti-PD-L1 antibody); BMS 936558 (anti-PD-1 antibody), BMS-936559 (anti-PD-1 antibody), atezolizumab (anti-PD-L1 antibody), AMP 224 (a fusion protein of the extracellular domain of PD-L2 and an IgG1 antibody designed to block the PD-L2 / PD-1 interaction), MEDI4736 (durvalumab; anti-PD-L1 antibody), MSB0010718C (anti-PD-L1 antibody) and those described in U.S. Patent Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149 and PCT Published Patent Application International Publication Nos. 03042402; 2008156712; 2010089411; 2010036959; 2011066342; 2011159877; 2011082400 and 2011161699 (each incorporated herein by reference), for example, a blocker or inhibitor of a molecule that inhibits the interaction with a PD-L1 and / or PD-L2 inhibitor.Examples of additional anti-PD-1 antibodies include PDR-001, SHR-1210, BGB-A317, BCD-100, JNJ-63723283, PF-06801591, BI-754091, JS-001, AGEN-2034, MGD-013, LZM-009, GLS-010, MGA-012, AK-103, genolimzumab, dostarlimab, semiprimab, IBI-308, camrelizumab, AMP-514, TSR-042, Sym-021, HX-008, and ABBV-368.

[0067] BMS 936558 is a fully human IgG4 monoclonal antibody that targets PD-1. In a phase 1 clinical trial, bi-weekly administration of BMS-936558 in subjects with advanced refractory malignancies demonstrated durable partial or complete regression. The most prominent response rates were observed in subjects with melanoma (28%) and renal cell carcinoma (27%), but substantial clinical activity was also observed in subjects with non-small cell lung cancer (NSCLC), and some responses persisted for over one year.

[0068] BMS 936559 is a fully human IgG4 monoclonal antibody that targets the PD-1 ligand PD-L1. Phase 1 trial results showed that bi-weekly administration of this agent resulted in durable responses, particularly in subjects with melanoma. Objective response rates ranged from 6% to 17% depending on cancer type in subjects with advanced NSCLC, melanoma, RCC, or ovarian cancer, and some subjects experienced responses lasting over one year.

[0069] AMP 224 is a fusion protein of the extracellular domain of PD-L2, a second PD-1 ligand, and IgG1, and has the ability to block the PD-L2 / PD-1 interaction. AMP-224 is currently in a phase 1 clinical trial as a single agent in subjects with advanced cancer.

[0070] MEDI4736 is an anti-PD-L1 antibody that demonstrated an acceptable safety profile and durable clinical activity in this dose-escalation trial. Development and investigation of MEDI4736 as monotherapy and in combination in multiple cancers are ongoing.

[0071] Method of treating a disease or disorder The present invention also relates to a method of treating a disease or disorder such as cancer using an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12). The oncolytic virus of the present invention (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) can be used to treat any injectable cancer (i.e., any tumor that can be injected using a needle, with or without guidance (e.g., visual or ultrasonic guidance)). In some embodiments, the cancer is B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, small cell lung cancer, basal cell carcinoma, cutaneous squamous cell carcinoma, colorectal cancer, melanoma (e.g., uveal melanoma), head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, sarcoma (e.g., soft tissue sarcoma, Ewing's sarcoma, osteosarcoma or rhabdomyosarcoma), gastroesophageal cancer, renal cell carcinoma, glioblastoma, pancreatic cancer, bladder cancer, prostate cancer, breast cancer (e.g., triple negative breast cancer), cutaneous T-cell lymphoma, Merkel cell carcinoma or multiple myeloma.

[0072] The term "metastatic cancer" refers to cancer that has spread from the part of the body where it originated (i.e., the primary site) to another part of the body. When cancer has spread to a new area (i.e., metastasized), it is named after the part of the body where it originated. For example, colon cancer that has spread to the pancreas is not pancreatic cancer but is referred to as "metastatic colon cancer to the pancreas". Treatment is also based on where the cancer originated. Even when colon cancer metastasizes to the bone, this is still colon cancer, and the treating physician will recommend a treatment that has been proven effective for metastatic colon cancer.

[0073] The present invention relates to the use of a combination of an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) and another agent (e.g., a checkpoint inhibitor) for treating cancer as discussed above.

[0074] The present invention further relates to a method of treating a disease or disorder such as cancer by administering (i) a therapeutically effective amount of an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12); and (ii) a therapeutically effective amount of another agent (e.g., a checkpoint inhibitor).

[0075] In certain embodiments, the present invention relates to a combination of an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) and an anti-PD-1 antibody, a combination of an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) and an anti-PD-L1 antibody, or a combination of an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) and an anti-CTLA-4 antibody. In certain embodiments, the oncolytic virus is HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12.

[0076] Often, cancer is present in a patient both as a primary tumor (i.e., a tumor that grows at the anatomical site where tumor progression begins and continues to produce a cancerous mass) and as secondary tumors or metastases (i.e., the spread of the tumor from its primary site to other parts of the body). The oncolytic viruses of the present invention can be effective in the treatment of tumors via a lytic effect and a systemic immune effect. For example, HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12 physically lyses tumor cells, inducing cell death of the primary tumor and the release of tumor-derived antigens that are later recognized by the immune system. Furthermore, HSV1 / ICP34.5 - / ICP47 - The replication of / FLT3L / IL12 results in the production of FLT3L and IL12, which help increase and maintain the (both local and systemic) anti-tumor immune response so that the immune system can recognize and attack both primary and secondary tumors / metastases. Accordingly, the present invention contemplates the treatment of primary tumors, metastases (i.e., secondary tumors), or both with an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) either alone or in combination with a secondary agent (e.g., a checkpoint inhibitor).

[0077] In some embodiments, the treatment methods or uses described herein include, for example, combination therapies with targeted cancer therapies using MEK inhibitors such as cobimetinib, trametinib, and binimetinib. In other embodiments, the treatment methods or uses described herein include, for example, treatments with cytokines such as pegylated IL2 (e.g., bempegaldesleukin) or pegylated IL10 (e.g., pegilodecakin). In still other embodiments, the treatment methods or uses described herein include treatments by combinations of targeted therapies and immunomodulatory agents.

[0078] The method of the present invention can be used to treat cancers at several different stages. Most staging systems include information regarding whether the cancer has spread proximal to the lymph nodes, where the tumor is located in the body, the cell type (e.g., squamous cell carcinoma), whether the cancer has spread to different parts of the body, the size of the tumor, and the malignancy of the tumor (i.e., the level of cell abnormality, the likelihood that the tumor will grow and spread). For example, stage 0 refers to the presence of abnormal cells - i.e., cells that have the potential to become cancerous - that have not spread to proximal tissue. Stages I, II, and III cancers refer to the presence of cancer. The higher the stage, the larger the cancerous tumor and the more it has spread to proximal tissue. Stage IV cancer is cancer that has spread to distant parts of the body. In some embodiments, the method of the present invention can be used to treat metastatic cancer.

[0079] Pharmaceutical composition The present invention further relates to a pharmaceutical composition, a targeted cancer therapy, and / or other immunomodulatory agents comprising an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12), or a combination of an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) and a checkpoint inhibitor. The pharmaceutical composition can contain, for example, formulation materials for changing, maintaining, or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. The pharmaceutically active agent can be administered to a patient, for example, by various routes including oral or parenteral such as intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrathecal, rectal, intratumoral, intravascular, intradermal, etc., or by passive or enhanced absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. In one embodiment, the oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 -( / FLT3L / IL12) is injected into the tumor (i.e., via intratumoral injection). In another embodiment, a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody) is administered systemically (e.g., intravenously). In another embodiment, a targeted therapy (e.g., a MEK small molecule kinase inhibitor such as cobimetinib, trametinib, or binimetinib) is administered systemically via the oral route. In still other embodiments, cytokines such as, for example, pegylated IL2 (e.g., bempegaldesleukin) or pegylated IL10 (e.g., pegilodecakin) are administered systemically.

[0080] One of ordinary skill in the art will be able to determine dosages and treatment durations according to any aspect of the present disclosure. For example, one of ordinary skill in the art can monitor a patient to determine whether treatment should be initiated, continued, interrupted, or restarted. The effective amount for a particular patient can vary depending on factors such as the condition being treated, the overall health of the patient, and the method, route, and dosage of administration. A clinician will determine an appropriate dosage using parameters known in the art. The effective amount of a pharmaceutically composition used therapeutically will depend, for example, on the context and purpose of the treatment. One of ordinary skill in the art will understand that appropriate dosage levels for treatment will vary in part depending on the molecule thus delivered, the indication for which the binding agent molecule is used, the route of administration, and the size (body weight, body surface area, or organ size) and condition (age and general health) of the patient. Accordingly, a clinician can titrate the dosage and change the route of administration to obtain an optimal therapeutic effect.

[0081] Clinical studies have demonstrated that oncolytic viruses can be injected directly into visible and palpable skin lesions, subcutaneous lesions, or lymph node lesions or can be injected using ultrasound guidance. Accordingly, in one aspect, HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12-containing pharmaceutical compositions are administered via intralesional injection. In some embodiments, HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12 is provided in 1 mL disposable vials at a fixed dosage concentration: 10 6 PFU / mL for the initial dose and 10 8 PFU / mL for subsequent doses. The volume to be injected may vary depending on the type of tumor. For example, HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12 is administered by intratumoral injection at a dose of up to 4.0 mL of 10 6 PFU / mL (plaque forming units / mL) on day 1 of week 1, followed by up to 4.0 mL of 10 8 PFU / mL on day 1 of week 4, and then every two weeks (±3 days) in injectable cutaneous tumors, subcutaneous tumors, and lymph node tumors. In another embodiment, HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12 is administered by intratumoral injection at a dose of up to 4.0 mL of 10 6 PFU / mL on day 1 of week 1, followed by up to 4.0 mL of 10 7 PFU / mL on day 1 of week 4, and then every two weeks (±3 days).

[0082] The composition of the present invention can include one or more additional components including a physiologically acceptable carrier, excipient, or diluent. For example, the composition can include one or more of a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (e.g., having fewer than 10 amino acids), a protein, an amino acid, a carbohydrate such as glucose, sucrose, or dextrin, a chelating agent such as EDTA, glutathione, a stabilizer, and an excipient. Acceptable diluents include, for example, neutral buffered saline or saline mixed with a specific serum albumin. A preservative such as benzyl alcohol can also be added. The composition can be formulated as a lyophilized product using a suitable excipient solution (e.g., sucrose) as a diluent.

[0083] In certain embodiments, the checkpoint inhibitor is administered at a dose of 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 0.7 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg or any combination thereof. In certain embodiments, the checkpoint inhibitor is administered once a week, twice a week, three times a week, once every two weeks or once a month. In certain embodiments, the checkpoint inhibitor is administered as a single dose, two doses, three doses, four doses, five doses or six or more doses.

[0084] In certain embodiments, the anti-PD-1 antibody is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1-30 mg / kg, such as about 5-25 mg / kg, about 10-20 mg / kg, about 1-5 mg / kg or about 3 mg / kg. The dosing schedule can be changed, for example, from once a week to once every 2, 3 or 4 weeks. In one embodiment, the anti-PD-1 antibody is administered at a dose of about 10-20 mg / kg every other week.

[0085] In one embodiment, the anti-PD-1 antibody molecule, such as nivolumab, is administered intravenously at a dose of about 1 mg / kg to 3 mg / kg every two weeks, such as about 1 mg / kg, 2 mg / kg or 3 mg / kg. In one embodiment, the anti-PD-1 antibody molecule, such as nivolumab, is administered intravenously at a dose of about 2 mg / kg at 3-week intervals. In one embodiment, nivolumab is administered in an amount of about 1 mg / kg to 5 mg / kg, such as 3 mg / kg, and can be administered over 60 minutes, approximately once a week to once every 2, 3 or 4 weeks.

[0086] In one embodiment, an anti-PD-1 antibody molecule, such as pembrolizumab, is administered intravenously at a dose of about 1 mg / kg to 3 mg / kg, such as about 1 mg / kg, 2 mg / kg, or 3 mg / kg every three weeks. In one embodiment, an anti-PD-1 antibody molecule, such as pembrolizumab, is administered intravenously at a dose of about 2 mg / kg at three-week intervals. In another embodiment, an anti-PD-1 antibody molecule, such as pembrolizumab, is administered intravenously at a dose of about 100 mg / kg to 300 mg / kg, such as about 100 mg / kg, 200 mg / kg, or 300 mg / kg every three weeks. In one embodiment, an anti-PD-1 antibody molecule, such as pembrolizumab, is administered intravenously at a dose of about 200 mg / kg at three-week intervals.

[0087] In certain embodiments, an anti-CTLA-4 antibody (e.g., ipilimumab) is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 3 mg / kg IV Q3W for up to 4 doses, at a dose of about 3 mg / kg IV Q6W for up to 4 doses, at a dose of about 3 mg / kg IV Q12W for up to 4 doses, at a dose of about 10 mg / kg IV Q3W for up to 4 doses, or at a dose of about 10 mg / kg IV Q12W for up to 4 doses. In certain embodiments, an anti-CTLA-4 antibody (e.g., tremelimumab) is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 10 mg / kg Q4W or at a dose of about 15 mg / kg every three months.

[0088] In certain embodiments, an anti-PD-L1 antibody (e.g., atezolizumab) is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1200 mg IV Q3W until disease progression or unacceptable toxicity occurs.

[0089] Accordingly, in one embodiment, the present invention relates to the use of a pharmaceutical composition for use in any injectable cancer treatment method. In some embodiments, the cancer is B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, small cell lung cancer, basal cell carcinoma, cutaneous squamous cell carcinoma, colorectal cancer, melanoma (e.g., uveal melanoma), head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, sarcoma (e.g., soft tissue sarcoma, Ewing sarcoma, osteosarcoma or rhabdomyosarcoma), gastroesophageal cancer, renal cell carcinoma, glioblastoma, pancreatic cancer, bladder cancer, prostate cancer, breast cancer (e.g., triple negative breast cancer), cutaneous T-cell cancer, Merkel cell carcinoma or multiple myeloma, where the pharmaceutical composition is a oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) or an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) and a second agent (e.g., a checkpoint inhibitor).

[0090] In other embodiments, the present invention provides a therapeutically effective amount of an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 -Relates to ( / FLT3L / IL12). In yet other embodiments, the present invention is for use in the treatment of B-cell lymphoma (e.g., diffuse large B-cell lymphoma), non-small cell lung cancer, small cell lung cancer, basal cell carcinoma, cutaneous squamous cell carcinoma, colorectal cancer, melanoma (e.g., uveal melanoma), head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric cancer, sarcoma (e.g., soft tissue sarcoma, Ewing sarcoma, osteosarcoma or rhabdomyosarcoma-like tumor), gastroesophageal cancer, renal cell carcinoma, glioblastoma, pancreatic cancer, bladder cancer, prostate cancer, breast cancer (e.g., triple-negative breast cancer), cutaneous T-cell lymphoma, Merkel cell carcinoma or multiple myeloma, a therapeutically effective amount of an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) and a second agent (e.g., a checkpoint inhibitor).

[0091] Kit In another aspect, the present invention relates to a kit comprising [1] an oncolytic virus (e.g., HSV1 / ICP34.5 optionally combined with a second agent (e.g., a checkpoint inhibitor) - / ICP47 - / FLT3L / IL12); and [2] instructions for administration to a patient. For example, the kit of the present invention may comprise an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12) and instructions for treating a patient with cancer (e.g., in the package insert or label). In some embodiments, the cancer is metastatic cancer. In another embodiment, the kit of the present invention may comprise an oncolytic virus (e.g., HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12), a checkpoint inhibitor (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody or an anti-CTLA-4 antibody), and instructions for treating a patient with cancer (e.g., in the package insert or label).

[0092] In some embodiments, the second agent is a targeted cancer therapy (e.g., a MEK inhibitor such as cobimetinib, trametinib, and binimetinib) or a cytokine (e.g., pegylated IL2 (e.g., bempegaldesleukin) or pegylated IL10 (e.g., pegilodecakin)).

[0093] In some embodiments, HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12 kits contain, on day 1 of week 1, a maximum dose of 4.0 mL at 10 6 PFU / mL, followed by a maximum dose of 4.0 mL at 10 8 PFU / mL on day 1 of week 4, and then (e.g., until complete response) include instructions (e.g., in the package insert or label) regarding administration by intratumoral injection every two weeks. In some embodiments, HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12 kits contain, on day 1 of week 1, a maximum dose of 4.0 mL at 10 6 PFU / mL, followed by a maximum dose of 4.0 mL at 10 7 PFU / mL on day 1 of week 4, and then (e.g., until complete response) include instructions (e.g., in the package insert or label) regarding administration by intratumoral injection every two weeks.

[0094] In embodiments where the kit contains an anti-PD-1 antibody, the kit includes instructions (e.g., in the package insert or label) for intravenous administration at the dosages described herein. Examples of anti-PD-1 antibodies include pembrolizumab and nivolumab.

[0095] In embodiments where the kit contains an anti-PD-L1 antibody, the kit includes instructions (e.g., in the package insert or label) for intravenous administration at the dosages described herein. An example of an anti-PD-L1 antibody is atezolizumab.

[0096] In embodiments where the kit comprises an anti-CTLA-4 antibody, the kit includes instructions (e.g., in the package insert or label) for intravenous administration at the dosages described herein. Examples of anti-CTLA-4 antibodies include ipilimumab.

[0097] In another embodiment, a method of manufacturing the kit of the present invention is provided.

Examples

[0098] The following examples are provided for the purpose of exemplifying specific embodiments or features of the present invention and are not intended to limit its scope.

[0099] Example 1: Interleukin 12 (IL12) generated as a single-chain protein with a p40 subunit at the 5' position and a p35 subunit at the 3' position and connected via a single G4S linker is active in vitro and in vivo The single-chain IL12 molecule recombinantly produced using specific genetic recombination criteria results in optimal expression and activity of the cytokine.

[0100] The optimal conformation of the p40 and p35 subunits of IL12 was evaluated by analyzing the crystal structure of IL12 (PDB ID 3HMX). The single-chain protein is predicted to have a higher degree of heterodimerization efficiency because these subunits are proximal for assembly. The geometric arrangement of p40-p35 (Figure 1A; dashed line) is structurally preferred compared to the geometric arrangement of p35-p40 because it is proximal to the point of attachment of the C-terminus and the N-terminus. This results in a linker that bridges a gap of approximately 36 Å (angstroms) (connecting the carboxy terminus of p40 to the amino starting end of p35). In contrast, the generation of the p35-p40 peptide results in a less favorable gap of approximately 60 Å that requires a longer linker.

[0101] To model the linker between the p40 and p35 subunits, the P40 and p35 subunits (PDB 3HMX) of the crystal structure of IL12 were prepared using FastRelax with 0.5 Å coordinate constraints in RosettaScripts (S.J. Fleishman, A. Leaver-Fay, J.E. Corn, E.-M. Strauch, S.D. Khare, N. Koga, J. Ashworth, P. Murphy, F. Richter, G. Lemmon, J. Meiler and D. Baker. RosettaScripts: A Scripting Language Interface to the Rosetta Macromolecular Modeling Suite. PLoS ONE. 2011, 6, 6, e20161). The resulting PDB file was ligated into a single chain with the p40-p35 geometry, and then Rosetta Remodel was used to model the following linkers between the two domains: the previously described elastin-like linker (VPGVGVPGVGGS), G4S (Figure 1B), 2×(G4S) (Figure 1C), 3×(G4S) and no linker. The unresolved C-terminal residue of p40 (S340) and the first 11 residues of mature p35 (RNLPVATPDPG) were included in the Remodel runs. Controls lacking the unresolved residues were also run. The computational loop closure rate using the simulation of Rosetta loop modeling was significantly improved when the linker was incorporated, so a linker was predicted to be required. For each linker, 2880 Remodel trajectories were run using fragment insertion from loop fragments for sampling and CCD-based inverse kinematics for leap closure. The models were scored using the set Remodel weights, and models that ended successfully in loop closure (chain break score < 0.07) were output as PDB files. The loop closure rate was determined by evaluating the percentage of trajectories that met the loop closure criteria.For each linker, conformational convergence was measured by plotting the root-mean-square deviation (RMSD) of each model to the lowest scoring model using RosettaScripts without using superposition. The top 10 models for each linker were evaluated by Rosetta energy units per residue (REU) and by the backbone score terms for the linker residues (Table 1). Models with Ramachandran outliers were identified in MOE (Chemical Computing Group, Inc.).

[0102] Remodel runs without using a linker or using the uncleaved p40 and p35 termini had a loop closure rate of less than 10%, suggesting that a linker is required to connect the p40 and p35 subunits as single strands. In contrast, Remodel runs with a linker had successful loop closure rates for all four linker sequences. The top scoring models for all four linkers were well scored without backbone strain or Ramachandran outliers. Longer elastin linkers and 3×(G 4 S) linkers showed greater RMSD divergence from the top scoring models for models from the former than for those from the latter, so G 4 S and 2×(G 4 S) linkers are likely to be conformationally more flexible. Rosetta Remodel was used to identify linkers for the p40-linker-p35 payload. The top scoring models of the G4S linker construct and the 2×G4S linker construct suggest that both linkers are suitable, similar to the elastin-based linker (Figure 2).

[0103] The loop closure rates are summarized in Table 1 below.

[0104]

Table 1

[0105] To confirm the function of single-chain IL12 from in silico modeling, single-chain IL12 constructs in various formats were cloned into the pΔ34.5(XS) vector, a pcDNA3.1-based vector with a construct introduced between the CMV promoter and the BGH poly(A) tail (see Figure 3A for the depiction of the constructs). The HSV-1 inverted repeat sequences flanking the CMV promoter and the BGH poly(A) tail facilitate recombination of the single-chain IL12 construct, CMV, and the BGH poly(A) tail into the HSV-1 virus. The pΔ34.5(XS) vector was linearized by restriction enzymes Hind III and Xho I located after the CMV promoter and before the BGH poly(A) tail, respectively. Overlapping DNA fragments encoding the single-chain IL12 construct were ordered and cloned into the linearized pΔ34.5(XS) vector using the Gibson assembly method. The integrity of the single-chain IL12 construct was confirmed by DNA sequencing. Using these constructs, HEK 293 cells were transfected in vitro and IL12 protein production was compared. Cells were transfected with 4 μg of DNA with 8 μL of Lipofectamine 2000 in Optimem medium and incubated at 37 °C for 48 h with 5% CO 2 2. The supernatant was removed and the expression of IL12 was quantified using the Biolegend Human IL12 p70 ELISA assay. The position of the peptide chain significantly altered the expression. The construct containing p35-elastin-p40 did not produce detectable levels of IL12, while the construct containing p40-elastin-p35 produced IL12 (Figure 3B).

[0106] In its native form, IL12 is produced as two independent chains, both containing a signal peptide required for protein secretion. In the modified version, the need for a second signal peptide was evaluated. Fusion [IL12(p40-elastin-SP None -p3 5)Constructs containing a single signal peptide located at the 5'-end were compared to constructs encoding signal peptides in both the p35 and p40 subunits [IL12(p40 - elastin - p35)]. Removal of the second signal increased the total yield of IL12 produced as a result of transfection (Figure 3B). Finally, expression of IL12 using an elastin linker was compared to a single G4S linker (Figure 3B). Based on these observations, a single - stranded IL12 cassette incorporating p40 - G4S linker - p35 along with the signal peptide removed from the p35 subunit was selected for inclusion within the recombinant virus.

[0107] Example 2: Bioactive FLT3L and IL12 can be co - expressed by the addition of a P2A linker These experiments relate to genetic manipulations performed to generate bioactive FLT3L and IL12 in a bicistronic format under the control of a single promoter using the porcine teschovirus 2A sequence.

[0108] Expression of a number of rationally selected proteins from the virus should enhance the immunostimulatory capacity of the virus to induce an antitumor response. FLT3L and IL12 were selected as immunostimulatory cytokines. Both cytokines were produced using a single promoter (CMV promoter). This approach had the advantage of producing both cytokines at the same rate and simultaneously within the same infected cells. The applicants selected two means for expressing multiple proteins from a single promoter: internal ribosome entry site (IRES) and 2A sequences. DNA constructs were designed to incorporate FLT3L - IRES - IL12, IL12 - IRES - FLT3L or FLT3L - P2A - IL12. These DNA constructs were tested in vitro as previously described (Figure 4A). The DNA constructs were transfected into 293T cells and the supernatants were tested by ELISA (Biolegend IL12 p70 assay for IL12 and Thermo FLT3L assay for FLT3L).

[0109] Either geometric arrangement (FLT3L as the first gene and IL12 as the second gene) or (IL12 as the first gene and FLT3L as the second gene), the production of the second gene was decreased when using IRES (Figures 4B and 4C). For this reason, the P2A sequence was selected as a functional unit to provide the production of two proteins from a single promoter.

[0110] In a separate experiment using an alternative payload (GMCSF), the effect of the consensus Kozak sequence was evaluated. The Kozak sequence is known to enhance mammalian translation and was expected to improve the translation of the complete cassette. Consistent with this, the expression of the 5' protein (GMCSF) was significantly increased by the incorporation of the Kozak sequence upstream of the translation start site regardless of the use of P2A or IRES (Figure 5; average ng / ML with Kozak = 660.9; average ng / mL without Kozak = 102.5).

[0111] A possible result when adding the P2A site is to add several amino acids to the end of the FLT3L protein. P2A is a sequence that results in the production of two separate polypeptide chains in most mammalian cells, and the first peptide produced contains the addition of the amino acid sequence GSGATNFSLLKQAGDVEENPG. In silico modeling was performed to determine whether the addition of amino acids to the carboxy terminus of FLT3L affects its interaction with its receptor, FLT3. PyMOL v.1.8.6.0 was used to evaluate the structure of the Flt3L / Flt3 complex for the purpose of selecting the geometric arrangement of the constructs in the payload1-P2A-payload2 cassette, a dual payload vector. P2A results in an 18-amino acid peptide fused to the C-terminus of payload1. The structure of Flt3L / Flt3 reveals that the C-terminus of Flt3L is exposed and distal to the interface between the receptor-binding site and Flt3L dimerization. Thus, Flt3L is likely to tolerate the P2A tag and was selected as the payload upstream of the P2A sequence (Figure 6). However, to verify the activity, proof of biological activity of both FLT3L and IL12 was performed.

[0112] For IL12, the supernatant previously described and used in an ELISA assay to quantify total expressed IL12 was used in the IL12 cell receptor assay. The biological activity of IL12 was measured using HEK-Blue IL12 cells (Invivogen #hkb-il12). Bioactive IL12 induces dose-dependent production of secreted embryonic alkaline phosphatase (SEAP) by the HEK-Blue IL12 cell line, and the level of SEAP can be evaluated using the chromogenic reagent QUANTI-Blue (Invivogen #rep-qb1). The supernatant from DNA-transfected 293T cells was added directly to a 96-well flat-bottom plate at 3-fold serial dilutions twice using HEK-Blue IL12 cells, and 5% CO at 37°C 2It was incubated overnight. The next day, the QUANTI-Blue reagent was freshly prepared according to the manufacturer's instructions, pre-warmed to 37°C for 15 minutes, and incubated with 20 μL of the overnight cell culture supernatant at 37°C for 1 hour. The SEAP level was detected by measuring the absorbance at 620 - 630 nm using a BioTek Synergy Neo2 microplate reader (BioTek; Gen5 software v3.04). The supernatant demonstrated activity comparable to that of recombinant human IL12 protein (R&D #219-IL-005; Figure 7) purchased from a commercial supplier in the IL12 reporter assay.

[0113] For FLT3L, the supernatant was further tested in a BaF3 cell proliferation assay described in the literature as a FLT3L-sensitive cell line. BaF3 cells were plated at 30,000 cells per well in RPMI + 10% FBS + Geneticin in 24-well plates overnight at 37°C. Supernatants from cells transfected with a DNA construct containing the gene recombinant payload or recombinant human FLT3L were added to the cells, and the total volume was adjusted to 500 μL for all wells, followed by incubation at 37°C in 5% CO 2 for 14 days. On day 14, the BaF3 cells were gently resuspended by pipetting, samples were taken from each well, and cell counting was performed using a Vi-CELL XR cell viability analyzer (Beckman Coulter). The total number of live cells in the well was calculated from the live cell concentration provided by the Vi-CELL XR. Human recombinant FLT3L was included as a control, and the supernatant from transfected 293T cells showed a comparable effect on cell proliferation (Figure 8).

[0114] Based on these observations, the final construct to be integrated into the HSV1 genome was selected as human FLT3L-P2A-huIL12(p40-G4S-p35) using the genetic manipulations described above.

[0115] Example 3: HSV1 / ICP34.5 - / ICP47 - Generation of / FLT3L / IL12 virus HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12 was generated as follows.

[0116] Description of the viral genome: HSV-1 was derived from the JS1 strain deposited with the European Collection of Authenticated Cell Cultures (ECAAC) under accession number 01010209. In HSV-1 / ICP34.5- / ICP47- / FLT3L / IL12, the HSV-1 viral genes encoding ICP34.5 and ICP47 are functionally deleted as described above. See Liu et al., Gene Ther., 10:292-303, 2003; U.S. Patent Nos. 7,223,593 and 7,537,924. In HSV-1 / ICP34.5- / ICP47- / FLT3L / IL12, the functional deletion of the genes encoding ICP34.5 and ICP47 in combination with the early expression of US11 improves tumor replication while maintaining safety. The coding genes for human FLT3L and IL12 were inserted into the viral genome at two Original sites of the ICP34.5 gene of HSV-1 / ICP34.5- / ICP47- / FLT3L / IL12 (Figure 9). The human FLT3L and IL12 expression cassettes replace almost all of the ICP34.5 gene, ensuring that any potential recombination events between HSV-1 / ICP34.5- / ICP47- / FLT3L / IL12 and the wild-type virus produce only non-pathogenic viruses rendered impotent and do not result in the generation of wild-type viruses carrying the genes for human FLT3L and IL1. The HSV thymidine kinase (TK) gene remains intact in HSV-1 / ICP34.5- / ICP47- / FLT3L / IL12, which renders the virus sensitive to antiviral agents such as acyclovir. Thus, acyclovir can be used, if necessary, to block the replication of HSV-1 / ICP34.5- / ICP47- / FLT3L / IL12.

[0117] Generation of the pΔ34.5 transfer plasmid: The transfer plasmid containing the human FLT3L and IL12 expression cassettes was generated from the modified SP72 vector (Promega) as previously described (see Liu et al., Gene Ther., 10:292 - 303, 2003; U.S. Patent Nos. 7,223,593 and 7,537,924). This plasmid contains a NotI fragment (nucleotides 124948 - 125713) encoding most of the deleted ICP34.5 and a modified Sau3AI fragment of HSV - 1 17syn+ (nucleotides 123462 - 126790). The expression cassette containing CMV - KOZAK - FLT3L - P2A - IL12 - BGHPolyA was inserted proximal to the original Not1 site within the plasmid. This insertion results in an expression cassette adjacent to the HSV - 1 17syn+ region cut by the Sau3AI fragment (Figure 9).

[0118] HSV - 1 / ICP34.5 of the therapeutic gene - / ICP47 - / Insertion into FLT3L / IL12: The gene was inserted into the viral genome by the process of homologous recombination. Vero cells were transfected with the pΔ34.5 transfer plasmid. The transfected cells were then infected with HSV - 1 / ICP34.5 - / ICP47 -Infected with / GFP (JS1 strain). This virus contained GFP within the ICP34.5 coding region of the genome into which the CMV-FLT3L-P2A-IL12-BGHPolyA expression cassette was inserted. The transfection-infection reaction was maintained until complete CPE (cytopathic effect) was observed. Cells and supernatants from the transfection-infection reaction were diluted and used to infect Vero cells in 96-well plates. Two days later, the supernatants were evaluated by ELISA to identify wells containing virions expressing IL12 and FLT3L. Cells and supernatants from IL12- and FLT3L-positive wells were collected and plated in a plaque assay using Vero cells. Two days later, the recombinant virus was identified by the disappearance of the GFP marker gene. The disappearance of the GFP marker gene suggested that GFP at the ICP34.5 site was replaced by the [CMV]-[Kozak]-[Flt3L]-[P2A]-[IL12]-[BGHpA] expression cassette (Figure 9). Non-GFP plaques were identified under a fluorescence microscope and transferred using a sterile pipette tip to an Eppendorf test tube containing fresh growth medium. Since the virus was released from the cells by freeze-thawing, these viruses were plated on fresh cells. This process was repeated every 2 - 3 days until a homogeneous population was achieved (i.e., the plaques were no longer green). The validity of the insertion of the CMV-FLT3L-P2A-IL12-BGHPolyA expression cassette was verified by PCR and sequencing.

[0119] Example 4: HSV-1 / ICP34.5 - / ICP47 - The / FLT3L / IL12 virus can infect, replicate internally, and kill tumor cell lines and can produce biologically active FLT3L and IL12 in vitro. The ability of the recombinant virus to produce biologically active FLT3L and IL12 while maintaining cell infection, replication, and lysis was evaluated.

[0120] To confirm that the recombinant virus can replicate in human cells, two human cell lines were infected and the total amount of virus after infection was quantified. One million A375 or Vero cells were plated in a 6-well dish and incubated overnight at 37 °C in 5% CO₂ in DMEM containing 5% FBS. The cells were infected three times with HSV-1 / ICP34.5 2 / ICP47 - / ICP47 at an MOI of 0.1 and returned to the incubator. Forty-eight hours after infection, the cells and supernatant were collected and the virus titer was evaluated by plaque assay on Vero cells. The recombinant HSV-1 / ICP34.5 - / ICP47 - / FLT3L / IL12 virus and HSV-1 / ICP34.5 - / ICP47 - / GMCSF virus were evaluated (Figure 10). - To confirm that the modifications introduced into the virus did not affect its ability to infect and lyse virus cells, an in vitro killing assay was performed. Various cell lines of both mouse (CT26) and human (HT-29, SK-MEL-5, FADU, and BxPC3) origin were cultured with virus particles at various multiplicities of infection (MOI) (Figures 11A–E). The results are described below.

[0121] Mouse colorectal cancer (CT26)

[0122] CT26 cells were plated at 6,000 cells per well in a 96-well plate and incubated overnight at 37 °C. HSV-1 / ICP34.5 / ICP47 - / ICP47 - / FLT3L / IL12 and HSV-1 / ICP34.5 - / ICP47 - / GMCSF was started at 100 MOI and serially diluted (4-fold, 10 wells). After 72 hours of incubation, the number of cells remaining in each well was quantified using the CellTiter-Glo Luminescent cell viability assay (Promega, Madison, WI).

[0123] Human cancer cell lines (HT-29, SK-MEL-5, FADU, and BxPC-3) Various human solid tumor cell lines (colorectal cancer, melanoma, head and neck squamous cell carcinoma, and pancreatic cancer) were plated in 96-well plates at 7,000 - 10,000 cells per well and incubated overnight at 37°C. HSV-1 / ICP34.5 - / ICP47 - / FLT3L / IL12 and HSV-1 / ICP34.5 - / ICP47 - / GMCSF was started at 100 MOI and serially diluted (4-fold, 10 wells). After 72 hours of incubation, the number of cells remaining in each well was quantified using the CellTiter-Glo Luminescent cell viability assay on a SpectraMax M5 microplate reader (Molecular Devices Corporation) using the CellTiter-Glo Luminescent cell viability assay (Promega, Madison, WI).

[0124] HSV-1 / ICP34.5 - / ICP47 - / FLT3L / IL12 was effective against all cancer cell lines tested. All cell lines tested showed an MOI IC 50 value below 1. Figure 11 shows the degree of cell growth inhibition achieved by increasing the concentration of HSV-1 / ICP34.5 - / ICP47 - / FLT3L / IL12 along with the MOI IC 50 values for each of the five cell lines. These results show that HSV-1 / ICP34.5 - / ICP47 -Treatment with / FLT3L / IL12 against HSV-1 / ICP34.5 - / ICP47 - / GMCSF similar MOI IC 50 has been shown to cause a strong inhibition of tumor cell proliferation with values comparable to those of / GMCSF.

[0125] HSV-1 / ICP34.5 - / ICP47 - The in vitro bioactivity of FLT3L and IL12 as a result of HSV-1 / ICP34.5 / ICP47 / FLT3L / IL12 infection was evaluated. ELISA expression, IL12 reporter assay and FLT3L cell proliferation assay were repeated using the supernatants from virus-infected cells. The supernatants from A375 and Vero cells used to confirm replication were screened as previously described. IL12p70 ELISA confirmed the expression of IL12 from all cancer cell lines tested (VERO, A375 and SK-MEL-5) (Figure 12A). Furthermore, FLT3L ELISA demonstrated the expression of FLT3L from all cell lines tested (Figure 12B). The proof of IL12 bioactivity was determined using the previously described IL12 reporter assay and BaF3 cell line proliferation assay. The supernatants of virus-infected cells showed active IL12 in a dose-dependent manner in both SK-MEL-5 (Figure 13A) and A375 cells (Figure 13B). The proof of FLT3L bioactivity was demonstrated using BaF3 cell lines stimulated with supernatants from either the SK-MEL-5 (Figure 14A) or A375 (Figure 14B) cell line.

[0126] In all cases tested, the supernatants from virus-infected cells contained bioactive IL12 and FLT3L as predicted based on the genetic engineering specifications.

[0127] Example 5: HSV-1 / ICP34.5 - / ICP47 - The / mFLT3L / mIL12 virus can produce bioactive FLT3L and IL12 in vivo in the treatment of animals with B cell lymphoma tumors (A20 cell line). HSV-1 / ICP34.5 in the mouse A20 tumor model - / ICP47 - The expression of the dual cytokine payload encoded by / mFLT3L / mIL12 was evaluated.

[0128] A20 tumor cells (2×10 6 cells) were subcutaneously injected into the right flank of female BALB / c mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When the tumors reached an average of approximately 230 mm 3 , the animals were randomly assigned to five groups (4 mice per group) such that the average tumor volume and the variability in tumor volume at the start of treatment administration were uniform across the treatment groups. The mice were given a single intratumoral injection of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, HSV-1 / ICP34.5 - / ICP47 - / mGMCSF, HSV-1 / ICP34.5 - / ICP47 - / mFLT3L or HSV-1 / ICP34.5 - / ICP47 - / mIL12 (1×10 6 PFU per injection), respectively. Sixteen hours later, tumors and plasma were collected. The levels of mGM-CSF, mFLT3L, and mIL12 were measured in tumor lysates and plasma from each treatment group using an MSD assay (mGM-CSF and mIL12 (mIL-12 nucleic acid shown in SEQ ID NO: 15; mIL-12 amino acid shown in SEQ ID NO: 16)) or an R&D Quantikine ELISA (mFLT3L).

[0129] The results (Figure 15) show that a single intratumoral dose of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 results in the expression of both mFLT3L and mIL12 in A20 tumor lysates and plasma 16 hours later.

[0130] Example 6: HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 virus produces bioactive FLT3L and IL12 in vivo after treatment of animals bearing melanoma tumors (B16F10 cell line). HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 was evaluated for expression of the dual cytokine payload encoded by it.

[0131] B16F10-mNectin1 tumor cells (3×10 5 cells) were subcutaneously injected into the right flank of female C57Bl / 6 mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When the tumors reached an average of approximately 210 mm 3 , the animals were randomly assigned to 5 groups (4 mice per group) such that the average tumor volume and tumor volume variability at the start of treatment administration were uniform across the treatment groups. Mice were given a single intratumoral injection of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, HSV-1 / ICP34.5 - / ICP47 - / mGMCSF, HSV-1 / ICP34.5 - / ICP47 - / mFLT3L or HSV-1 / ICP34.5 - / ICP47 - / mIL12 (5×10 6 PFU per injection), respectively. Then, tumors and plasma were collected 16 hours later. Levels of mGM-CSF, mFLT3L, and mIL12 were measured in tumor lysates and plasma from each treatment group using an MSD assay (mGM-CSF and mIL12) or an R&D Quantikine ELISA (mFLT3L).

[0132] Results (Figure 16) show that HSV-1 / ICP34.5 - / ICP47 -A single intratumoral dose of / mFLT3L / mIL12 has been shown to result in the expression of both mFLT3L and mIL12 in A20 tumor lysates and plasma 16 hours later.

[0133] Example 7: HSV-1 / ICP34.5 - / ICP47 - The / mFLT3L / mIL12 virus induces a systemic anti-tumor immune response after intratumoral injection in vivo HSV-1 / ICP34.5 - / ICP47 - The systemic anti-tumor T cell response induced by treatment with / mFLT3L / mIL12 was evaluated.

[0134] A20 tumor cells (2×10 6 cells) were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When the tumors reached an average of approximately 100 mm 3 (day 11), the animals were randomly assigned to three groups (12 mice per group) such that the average tumor volume and tumor volume variability at the start of treatment administration were uniform across the treatment groups. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF (3×10 4 PFU / dose) or the preparation buffer was administered intratumorally (to the right side of the animals) on days 11, 14, and 17 of the study. The contralateral (left side of the animals) tumors received no injection. The study was terminated on day 21 and the spleens were harvested. Splenocytes were isolated from individual spleens and used in a whole cell ELISpot assay (CTL, Shaker Heights, OH) to measure the number of T cells that secrete mIFN-γ when mixed with A20 tumor cells. Briefly, 7.5×10 4 splenocytes were mixed with 1.5×10 4They were mixed with individual A20 tumor cells and incubated at 37 °C for 20 hours. The assay was read using a CTLS6 Fluorospot analyzer (CTL, Shaker Heights, OH), and IFN-γ+ spots were counted.

[0135] The results (Figure 17A) show that treatment with HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 resulted in a significant increase in systemic anti-A20 tumor activity compared to treatment with HSV-1 / ICP34.5 - / ICP47 - / mGMCSF (427 spots per 7.5×10 4 spleen cells vs. 152 spots, respectively; p = 0.0008). In addition to total tumor cells, EliSpot was performed using viral antigens identified in relation to the A20 cell line, AH1 (Figure 17B), and neoantigen mutations identified within the A20 cell line, UV Rag (Figure 17C).

[0136] Example 8: HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 induces antitumor efficacy in a syngeneic mouse B cell lymphoma tumor model (A20 cells) This study was designed to evaluate the tolerability and antitumor activity of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF in a contralateral mouse A20 tumor model.

[0137] A20 tumor cells (2×10 6 cells) were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using vernier calipers. When the tumors reached an average of approximately 100 mm 3Once that was reached, the animals were randomly assigned to six groups (10 mice per group) such that the average tumor volume and the variability of the tumor volume at the start of treatment administration were uniform across the treatment groups. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF (3×10 4 PFU / dose) or the preparation buffer control was administered into the tumor (on the right side of the animal) every three days for all three injections. The tumor on the contralateral side (left side of the animal) did not receive an injection. Clinical signs, body weight changes, and survival time (mice were excluded from the study when the tumor reached 800 mm 3 were measured twice a week until the end of the study).

[0138] All animals survived throughout the experiment and, as evident from the body weight, showed no evidence of harmful health effects related to the treatment and had no noted adverse clinical signs identified by the daily health monitoring examinations.

[0139] Inhibition of tumor growth was observed in both the treated group tumors (right side) and the untreated group tumors (left side) in both the HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF treatment groups (Figure 18). However, an increase in complete response was observed in the treated tumors (10 / 10 vs. 7 / 10) and contralateral tumors (5 / 10 vs. 2 / 10) in the HSV-1 / ICP34.5- / ICP47- / mFLT3L / mIL12-treated animals compared to the animals treated with HSV-1 / ICP34.5- / ICP47- / mGMCSF. The median survival was HSV-1 / ICP34.5 - / ICP47 - compared to HSV-1 / ICP34.5 - / ICP47 -Significantly increased in the / mFLT3L / mIL12 treatment group (53 days vs. 32 days respectively; p = 0.048).

[0140] These data show that HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment leads to improvement in contralateral tumor clearance and overall survival rate.

[0141] Example 9: HSV-1 / ICP34.5 in a mouse neuroblastoma (Neuro2A) tumor model - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF efficacy evaluation test This study was designed to evaluate the tolerance and antitumor activity of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF in a contralateral mouse Neuro2A tumor model.

[0142] Neuro2A tumor cells (1×10 6 cells) were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When the tumors reached approximately 100 mm 3 on average, the animals were randomly assigned to groups (10 mice per group) such that the average tumor volume (at both flanks) and the variation in tumor volume at the start of treatment administration were uniform across all treatment groups. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF (5×10 5 or 5×10 4The PFU / reagent or buffer control for preparation was administered into the tumor (on the right side of the animal) every three days for all three injections. The non-injected tumor (contralateral; on the left side of the animal) did not receive an injection. Clinical signs, body weight changes, and survival time (mice were excluded from the study when the tumor reached 800 mm 3 were measured twice a week until the end of the study.)

[0143] All animals survived throughout the experiment and showed no evidence of harmful health effects related to the treatment, as evident from their body weights, and there were no noted harmful clinical signs identified in the daily health monitoring examinations.

[0144] For 50 5 PFU per administration, both the HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment group and the HSV-1 / ICP34.5 - / ICP47 - / mGMCSF treatment group were statistically significant compared to the control treatment group animals. For 50 4 PFU per administration, compared to HSV-1 / ICP34.5 - / ICP47 - / mGMCSF, the overall survival rate of the HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment group increased (although the median survival time for both groups was 20 days; p = 0.0056).

[0145] These data indicate that the HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment results in an improvement in contralateral tumor clearance and overall survival rate.

[0146] Example 10: HSV-1 / ICP34.5 in a mouse neuroblastoma (CT26) tumor model - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / Test for evaluating the efficacy of mGMCSF This test is for HSV-1 / ICP34.5 in a contralateral mouse CT26 (also known as colon26) tumor model - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / Designed to evaluate the tolerance and antitumor activity of mGMCSF

[0147] CT26 tumor cells (3×10 5 cells) were subcutaneously injected into the right and left flanks of female BALB / c mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When the tumors reached an average of approximately 100 mm 3 , the animals were randomly assigned to groups (10 mice per group) such that the average tumor volume (in both flanks) and the variation in tumor volume at the start of treatment administration were uniform across the treatment groups. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF (5×10 6 PFU / dose) or the preparation buffer control was administered intratumorally (in the right flank of the animal) every three days for a total of three injections. The non-injected tumors (contralateral; left flank of the animal) were not injected. Clinical signs, weight changes, and survival time (mice were excluded from the study when the tumors reached 800 mm 3 ) were measured twice a week until the end of the study

[0148] All animals survived throughout the experiment and showed no evidence of harmful health effects related to the treatment, as evident from their weights, and there were no noted harmful clinical signs identified in the daily health monitoring examinations

[0149] At 5×10 6 PFU per dose, HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment group and HSV-1 / ICP34.5 - / ICP47 - / mGMCSF treatment group, the survival rates of both were significantly increased compared to the control treatment group animals (control vs. HSV-1 / ICP34.5 - / ICP47 - / mGMCSF; p = 0.0017 and control vs. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12; p = 0.0008). Furthermore, HSV-1 / ICP34.5 - / ICP47 - / mGMCSF compared to HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment group, the overall survival rate increased (HSV-1 / ICP34.5 - / ICP47 - / mGMCSF compared to HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, the median survival period was not defined; p = 0.0059). See Figure 20.

[0150] These data indicate that HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment results in improved contralateral tumor clearance and overall survival compared to either control treatment or HSV-1 / ICP34.5 - / ICP47 - / mGMCSF treatment.

[0151] Example 11: Evaluation of the efficacy of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 in combination with checkpoint inhibition (anti-PD1 mAb) efficacy in a mouse colorectal cancer (MC38) tumor model This study was conducted on the HSV-1 / ICP34.5 in a contralateral mouse MC38 tumor model- / ICP47 - Designed to evaluate the tolerance and antitumor activity when combined with / mFLT3L / mIL12 alone or with an anti-programmed cell death protein 1 (PD1) monoclonal antibody (mAb).

[0152] MC38 tumor cells (3×10 5 cells) were subcutaneously injected into the right and left flanks of female C57BL / 6 mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using a vernier caliper. When the tumors reached an average of approximately 100 mm 3 , the animals were randomly assigned to groups (10 mice per group) such that the average tumor volume (in both flanks) and the variation in tumor volume at the start of treatment administration were uniform across the treatment groups. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 (5×10 6 PFU / dose) or a preparation buffer control was administered into the tumor (on the right side of the animal) every 3 days for a total of 3 injections. The non-injected tumor (contralateral; on the left side of the animal) was not injected. The anti-PD1 monoclonal antibody (200 μg / dose) was administered by intraperitoneal injection on the same schedule (every 3 days for a total of 3 injections). Clinical signs, body weight changes, and survival time (mice were excluded from the study when the tumors reached 800 mm 3 ) were measured twice a week until the end of the study.

[0153] All animals survived throughout the experiment and showed no evidence of harmful health effects related to the treatment, as evident from their body weights, and there were no noted harmful clinical signs identified by daily health monitoring examinations.

[0154] Anti-PD1 mAb alone and 5×10 6 PFU of HSV-1 / ICP34.5 - / ICP47 -Single administration of either / mFLT3L / mIL12 alone demonstrated a significant increase in survival rate compared to control-treated animals (p < 0.0001 for each comparison). The survival rate of animals treated with anti-PD1 mAb alone was 6 5 × 10 - PFU of HSV-1 / ICP34.5 - / ICP47, which was not statistically significant compared to / mFLT3L / mIL12 alone (p = 0.246). The combination of both treatments, anti-PD1 mAb and 5 × 10 6 PFU of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, demonstrated a significant increase in survival rate compared to all other treatment groups (p = 0.0016 compared to / mFLT3L / mIL12 alone, p < 0.0001 compared to anti-PD1 mAb alone, and p < 0.0001 compared to control treatment). See Figure 21. 6 5 × 10 - PFU of HSV-1 / ICP34.5 - / ICP47

[0155] These data indicate that HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 or anti-PD1 mAb treatment alone results in a significant improvement in overall survival compared to control treatment, and the combination of both treatments results in a significant improvement in overall survival compared to either treatment alone.

[0156] Example 12: Study to Evaluate the Kinetics of Cytokine Expression by HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 in a Mouse Colorectal Cancer (CT26) Tumor Model This study was designed to evaluate the kinetics of cytokine expression by HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 when injected in a mouse CT26 tumor model.

[0157] CT26 tumor cells (3×10 5 cells) were subcutaneously injected into the right flank of female BALB / c mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using a digital caliper. When the tumors reached an average volume of approximately 100 mm 3 , the animals were randomly assigned to groups (5 mice per group for the control, 25 mice per group for HSV-1 / ICP34.5 - / ICP47 - and 25 mice per group for HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12). The average tumor volume and the variation in tumor volume at the start of treatment administration were uniform across all treatment groups. HSV-1 / ICP34.5 - / ICP47 - (5×10 6 PFU / virus per injection; virus without cytokine payload), HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 (5×10 6 PFU / virus per injection) and preparation buffer control were each administered intratumorally every 3 days for a total of 3 injections. Clinical signs and changes in body weight were measured twice a week until the end of the study. Five mice per virus treatment group were euthanized at 4, 24, 72, 168, and 240 hours after virus administration. Five mice per control treatment group were sacrificed immediately after preparation buffer control injection. Blood was isolated and prepared as serum, and tumors were excised from the animals and prepared as protein lysates.

[0158] All animals survived throughout the experiment and showed no evidence of treatment-related adverse health effects as evidenced by body weight and no noted adverse clinical signs identified by daily health monitoring examinations.

[0159] Serum and tumor protein lysates were analyzed for the virus HSV-1 / ICP34.5 - / ICP47- The presence of murine FLT3L and IL-12, two cytokines encoded by / mFLT3L / mIL12, was analyzed. To control for endogenous cytokine expression, a virus lacking the cytokines (HSV-1 / ICP34.5 - / ICP47 - ) was used.

[0160] In the tumor lysate, all animals injected with HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 showed expression of IL-12 in the tumor lysate on day 7 (168 hours) after injection. Two out of five animals showed expression of IL-12 on day 10 (240 hours) after injection (Figure 22A). All animals injected with either the control or HSV-1 / ICP34.5 - / ICP47 - virus had IL-12 levels below the limit of detection (LLOD). In plasma, IL-12 was detected 4 hours after injection in all five animals injected with HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12. Twenty-four hours after injection, four out of five animals injected with HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 had detectable IL-12. All time points sampled 24 hours later were below the LLOD (Figure 22B).

[0161] In the tumor lysate, all animals injected with HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 showed a statistically significant increase in the expression of FLT3L in the tumor lysate on day 3 (72 hours) after injection (HSV-1 / ICP34.5 - / ICP47 - vs. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, p = 0.0197; HSV-1 / ICP34.5 after 24 hours - / ICP47 - against HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, p = 0.0043, HSV-1 / ICP34.5 after 72 hours - / ICP47 - against HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, p = 0.0012; HSV-1 / ICP34.5 after 168 hours - / ICP47 - against HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, p = 0.2281; HSV-1 / ICP34.5 after 240 hours - / ICP47 - against HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, p = 0.4890; Figure 22C). In plasma, FLT3L was detectable in all samples from all mice in all groups. No statistically significant difference was found between any groups at any time point (Figure 22D).

[0162] In tumor lysates, HSV-1 / ICP34.5 - / ICP47 - and HSV-1 / ICP34.5 - / ICP47 - Only the animals injected with / mFLT3L / mIL12 showed a significant increase in the expression of IFN-γ in tumor lysates compared to the control 4 hours after injection (P = 0.0057). No detectable IFN-γ was present in the control-treated tumors 24, 72, 168, and 240 hours after injection. 24 hours after injection, the animals inoculated with HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 - / ICP47 -showed a significant increase in FN-γ levels compared to (p = 0.0253). At 72, 168, and 240 hours after injection, HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12, the levels of IFN-γ were higher in HSV-1 / ICP34.5 - / ICP47 - but did not reach statistical significance (p = 0.2306, 0.1155, and p = 0.0693; respectively; Figure 22E). The persistent IFN-γ production 24 hours after injection was consistent with the production of IL-12 and should initiate an enhanced anti-tumor immune response. In plasma, IFN-γ was not detected in animals treated with control injection. In HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and HSV-1 / ICP34.5 - / ICP47 - -treated animals, there was no statistical difference in plasma IFN-γ 4 hours after injection (p = 0.4803), but there was a significant increase 24 hours after injection (p = 0.0140) and IFN-γ was detected in HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 at 72 hours. For this assay, all other time points and conditions were below the limit of detection (LLOD) (Figure 22F).

[0163] Example 13: Test to evaluate the ability of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 to generate an anti-tumor T cell response In this test, the anti-tumor immune response generated by injection of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 was evaluated in a contralateral mouse MC38 tumor model.

[0164] MC38 tumor cells (3×10 5(number) were subcutaneously injected into the right and left flanks of female C57BL / 6 mice on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using an electronic caliper. Once the tumors reached an average of approximately 100 mm 3 , the animals were randomly assigned to groups (12 mice per group) such that the average tumor volume (in both flanks) and the variability in tumor volume at the start of treatment administration were uniform across the treatment groups. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 (5×10 6 PFU / dose) or the preparation buffer control was administered into the tumors (on the right side of the animals) every three days for all three injections. The non-injected tumors (contralateral; on the left side of the animals) were not injected. The anti-PD1 monoclonal antibody (200 μg / dose) was administered by intraperitoneal injection on the same schedule (every three days for all three injections). Clinical signs, body weight changes, and tumor volume were measured twice a week until the end of the 21-day study.

[0165] All animals survived throughout the experiment and showed no evidence of treatment-related adverse health effects as evident from their body weights and had no noted adverse clinical signs identified by daily health monitoring examinations.

[0166] On day 21, the mice were euthanized, the spleens were excised, and an IFN-γ ELISpot assay (peptide restimulation and whole cell) was performed on single cell suspensions of splenocytes. For the peptide restimulation assay, 5×10 5 splenocytes were plated and stimulated overnight with a single 9-mer peptide (MC38 neoantigen or virus-derived tumor antigen) at a final concentration of 1 μM. The whole cell assay was set up by plating 1.25×10 4 splenocytes together with 1.25×10 5 MC38 cells. In each assay, the counting of spots indicates the total number of immune cells expressing IFN-γ.

[0167] In the peptide restimulation assay, HSV-1 / ICP34.5 - / ICP47 - Treatment with mFLT3L / mIL12 alone resulted in a significant increase in immune responsiveness to MC38 tumor cells; in whole cell assays, HSV-1 / ICP34.5 - / ICP47 - Treatment with mFLT3L / mIL12 resulted in a significant increase in anti-MC38 activity compared to both control and anti-PD1 treated animals (p < 0.0001 for both; Figure 23A). The immune responsiveness to the viral tumor antigen P15E was also significantly increased in the HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment group (p = 0.0008; Figure 23B).

[0168] MC38 contains several genomic mutations that generate neoantigens. The immune responsiveness to these tumor-specific mutations was quantified. In HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treated animals, the responsiveness to Adpgk (Figure 23C), 2410127L17Rik (Figure 23D) and Aatf (Figure 23E) was significantly increased compared to control-treated mice (p = 0.003, p = 0.0416 and p = 0.0035, respectively). Furthermore, the combination of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 and anti-PD1 inhibition resulted in a significant increase in immune responsiveness to Adpgk (p = 0.002), Aatf (p = 0.040), Cpne1 (p = 0.030) and P15E (p = 0.0008) compared to HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 treatment alone. These data indicate that HSV-1 / ICP34.5 - / ICP47 -Treatment with / mFLT3L / mIL12 has been shown to increase the anti-tumor response in the MC38 tumor model. This increase can be further enhanced by the addition of anti-PD1. The generation and enhancement of a systemic anti-tumor response by checkpoint inhibition should contribute to anti-tumor immunity against both injected and non-injected lesions, as demonstrated in the efficacy studies described herein.

[0169] Example 14: HSV-1 / ICP34.5 in combination with a 4-1BB agonist mAb in a murine colorectal cancer (MC38) tumor model - / ICP47 - Study to evaluate the efficacy of / mFLT3L / mIL12 This study was designed to evaluate the tolerance and anti-tumor activity of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 alone or in combination with an agonist antibody targeting 4-1BB (aka CD137).

[0170] MC38 tumor cells (3×10 5 cells) were subcutaneously injected into the right and left flanks of female C57BL / 6 mice on day 0. Tumor volume (mm 3 ) was measured twice weekly (Q2W) using calipers. When the tumors reached an average of approximately 100 mm 3 , the animals were randomly assigned to groups (10 mice per group) such that the average tumor volume (in both flanks) and the variability in tumor volume at the start of treatment administration were uniform across the treatment groups. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 (5×10 6The PFU / replicate or buffer control for preparation was administered into the tumor (on the right side of the animal) every three days for all three injections. The non-injected tumor (contralateral; on the left side of the animal) did not receive an injection. The anti-4-1BB monoclonal antibody (150 μg / replicate) was administered by intraperitoneal injection on the same schedule (every three days for all three injections). Clinical signs, body weight changes, and survival time (mice were excluded from the study when the tumor reached 800 mm 3 were measured twice a week until the end of the study.)

[0171] All animals survived throughout the experiment and showed no evidence of harmful health effects related to the treatment, as was evident from their body weights, and there were no noted harmful clinical signs identified by daily health monitoring examinations.

[0172] Anti-4-1BB mAb alone and 5×10 6 PFU of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 alone both single treatments demonstrated a significant increase in survival rate compared to control-treated animals (p = 0.0048 and p < 0.0001 for each comparison respectively). The survival rate of animals treated with 5×10 6 PFU of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 was statistically significant compared to anti-4-1BB mAb alone (p = 0.0175). Both treatments, anti-4-1BB mAb and 5×10 6 PFU of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 in combination were shown to significantly increase the survival rate compared to all other treatment groups (p = 0.0246 compared to 5×10 6 PFU of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 alone, p = 0.0004 compared to anti-4-1BB mAb alone, and p < 0.0001 compared to control treatment). See Figure 24.

[0173] These data are from HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 or anti-4-1BB mAb treatment alone resulted in a significant improvement in overall survival compared to the control treatment, but the combination of both treatments showed a significant improvement in overall survival compared to either treatment alone.

[0174] Example 15: HSV-1 / ICP34.5 in combination with a bispecific T cell engager (BiTE®) molecule in a murine colorectal cancer (MC38) tumor model - / ICP47 - A study to evaluate the efficacy of / mFLT3L / mIL12 This study was designed to evaluate the tolerance and antitumor activity of HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 alone or in combination with a bispecific T cell engager (BiTE®) molecule in a murine MC38 tumor model overexpressing human epithelial cell adhesion molecule (EpCAM).

[0175] MC38 tumor cells (3×10 5 cells) recombinantly engineered to express human EpCAM were subcutaneously injected into the right and left flanks of female C57BL / 6 mice recombinantly engineered to express human CD3 from the endogenous murine CD3 locus on day 0. Tumor volume (mm 3 ) was measured twice a week (Q2W) using vernier calipers. When the tumors reached an average of approximately 100 mm 3 , the animals were randomly assigned to groups (10 mice per group) such that the average tumor volume (in both flanks) and the variability in tumor volume at the start of treatment administration were uniform across the treatment groups. HSV-1 / ICP34.5 - / ICP47 - / mFLT3L / mIL12 (5×10 6The PFU / well or preparation buffer control was administered intratumorally (on the right side of the animal) every three days for all three injections. The non-injected tumors (contralateral; left side of the animal) received no injection. The BiTE® molecule containing the anti-human CD3 and anti-human EpCAM binding domains (150 μg / kg) was administered by intravenous injection once a week for all two injections. Clinical signs, body weight changes, and survival (mice were excluded from the study when the tumor reached 800 mm 3 3) were measured twice a week until the end of the study.

Claims

1. 1. An oncolytic herpes simplex virus, comprising: The oncolytic herpes simplex virus lacks a functional gene encoding ICP34.5 and lacks a functional gene encoding ICP47; the oncolytic herpes simplex virus comprises a nucleic acid sequence encoding Fms-related tyrosine kinase 3 ligand (FLT3L), a nucleic acid sequence encoding interleukin 12 (IL12), and a nucleic acid sequence encoding a Kozak sequence; the nucleic acid sequence encoding FLT3L and the nucleic acid sequence encoding IL12 are both under the control of a cytomegalovirus (CMV) promoter; The nucleic acid sequence encoding FLT3L and the nucleic acid sequence encoding IL12 are linked by a polycistronic linker element, porcine tescho virus 2a (P2A). Oncolytic herpes simplex virus.

2. The oncolytic herpes simplex virus according to claim 1, which is herpes simplex virus type 1 (HSV-1).

3. 3. The oncolytic herpes simplex virus according to claim 1 or 2, which is a modified version of the JS1 strain deposited with the ECAAC under accession number 01010209.

4. The oncolytic herpes simplex virus according to any one of claims 1 to 3, wherein the nucleic acid sequence encoding IL12 and the nucleic acid sequence encoding FLT3L are present in the anterior region of the gene encoding ICP34.

5.

5. The oncolytic herpes simplex virus of any one of claims 1 to 4, further comprising a bovine growth hormone polyadenylation signal sequence (BGHpA).

6. The oncolytic herpes simplex virus of any one of claims 1 to 5, wherein the nucleic acid sequences encoding CMV, Kozak, Flt3L, P2A, IL12 and BGHpA are present as [CMV]-[Kozak]-[Flt3L]-[P2A]-[IL12]-[BGHpA].

7. The oncolytic herpes simplex virus according to any one of claims 1 to 6, wherein the IL12 exists as [P40 subunit]-[GGGGS]-[P35 subunit].

8. The oncolytic herpes simplex virus of claim 7, wherein there is no signal peptide in the IL12 P35 subunit.

9. FLT3L sequence comprising SEQ ID NO:1; and IL12 sequence comprising SEQ ID NO:7 The oncolytic herpes simplex virus according to any one of claims 1 to 8, comprising:

10. the oncolytic herpes simplex virus is HSV-1; The oncolytic herpes simplex virus is CMV promoter comprising SEQ ID NO:24; Kozak sequence comprising SEQ ID NO:20; FLT3L sequence comprising SEQ ID NO:1; P2A sequence comprising SEQ ID NO:17; IL12 sequence comprising SEQ ID NO:7; and BGHpA sequence comprising SEQ ID NO:21 The oncolytic herpes simplex virus according to any one of claims 1 to 8, comprising:

11. HSV1 / ICP34.5 － / ICP47 － 1. An oncolytic herpes simplex virus, HSV1 / ICP34.5 - / ICP47 - / FLT3L / IL12, comprising a modified HSV-1 genetically engineered from JS1 strain, wherein the modified HSV-1 lacks a functional ICP34.5 coding gene, lacks a functional ICP47 coding gene, and comprises the following inserted into the original site of the ICP34.5 gene: [CMV]-[Kozak]-[FIt3L]-[P2A]-[IL12(p40-GGGGS-no SP-p35)]-[BGHpA].

12. 12. An oncolytic herpes simplex virus according to any one of claims 1 to 11 for use in the manufacture of a medicament for the treatment of cancer.

13. A pharmaceutical composition for use in the treatment of cancer, comprising an oncolytic herpes simplex virus according to any one of claims 1 to 11.

14. The pharmaceutical composition of claim 13 for use in combination with a checkpoint inhibitor.

15. A kit comprising an oncolytic herpes simplex virus according to any one of claims 1 to 11.

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