Oncolytic viruses as therapeutic agents for treatment of cancer

Abstract

The present invention relates to compositions and methods for treating cancer using oncolytic viruses, components thereof, and / or derivatives thereof. In particular, the present invention relates to modified picornaviruses comprising changes in one or more of its capsid proteins that confer enhanced binding capacity to cancer cells.

Description

Technical Field

[0001] The present invention generally relates to the field of cancer treatment. More specifically, the present invention relates to compositions and methods for treating cancer using oncolytic viruses, their components and / or their derivatives. The compositions and methods may optionally include one or more additional anticancer agents or be combined with one or more anticancer treatments, including but not limited to immune checkpoint inhibitors, CAR-T cells, natural killer (NK) cells, chemotherapy and / or radiotherapy. Background Art

[0002] Viral oncolytic therapy is emerging as a promising treatment for many human and animal cancers.

[0003] The Picornaviridae family is one of the largest virus families, named after the Greek word "pico" (very small) and the "RNA" of their RNA genome. The family contains many clinically significant human and animal pathogens, including poliovirus, rhinovirus, and hepatitis A. According to the physical properties of the virions, RNA sequence similarity, and viral RNA genome structure, the Picornaviridae family is divided into nine genera (Stanway, G., et al., Molecular and Biological Basis of Picornavirus Taxonomy, in Molecular Biology of Picornaviruses, in B. Semler and E. Wimmer, Editors. 2002, ASM Press: Washington DC. p. 17-24). They are small, non-enveloped icosahedral viruses, and their capsids contain sixty copies of each of four viral proteins, VP1, VP2, VP3, and VP4, which form an icosahedral shell (approximately 300 angstroms in diameter) that accommodates the single-stranded positive-sense RNA genome. A unique feature of the capsid surface is a depression ("canyon") around the five-fold symmetry axis, which is the receptor binding site for many picornaviruses (including echoviruses) that utilize beta-integrins. The receptor molecules that bind in the canyon have been identified as members of the immunoglobulin superfamily, which, upon binding, dislodge factors from a pocket immediately below the canyon surface. When a receptor binds within the canyon, it depresses the canyon bottom, which corresponds to the top of the pocket. Similarly, when an antiviral compound or lipid binds to the pocket, it expands the pocket top, which corresponds to the canyon bottom. Thus, the receptors that bind the pocket factors and the canyon compete with each other for binding to the virus. The absence of the hydrophobic pocket factors destabilizes the virus and begins to transform into an altered form that is a precursor to virion uncoating. Examples of cellular receptors that bind to the canyon include immunoglobulin superfamily receptors (e.g., VCAM-1, ICAM-1, PVR, and CAR).

[0004] Many receptors bind to picornaviruses without using canyons. For example, a subgroup of human rhinoviruses (HRVs) bind to the low-density lipoprotein receptor family. Other picornaviruses, including certain coxsackieviruses and echoviruses, use decay-accelerating factor (DAF) as a cellular receptor, usually in combination with another receptor. DAF is a member of the complement activating protein family that binds to and accelerates the decay of the classical and alternative pathway C3 and C5 convertases (central amplifiers of the complement cascade). DAF is expressed on the surface of almost all cells and protects host cells from the immune system by rapidly dissociating any convertase assembled, thereby preventing the progression of complement attack against self cells. The functional region of DAF consists of four short common repeat sequences (SCR1-4), each containing approximately 60 residues and folded into a beta structure stabilized by disulfide bridges. The four SCR domains form a relatively rigid extended rod. Closely related picornaviruses have adapted to bind to DAF at different sites on the receptor surface.

[0005] Oncolytic virotherapy is based on the ability of viruses to infect and kill tumor cells without destroying normal tissue. Although some viruses have a natural preference for tumor cells, most require at least some modification of their receptor tropism to specifically enter cancer cells and replicate in them. The oncolytic picornavirus family has been successfully used to treat certain cancers. For example, Coxsackievirus A21 (CVA21) is a naturally occurring picornavirus that has the ability to preferentially infect and destroy malignant cells that carry the virus-cell entry receptor intercellular adhesion molecule-1 (ICAM-1). The efficacy of CVA21 against melanoma cells has been demonstrated in a range of preclinical xenograft models using immunodeficient mice, as well as against other forms of cancer. There are many other cell receptor targets that provide possible avenues for the design of effective oncolytic picornaviruses.

[0006] The recent emergence of immune checkpoint inhibitor therapy has changed cancer treatment in a wide range of tumor types. Effective and lasting clinical responses have been observed in refractory cancer histology. However, despite these promising long-term responses, many patients have no response to immune checkpoint blockade, showing primary drug resistance. In addition, many patients who initially responded to treatment experienced recurrence secondary to acquired drug resistance. Both primary and acquired drug resistance are the result of the complex and evolving interaction between the immune system and cancer cells (see, for example, Fares et al., American Society of Clinical Oncology Educational Book 39, May 17, 2019, 147-164).

[0007] There remains a need for improved compositions and methods for treating, mitigating, or preventing cancer, including cancer types that respond poorly to immune checkpoint inhibitor therapy. Summary of the Invention

[0008] The present inventors have generated modified Picornaviridae viruses that have an enhanced ability to bind decay accelerating factor (DAF / CD55) on the surface of cancer cells compared to their wild-type counterparts. These have been shown to be effective oncolytic agents against cancer cells.

[0009] Without limitation to any particular form of cancer, the compositions and methods disclosed herein are effective in treating, alleviating, and / or preventing cancer, including cancer types that are poorly responsive to immune checkpoint therapy.

[0010] The compositions and methods described herein can be used in combination with other anti-cancer agents or treatments, including but not limited to immune checkpoint inhibitors / immune checkpoint inhibitor therapy, CAR-T cells / CAR-T cell therapy, natural killer (NK) cells, chemotherapeutic agents / chemotherapy, radiotherapeutic agents / radiotherapy, and the like.

[0011] Without limitation, the present invention relates to at least the following embodiments 1-71:

[0012] Implementation Plan 1 .A modified picornavirus comprising changes in any one or more of the capsid proteins VP1, VP2, and VP3 compared to a wild-type strain of the virus; the changes confer enhanced decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn) binding ability compared to the wild-type strain.

[0013] Implementation Plan 2 . The modified picornavirus according to embodiment 1, comprising said changes in each of the VP2 and VP3 capsid proteins.

[0014] Implementation Plan 3 . The modified picornavirus according to embodiment 1, comprising said changes in each of the VP1, VP2 and VP3 capsid proteins.

[0015] Implementation Plan 4 .The modified picornavirus according to any one of embodiments 1-3, which further comprises a change in any one or more of the non-structural proteins 2A, 3A, 3C, 3D compared to the wild-type strain of the virus; compared to the wild-type strain, the change confers enhanced decay accelerating factor (DAF / CD55) binding ability.

[0016] Implementation Plan 5The modified picornavirus of any one of embodiments 1-4, wherein the wild-type strain and the modified picornavirus have the same nucleotide sequence and / or the same amino acid sequence, except for the following:

[0017] (i) changes in any one or more of the capsid proteins VP1, VP2, VP3; and optionally

[0018] (ii) changes in any one or more of the nonstructural proteins 2A, 3A, 3C, 3D.

[0019] Implementation Plan 6 . The modified picornavirus of any one of embodiments 1-5, wherein the modified picornavirus is an enterovirus.

[0020] Implementation Plan 7 .The modified picornavirus of embodiment 6, wherein the enterovirus is selected from the group consisting of an echovirus, a poliovirus, an unclassified enterovirus, a rhinovirus, a double echovirus, a hepatovirus, and a cardiovirus.

[0021] Implementation Plan 8 . The modified picornavirus of any one of embodiments 1-7, wherein the modified picornavirus is not a coxsackievirus.

[0022] Implementation Plan 9 . The modified picornavirus of any one of embodiments 1-8, wherein the modified picornavirus is an echovirus, enterovirus B85, coxsackievirus A9, coxsackievirus A13, coxsackievirus 15, or coxsackievirus 21.

[0023] Implementation Plan 10 . The modified picornavirus of any one of embodiments 1-9, wherein the modified picornavirus is echovirus 1, echovirus 3, echovirus 6, echovirus 7, echovirus 9, echovirus 11, echovirus 12 (E12), echovirus 12, echovirus 13, echovirus 14, echovirus 15, echovirus 17, echovirus 25, echovirus 26, echovirus 29, or echovirus 30.

[0024] Implementation Plan 11 . The modified picornavirus according to any one of embodiments 1-10, wherein the change in capsid protein VP2 comprises or consists of an asparagine to threonine change at residue 142.

[0025] Implementation Plan 12. The modified picornavirus according to any one of embodiments 1-11, wherein the change in capsid protein VP3 comprises or consists of an alanine to valine change at residue 206.

[0026] Implementation Plan 13 . The modified picornavirus according to any one of embodiments 1-12, wherein the changes in the capsid protein VP2 comprise or consist of a histidine to tyrosine change at residue 154 and / or a serine to asparagine change at residue 168.

[0027] Implementation Plan 14 . The modified picornavirus according to any one of embodiments 1-13, wherein the change in capsid protein VP1 comprises or consists of a tyrosine to histidine change at residue 230.

[0028] Implementation Plan 15 . The modified picornavirus according to any one of embodiments 1-14, wherein the change in capsid protein VP1 comprises or consists of a phenylalanine to tyrosine change at residue 210.

[0029] Implementation Plan 16 . The modified picornavirus according to any one of embodiments 1-15, wherein the change in capsid protein VP1 comprises or consists of a glutamine to arginine change at residue 132.

[0030] Implementation Plan 17 .A modified picornavirus according to embodiment 16, wherein the changes confer enhanced neonatal Fc receptor (FcRn) binding.

[0031] Implementation Plan 18 The modified picornavirus of any one of embodiments 1-17, wherein the changes in nonstructural proteins 2A, 3A, 3C, 3D comprise or consist of any one or more of the following:

[0032] - a phenylalanine to tyrosine change at residue 47 of the nonstructural 2A protein;

[0033] - an isoleucine to tyrosine change at residue 6 of the nonstructural 3A protein;

[0034] - a histidine to arginine change at residue 39 of the nonstructural 3C protein;

[0035] - Isoleucine to leucine change at residue 123 of the unstructured 3D protein.

[0036] Implementation Plan 19. The modified picornavirus of any one of embodiments 1-18, wherein the wild-type strain is Echovirus 12 strain Travis.

[0037] Implementation Plan 20 . The modified picornavirus according to any one of embodiments 1-19, wherein the wild-type strain is Echovirus 12 strain Travis, which comprises or consists of the amino acid sequence as defined in SEQ ID NO:46.

[0038] Implementation Plan 21 .The modified picornavirus according to any one of embodiments 1-20, further comprising at least one nucleic acid sequence encoding an exogenous protein or a component thereof.

[0039] Implementation Plan 22 .A modified picornavirus according to embodiment 21, wherein the exogenous protein is an immunostimulatory protein or an agent (such as an antibody) that can bind to an immune checkpoint molecule or a ligand of an immune checkpoint molecule and inhibit its biological activity.

[0040] Implementation Plan 23 .A modified picornavirus according to embodiment 22, wherein the immunostimulatory protein is selected from any one or more of the following: interleukins, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, interferons, including but not limited to IFN-γ, TNF-α, prodrug converting enzymes, biologically active components thereof, and combinations thereof.

[0041] Implementation Plan 24 .The modified picornavirus according to embodiment 22, wherein the immune checkpoint molecule is selected from any one or more of the following: PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, LAG-3, ICOS, TIM3, GAL9, CD28, OX-40 and TIGIT, their biologically active components, and combinations thereof.

[0042] Implementation Plan 25 .The modified picornavirus according to any one of embodiments 1-24, further comprising one or more components for expressing tissue-specific miRNA, wherein the miRNA is capable of inhibiting the replication of the modified picornavirus in a tissue-specific manner.

[0043] Implementation Plan 26. The modified picornavirus of any one of embodiments 1-25, comprising enhanced oncolytic activity compared to a wild-type strain.

[0044] Implementation Plan 27 .The modified picornavirus according to embodiment 26, wherein the enhanced oncolytic activity is directed against any one or more of ovarian cancer cells, colorectal cancer cells, gastric cancer cells, liver cancer cells, pancreatic cancer cells, head and neck cancer cells, gastric cancer cells, breast cancer cells, sarcoma cells, lymphoma cells, and brain cancer cells.

[0045] Implementation Plan 28 .A modified picornavirus according to embodiment 27, wherein the cancer cell expresses decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

[0046] Implementation Plan 29 The modified picornavirus of any one of embodiments 1-28, wherein the picornavirus comprises:

[0047] (i) a VP1 capsid protein amino acid sequence comprising any one of SEQ ID NOs: 54-57, 74, 86, 98, 110, 122, 134, 146 or 158;

[0048] (ii) a VP2 capsid protein amino acid sequence comprising any one of SEQ ID NO: 58-61, 75, 87, 99, 111, 123, 135, 147 or 159;

[0049] (iii) a VP3 capsid protein amino acid sequence comprising any one of SEQ ID NO: 62, 63, 76, 88, 100, 112, 124, 136, 148 or 160;

[0050] (iv) a nonstructural protein 2A sequence comprising any one of SEQ ID NO: 64, 65, 77, 89, 101, 113, 125, 137, 149, or 161;

[0051] (v) a nonstructural protein 3A sequence comprising any one of SEQ ID NO: 66, 67, 78, 90, 102, 114, 126, 138, 150, or 162;

[0052] (vi) a nonstructural protein 3C sequence comprising any one of SEQ ID NO: 68, 69, 79, 91, 103, 115, 127, 139, 151, or 163;

[0053] (vii) a nonstructural protein 3D sequence comprising any one of SEQ ID NO: 70, 71, 80, 92, 104, 116, 128, 140, 152 or 164.

[0054] Implementation Plan 30 . The modified picornavirus according to any one of embodiments 1-29, comprising or consisting of an amino acid sequence as defined in SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:73, SEQ ID NO:85, SEQ ID NO:97, SEQ ID NO:109, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:145, or SEQ ID NO:157, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:73, SEQ ID NO:85, SEQ ID NO:97, SEQ ID NO:109, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:145, or SEQ ID NO:157.

[0055] Implementation Plan 31 The modified picornavirus according to any one of embodiments 1-30, which is encoded by a nucleotide sequence comprising an RNA or cDNA / DNA sequence defined in SEQ ID NO:41, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:72, SEQ ID NO:84, SEQ ID NO:96, SEQ ID NO:108, SEQ ID NO:120, SEQ ID NO:132, SEQ ID NO:144 or SEQ ID NO:156, or a combination thereof with an RNA or cDNA / DNA sequence defined in SEQ ID NO:41, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:72, SEQ ID NO:84, SEQ ID NO:96, SEQ ID NO:108, SEQ ID NO:120, SEQ ID NO:132, SEQ ID NO:144 or SEQ ID NO:156. A variant of the RNA or cDNA / DNA sequence defined in NO: 156 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, or consisting of said RNA or cDNA / DNA sequence or said variant.

[0056] Implementation Plan 32 . The modified picornavirus according to any one of embodiments 1-31, which is synthesized using recombinant methods.

[0057] Implementation Plan 33 A pharmaceutical composition comprising any one or more of the following:

[0058] - the modified picornavirus according to any one of embodiments 1 to 32,

[0059] - the modified picornavirus RNA according to any one of embodiments 1 to 32,

[0060] - a complementary DNA (cDNA) encoding all or part of the genome of the modified picornavirus according to any one of embodiments 1-32;

[0061] and a pharmaceutically acceptable carrier, excipient or diluent.

[0062] Implementation Plan 34 .A pharmaceutical composition according to embodiment 33, wherein the RNA of the modified picornavirus (e.g., synthetic RNA) and / or complementary DNA (cDNA) encoding all or a portion of the genome of the modified picornavirus is provided within a nanoparticle (e.g., a lipid nanoparticle).

[0063] Implementation Plan 35 .A pharmaceutical composition according to embodiment 33 or 34, further comprising CAR-T cells, natural killer (NK) cells, immunostimulatory proteins and / or agents (e.g., antibodies) capable of binding to immune checkpoint molecules or ligands of immune checkpoint molecules.

[0064] Implementation Plan 36 .A pharmaceutical composition according to embodiment 35, wherein the CAR-T cells are engineered to bind to any one or more of the following on the surface of cancer cells: EGFRVIII, interleukin 13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Muc1, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Muc1, EphA2, CD123, CD19, Claudin 18.2.

[0065] Implementation Plan 37.A pharmaceutical composition according to embodiment 35 or 36, wherein the immunostimulatory protein is selected from any one or more of the following: interleukins, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, interferons, including but not limited to IFN-γ, TNF-α, prodrug converting enzymes, biologically active components thereof, and combinations thereof.

[0066] Implementation Plan 38 .A pharmaceutical composition according to any one of embodiments 35-37, wherein the immune checkpoint molecule is selected from any one or more of the following: PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, LAG-3, ICOS, TIM3, GAL9, CD28, OX-40 and TIGIT, their biologically active components, and combinations thereof.

[0067] Implementation Plan 39 . A pharmaceutical composition according to any one of embodiments 33-38, wherein the composition comprises about 10 8 to about 10 15 Virus particles / mL, about 10 8 to 10 12 virus particles / mL or about 10 8 to 10 10 Virus particles / mL of picornavirus.

[0068] Implementation Plan 40 .A pharmaceutical composition according to any of embodiments 33-39, wherein the composition comprises a particle to infectivity ratio of less than or equal to about 3000:1, less than or equal to about 300:1, in the range of about 200:1 to about 5:1, in the range of about 50:1 to about 10:1, or less than about 20:1.

[0069] Implementation Plan 41 .A pharmaceutical composition according to any one of embodiments 33-40, wherein the composition comprises a host cell DNA level of less than or equal to about 200 ng / mL, in a range of 1 to about 100 ng / mL, in a range of about 1 to about 50 ng / mL, or in a range of about 1 to about 10 ng / mL.

[0070] Implementation Plan 42 A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of any one or more of:

[0071] - the modified picornavirus according to any one of embodiments 1-32;

[0072] - the modified picornavirus RNA according to any one of embodiments 1-32;

[0073] - a complementary DNA (cDNA) encoding all or part of the genome of the modified picornavirus according to any one of embodiments 1-32;

[0074] - The pharmaceutical composition according to any one of embodiments 33-41.

[0075] Implementation Plan 43 .The method of embodiment 42, wherein the modified picornavirus is bioselective for any one or more of the following: enhanced migration between cancer cells, enhanced binding to DAF and / or FcRn, enhanced replication capacity at mammalian body temperature (33°C–39°C), and enhanced lytic activity against cancer cells expressing any one or more of PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, ICOS, TIM3, LAG-3, GAL9, CD28, and / or OX-40.

[0076] Implementation Plan 44 .A method according to embodiment 43, wherein the modified picornavirus is bioselected in vivo using tumor xenografts.

[0077] Implementation Plan 45 .A method according to embodiment 44, wherein the tumor is from the subject.

[0078] Implementation Plan 46 .A method according to any one of embodiments 42-45, wherein the therapeutically effective amount is administered to the subject by parenteral, intravenous, intravesical, subcutaneous, oral, intratumoral, ophthalmic, topical or systemic administration.

[0079] Implementation Plan 47 .A method according to any one of embodiments 42-46, wherein the therapeutically effective amount is co-administered with CAR-T cells and / or natural killer (NK) cells.

[0080] Implementation Plan 48.A method according to embodiment 47, wherein the CAR-T cells are engineered to bind to any one or more of the following on the surface of cancer cells: EGFRVIII, interleukin 13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Muc1, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Muc1, EphA2, CD123, CD19, Claudin 18.2.

[0081] Implementation Plan 49 .A method according to embodiment 47 or 48, wherein the CAR-T cells and / or natural killer (NK) cells are co-administered before or after administration of the therapeutically effective amount to the subject.

[0082] Implementation Plan 50 .A method according to any one of embodiments 42-49, wherein the therapeutically effective amount is co-administered with an immunostimulatory protein and / or an agent (e.g., an antibody) that is capable of binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule.

[0083] Implementation Plan 51 .A method according to embodiment 50, wherein the immunostimulatory protein is selected from any one or more of the following: interleukins, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, interferons, including but not limited to IFN-γ, TNF-α, prodrug converting enzymes, biologically active components thereof, and combinations thereof.

[0084] Implementation Plan 52 .The method according to embodiment 50 or 51, wherein the immune checkpoint molecule is selected from: PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, ICOS, LAG-3, TIM3, GAL9, CD28, OX-40 and TIGIT.

[0085] Implementation Plan 53 .A method according to any one of embodiments 50-52, wherein the agent is a monoclonal antibody.

[0086] Implementation Plan 54 .A method according to any one of embodiments 50-53, wherein the immunostimulatory protein and / or agent is co-administered before or after administration of the therapeutically effective amount to the subject.

[0087] Implementation Plan 55 .A method according to any one of embodiments 42-54, wherein the cancer is classified as a cancer resistant to immune checkpoint therapy.

[0088] Implementation Plan 56 .A method according to any one of embodiments 42-55, wherein the cancer is ovarian cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, head and neck cancer, stomach cancer, breast cancer, sarcoma, lymphoma or brain cancer.

[0089] Implementation Plan 57 .A method according to embodiment 56, wherein the cancer comprises cancer cells expressing decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

[0090] Implementation Plan 58 Use of any one or more of the following in the preparation of a medicament for treating cancer in a subject:

[0091] - the modified picornavirus according to any one of embodiments 1-32;

[0092] - the RNA of the picornavirus according to any one of embodiments 1-32;

[0093] - a complementary DNA (cDNA) encoding all or part of the genome of the picornavirus according to any one of embodiments 1-32;

[0094] - The pharmaceutical composition according to any one of embodiments 33-41.

[0095] Implementation Plan 59 .The use according to embodiment 58, wherein the drug further comprises CAR-T cells, natural killer (NK) cells, immunostimulatory proteins and / or agents (such as antibodies) that can bind to immune checkpoint molecules or ligands of immune checkpoint molecules.

[0096] Implementation Plan 60 . The modified picornavirus according to any one of embodiments 1-32, the RNA of the picornavirus according to any one of embodiments 1-32, the complementary DNA (cDNA) encoding all or part of the genome of the picornavirus according to any one of embodiments 1-32, and / or the pharmaceutical composition according to any one of embodiments 33-41, for use in treating cancer.

[0097] Implementation Plan 61.The modified picornavirus, RNA and / or cDNA according to embodiment 60, for use in combination with CAR-T cells and / or natural killer (NK) cells and / or immunostimulatory agents in the treatment of cancer.

[0098] Implementation Plan 62 .The use according to embodiment 59 or the modified picornavirus, RNA and / or cDNA according to embodiment 61, wherein the CAR-T cell is engineered to bind to any one or more of the following on the surface of cancer cells: EGFRVIII, interleukin 13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Muc1, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Muc1, EphA2, CD123, CD19, Claudin18.2.

[0099] Implementation Plan 63 .The use according to embodiment 59 or 62 or the modified picornavirus, RNA and / or cDNA according to embodiment 60 or 61, wherein the immunostimulatory protein is selected from any one or more of the following: interleukins, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, interferons, including but not limited to IFN-γ, TNF-α, prodrug converting enzymes, biologically active components thereof, and combinations thereof.

[0100] Implementation Plan 64 .The use according to embodiment 63 or the modified picornavirus, RNA and / or cDNA according to embodiment 84, wherein the immune checkpoint molecule is selected from: PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, LAG-3, B7RP1, ICOS, TIM3, GAL9, CD28, OX-40 and TIGIT.

[0101] Implementation Plan 65The use of any one of embodiments 58, 59, or 62-64, or the modified picornavirus, RNA, and / or cDNA of any one of embodiments 60-64, wherein the modified picornavirus is bioselected for any one or more of: enhanced migration between cancer cells, enhanced binding to DAF and / or FcRn, enhanced replication capacity at mammalian body temperature (37°C-39°C), enhanced lytic activity against cancer cells expressing any one or more of PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, LAG-3, ICOS, TIM3, GAL9, CD28, and / or OX-40.

[0102] Implementation Plan 66 The use according to any one of embodiments 58, 59, or 62-65, or the modified picornavirus, RNA and / or cDNA according to any one of embodiments 60-65, wherein the modified picornavirus is bioselected in vivo using tumor xenografts.

[0103] Implementation Plan 67 .The use according to embodiment 66 or the modified picornavirus, RNA and / or cDNA according to embodiment 66, wherein the tumor is from the subject.

[0104] Implementation Plan 68 The use of any one of embodiments 58, 59, or 62-67, or the modified picornavirus, RNA, and / or cDNA of any one of embodiments 60-67, wherein the cancer is ovarian cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, head and neck cancer, stomach cancer, breast cancer, sarcoma, lymphoma, or brain cancer.

[0105] Implementation Plan 69 .The use according to embodiment 68 or the modified picornavirus, RNA and / or cDNA according to embodiment 68, wherein the cancer comprises cancer cells expressing decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

[0106] Implementation Plan 70The use according to any one of embodiments 58, 59 or 62-69, or the modified picornavirus, RNA and / or cDNA according to any one of embodiments 60-69, wherein the modified picornavirus comprises an amino acid sequence as defined in SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 73, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 109, SEQ ID NO: 121, SEQ ID NO: 133, SEQ ID NO: 145 or SEQ ID NO: 157, or a combination thereof with SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 73, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 109, SEQ ID NO: 121, SEQ ID NO: 133, SEQ ID NO: 145 or SEQ ID NO: 157. NO:157 comprises or consists of a variant having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0107] Implementation Plan 71 The use according to any one of embodiments 58, 59 or 62-70, or the modified picornavirus, RNA and / or cDNA according to any one of embodiments 60-70, wherein the modified picornavirus is encoded by a nucleotide sequence comprising an RNA or cDNA / DNA sequence as defined in SEQ ID NO: 41, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 144 or SEQ ID NO: 156, or a combination thereof with an RNA or cDNA / DNA sequence as defined in SEQ ID NO: 41, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 144 or SEQ ID NO: 156. A variant of the RNA or cDNA / DNA sequence defined in NO: 156 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, or consisting of said RNA or cDNA / DNA sequence or said variant.

[0108] definition

[0109] The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and do not limit the scope of the present invention to only these terms, but are provided for a better understanding of the following description.

[0110] As used in this application, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. For example, the term "rhinovirus" includes a variety of rhinoviruses and / or modified forms thereof. Therefore, in the context of this application, the singular also includes the plural, unless the particular context clearly indicates otherwise. For example, when stating that the present invention includes a method for treating cancer by administering an oncolytic virus or an oncolytic viral RNA, it will be understood that this includes administering one or more such viruses or viral RNAs. Similarly, unless the context requires otherwise or clearly indicates otherwise, integers, steps or elements of the invention recorded herein as singular integers, steps or elements clearly include both the singular and plural forms of the integers, steps or elements.

[0111] As used herein, the addition of "(s)" to a given term will be understood to convey the option of singular or plural. For example, "change(s)" will be understood to include a single change or a plurality of changes.

[0112] In the context of this application, when a numerical range is provided, it is to be understood that it includes the endpoints stated in the range and all values ​​between these endpoints, including any sub-ranges within these endpoints.

[0113] As used herein, the term "comprising" is intended to mean "including" in a non-exhaustive sense. Variations of the word "comprising," such as "comprise" and "comprises," have correspondingly varying meanings. Thus, for example, a composition "comprising" a picornavirus may consist solely of the picornavirus, or may include one or more additional components (e.g., a pharmaceutically acceptable excipient, carrier, or diluent).

[0114] As used herein, the term "therapeutically effective amount" includes within its meaning a non-toxic but sufficient amount of one or more given agents of the present invention (e.g., oncolytic picornaviruses or viral RNA from oncolytic picornaviruses) to provide the desired therapeutic effect. The exact amount of the required agent will vary with the subject, depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the specific agent being administered, and the mode of administration. Therefore, it is impossible to specify an accurate "effective amount." However, for any given situation, a suitable "effective amount" can be determined by one of ordinary skill in the art using only routine experiments.

[0115] As used herein, a nucleic acid or nucleic acid sequence "derived from" a picornavirus will be understood to include viral RNA isolated directly from a picornavirus, synthetic viral RNA, and cDNA encoding the viral genome or components thereof corresponding to the isolated sequence. Also included are synthetic polynucleotide sequences that contain one or more mutations in the sequence compared to the wild-type or parental sequence, including, for example, mutations in the capsid protein.

[0116] As used herein, "wild-type" strains, viruses, sequences or organisms will be understood as strains, viruses, sequences or organisms that exist in nature or that are isolated from naturally occurring sources, and also include forms that are artificially synthesized but otherwise identical to those that exist in nature. In some embodiments, they can represent the forms most commonly found in natural populations. The wild-type strains, viruses, sequences or organisms mentioned herein can be used as basic strains, viruses, sequences or organisms into which variations are introduced. Therefore, they can be used as references when comparing modified strains, viruses, sequences or organisms described herein.

[0117] As used herein, the term "polynucleotide" refers to a single- or double-stranded polymer of deoxyribonucleotides, ribonucleotide bases or known analogs or natural nucleotides, or mixtures thereof.

[0118] As used herein, the term "treatment" and related terms, such as "treating," "treated," and "treat" refer to any and all uses that ameliorate or alleviate a disease state or symptoms, prevent the occurrence of a disease, or otherwise prevent, hinder, slow, or reverse the progression of a disease or other unwanted symptoms in any way. To avoid misunderstanding, it is noted that "treatment" and related terms as used herein do not require a complete cure or alleviation of the disease being treated.

[0119] As used herein, the term "subject" or "patient" includes humans and individuals of any species of social, economic, or research importance, including but not limited to members of the genera ovine, bovine, equine, porcine, feline, canine, primate, and rodent. The subject or patient can be a mammal, such as a human.

[0120] As used herein, the term "test kit" refers to any delivery system for delivering materials. Such a delivery system includes a system that allows storage, transportation or delivery of reaction reagents (e.g., labels, reference samples, support materials, etc. in suitable containers) and / or support materials (e.g., buffer, written instructions for performing assays, etc.) from one location to another. For example, a test kit may include one or more accessories, such as a box, which contains relevant reaction reagents and / or support materials. The term "test kit" includes two types of test kits and combination test kits. A "test kit" refers to a delivery system comprising two or more separate containers, each of which contains a sub-portion of all test kit components. The containers can be delivered to the intended recipient together or separately. Any delivery system comprising two or more separate containers each containing a sub-portion of all test kit components is included in the meaning of the term "test kit." A "combination test kit" refers to a delivery system containing all components of a reaction assay (e.g., in a single box containing each required component) in a single container.

[0121] Any description of prior art documents herein or statements herein derived from or based upon these documents is not an admission that the documents or derived statements are part of the common general knowledge in the relevant art.

[0122] For the purposes of this description, all documents mentioned herein are incorporated by reference in their entirety unless otherwise indicated. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Non-limiting embodiments of the present invention are described below, by way of example only, with reference to the accompanying drawings, in which:

[0124] Figure 1 Depicted are novel capsid changes in the DAF-binding footprint that enhance the oncolytic activity of the IVX037 strain of the present invention compared to the prototype strain. Residues are colored according to their contribution to the total contact area with DAF: yellow (<5%), orange (<9%), and red (>9%).

[0125] Figure 2 Shown is the three-dimensional structure of the modified echovirus 12 (E12) IVX037 strain according to an embodiment of the present invention (front view).

[0126] Figure 3 The three-dimensional structure of the modified E12 IVX037 strain according to an embodiment of the present invention is shown (side view).

[0127] Figure 4 A series of graphs (AC) are provided comparing the oncolytic activity of modified E12 strains according to embodiments of the present invention with other pre-existing E12 strains.

[0128] Figure 5It was shown that the IVX 037 strain according to an embodiment of the present invention induced upregulation of DDX58 (RIG-I), CD274 (PD-L1), and IFN-g inducible protein 10 (CXCL10) and PD-L1 in in vitro cultures of human MSS-colorectal and ovarian epithelial adenocarcinoma cells 10 hours after infection.

[0129] Figure 6 The in vivo anti-tumor and oncolytic activities of the intratumoral IVX 037 strain according to an embodiment of the present invention were shown in human MSS-colorectal cancer (WiDr) xenografts.

[0130] Figure 7 The IVX 037 strain according to an embodiment of the present invention was shown to induce upregulation of DDX58 (RIG-I), CD274 (PD-L1), and IFN-g inducible protein 10 (CXCL10) and PD-L1 in human MSS-colorectal cancer (WiDr) xenografts.

[0131] Figure 8 Shown are infectivity data for the IVX-037OVO strain according to an embodiment of the present invention on CHO cells transfected with human FcRn using forward (A) and reverse (B) transfection methods.

[0132] Figure 9 Shown are the infectivity data of the IVX-037OVO strain according to an embodiment of the present invention on CHO cells transfected with a human FcRn plasmid or a pIND control plasmid (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0133] Figure 10 Shown are the mean (A) and individual (B) tumor volumes of mice treated with the IVX037 OVO strain of the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0134] Figure 11 Shown are the mean (A) and individual (B) tumor volumes of mice treated with IVX-037 OVO strain according to an embodiment of the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0135] Figure 12 Individual tumor volumes of mice treated with the IVX037 strain of the present invention are shown.

[0136] Figure 13Shown are the mean (A) and individual (B) tumor volumes of mice treated with IVX-037 OVO strain according to an embodiment of the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0137] Figure 14 IVX037 was shown to infect and replicate in human colorectal cancer xenografts, resulting in tumor growth inhibition. (A) A single injection of IVX037 (1x10 8 TCID 50 ) were administered intratumorally (it) into WiDr tumor xenografts. (B) Increasing doses of IVX037 (1×10 4 , 1×10 6 , 1×10 8 TCID 50 ) was injected intratumorally into WiDr tumor xenografts. (C) Mice bearing WiDr tumor xenografts were treated with four intravenous (iv) infusions of IVX037 spaced 3 to 4 days apart. (D) A single injection of IVX037 (1x10 8 TCID 50 ) were administered intratumorally into Caco-2 tumor xenografts. Tumor volumes are expressed as mean ± SD (mm 3 ). ****P<0.0001.

[0138] Figure 15 Expression of RIG-I, CD274, and CXCL10 was measured 1, 2, and 3 days after treatment. SCID mice were inoculated with WiDr cells. When tumors reached an average size (approximately 50 mm 3 ), mice were treated with virus IVX037 (n = 8 mice) or control formulation buffer (n = 6 mice) on day 0. Mice were sacrificed 1, 2, and 3 days after treatment. Tumors were harvested and RNA was extracted. Expression of RIG-I, CD274, and CXCL10 was measured using RT-PCR and calculated as fold change.

[0139] Figure 16 (A) Patient serum levels of IVX037 viral RNA. IVX037 viral levels were measured using semi-quantitative RT-qPCR. (B) Patient serum levels of neutralizing antibodies (nAbs) after IVX037 administration. (C) Patient serum CXCL10 levels. CXCL10 levels were measured using a multiplex flow cytometry assay. Statistical analysis used the paired sample "t-test" technique.

[0140] Figure 17 Phase 1a / b clinical protocol is shown.

[0141] Figure 18 CXCL10 serum levels in the patients studied are shown. P values ​​were calculated by T-test using Graphpad Prism. CXCL10 is expressed as pg / mL.

[0142] Figure 19 Dose-response curves of IVX037 are shown on a panel of susceptible hepatocellular carcinoma cell lines. Cell death induced by dose-dependent IVX037 infection is plotted as percentage of cell survival. Analyzed using GraphPad Prism, the normalized dose response (variable slope) was used as the best fit curve. Cell survival data were generated using the XTT cell viability assay. IVX037 dose was defined as MOI, as TCID 50 / cell representation. Micrographs are shown demonstrating the cytopathic effect of IVX037 on these cell lines. DETAILED DESCRIPTION

[0143] The invention will now be described in greater detail, including by way of illustration only with respect to the following Examples.

[0144] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those explicitly described. It will be understood that the invention encompasses all such variations and modifications. The invention also encompasses all steps, features, compositions, and compounds referenced or described herein, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0145] The present invention relates to Picornaviridae viruses and modified forms thereof that bind to decay accelerating factor (DAF) receptors on the surface of cancer cells. This binding promotes lytic infection of cancer cells by the viruses, which are therefore effective oncolytic agents in the treatment, alleviation and / or prevention of cancer.

[0146] Compared to unmodified or wild-type picornavirus strains, the oncolytic picornaviruses of the present invention can be modified to enhance their ability to bind to DAF. Increased ability to bind to DAF can promote more effective binding to cancer cells, thereby enhancing the effectiveness of the modified picornavirus strain in treating, alleviating, and / or preventing cancer.

[0147] Additionally or alternatively, the oncolytic picornaviruses of the present invention may be modified in other ways to enhance their effectiveness in treating, alleviating, and / or preventing cancer. By way of non-limiting example, the virus may be subjected to one or more biological selection procedures to increase its ability to target and / or dissolve cancer cells. Additionally or alternatively, and again by way of non-limiting example, the virus may be recombinantly modified or inserted with exogenous nucleic acids encoding proteins that increase its ability to target and / or dissolve cancer cells.

[0148] The oncolytic picornaviruses of the present invention and modified forms thereof can be administered in combination with other anti-cancer agents such as, by way of non-limiting example, immune checkpoint inhibitors, CAR-T cells, natural killer (NK) cells, chemotherapeutic agents, and the like.

[0149] Without any limitation on the specific form of cancer, oncolytic picornaviruses and their modified forms can effectively treat, alleviate and / or prevent cancer types that respond poorly to immune checkpoint therapy, such as cancers with an objective clinical response rate of <25%, non-limiting examples of which include liver cancer, pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, gastric cancer, breast cancer, sarcoma, lymphoma, brain cancer and ovarian cancer. Cancer can be characterized by cancer cells expressing decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

[0150] Picornavirus of the present invention can be any picornavirus, comprises the picornavirus of known and classification and the picornavirus of not yet classification.Picornavirus can be selected from prototype and clinical isolate.The representative type of human picornavirus comprises enterovirus, coxsackievirus, echovirus, poliovirus, unclassified enterovirus, rhinovirus, double echovirus, hepatovirus and cardiovirus.In some embodiments, picornavirus can comprise enterovirus, described enterovirus comprises coxsackievirus, echovirus, poliovirus, unclassified enterovirus or the virus from other genus of picornavirus, it can comprise rhinovirus, double echovirus, hepatovirus, cardiovirus, foot-and-mouth disease virus, equine rhinovirus (Erbovirus), crest virus (Kobovirus) and Teschovirus (Teschovirus).These viral modified forms are also included within the scope of the present invention.

[0151] In certain embodiments, the picornavirus and its modified forms can be a picornavirus that can bind to the decay accelerating factor (DAF, also known as CD55) receptor on the surface of cancer cells, such as an enterovirus (e.g., a coxsackievirus or an echovirus). Non-limiting examples include enterovirus 68, 70, coxsackie B virus serotypes 1, 3, and 5, and echovirus serotypes 6, 7, 12, 20, 21, and coxsackievirus A21. Picornaviruses and their modified forms can use the DAF receptor to mediate viral attachment to cancer cells and / or mediate viral entry into cancer cells.

[0152] Additionally or alternatively, the picornaviruses and modified forms thereof may be capable of binding to one or more other receptor types on the surface of cancer cells, such as intercellular adhesion molecule 1 (ICAM-1), intercellular adhesion molecule 1 (ICAM-5), integrin α2β1, integrin αVβ3, integrin αVβ6, neonatal Fc receptor (FcRn), coxsackievirus and adenovirus receptor (CAR / CXADR), sialic acid (e.g., α2,3 sialic acid), lysosomal integral membrane protein 2 (LIMP-2 / SCARB2), P-selectin glycoprotein ligand (SELPLG), low-density lipoprotein receptor (LDLR), poliovirus receptor (PVR) and / or cadherin-related family member 3 (CDHR3).

[0153] The picornaviruses used in the compositions and methods of the present invention may be naturally occurring or modified forms thereof.

[0154] A picornavirus is "naturally occurring" when it can be isolated from a source in nature and has not been intentionally modified by man in a laboratory. For example, a picornavirus can be obtained from a "field source," such as a human patient.

[0155] A picornavirus can be "modified" by having one or more characteristics altered compared to a naturally occurring picornavirus.

[0156] For example, the picornavirus can be a recombinant picornavirus derived from two or more types of picornaviruses with different pathogenic phenotypes, such that it contains different antigenic determinants, thereby reducing or preventing the immune response of mammals previously exposed to the picornavirus subtype. Such recombinant virus particles can be produced by co-infecting mammalian cells with picornaviruses of different subtypes, and then sorting and incorporating different subtype coat proteins into the resulting virus particle capsids.

[0157] Additionally or alternatively, the picornavirus can be modified by altering one or more of the structural proteins, such as VP1, VP2, VP3, and / or VP4. The structural proteins can be exposed on the outer surface of the capsid (e.g., one or more of VP1, VP2, VP3). Non-limiting examples of alterations in the VP1, VP2, and / or VP3 proteins include those listed in Table 1 below:

[0158] Table 1. Non-limiting amino acid changes in VP1, VP2, and VP3

[0159]

[0160] 1 Residue numbering is according to echovirus strain 12 Travis.

[0161] Without limitation, the picornavirus may comprise any one or more of the following: a VP1 capsid protein amino acid sequence comprising any one of SEQ ID NOs: 54-57, 74, 86, 98, 110, 122, 134, 146 or 158, a VP2 capsid protein amino acid sequence comprising any one of SEQ ID NOs: 58-61, 75, 87, 99, 111, 123, 135, 147 or 159, and / or a VP3 capsid protein amino acid sequence comprising any one of SEQ ID NOs: 62, 63, 76, 88, 100, 112, 124, 136, 148 or 160.

[0162] Additionally or alternatively, the picornavirus can be modified by making changes to one or more of the nonstructural viral proteins, e.g., 2A, 2B, 2C, 2BC, 3A, 3B, 3AB, 3C, 3D, and 3CD.

[0163] Table 2. Non-limiting amino acid changes in other picornavirus proteins

[0164]

[0165] 1 Residue numbering is according to echovirus strain 12 Travis.

[0166] Without limitation, the picornavirus may comprise any one or more of the following: a nonstructural protein 2A sequence comprising any one of SEQ ID NOs: 64, 65, 77, 89, 101, 113, 125, 137, 149, or 161, a nonstructural protein 3A sequence comprising any one of SEQ ID NOs: 66, 67, 78, 90, 102, 114, 126, 138, 150, or 162, a nonstructural protein 3C sequence comprising any one of SEQ ID NOs: 68, 69, 79, 91, 103, 115, 127, 139, 151, or 163, and / or a nonstructural protein 3D sequence comprising any one of SEQ ID NOs: 70, 71, 80, 92, 104, 116, 128, 140, 152, or 164.

[0167] With respect to any amino acid sequence or nucleotide sequence variation described herein, including the amino acid variations noted in Tables 1 and 2 above, it will be understood that the length of the amino acid sequence of a given individual protein may vary in different picornavirus strains. Thus, reference herein to an amino acid variation at a given residue position of a picornavirus protein sequence "A" will be understood to include the same amino acid variation at the equivalent position of the same picornavirus protein sequence "B," which, while different in length from the sequence of "A," still represents the same picornavirus protein. Equivalent amino acid positions of amino acid sequences of different lengths but representing the same picornavirus protein can be routinely identified using standard methods of sequence alignment known in the art.

[0168] Picornavirus proteins can be changed by amino acid replacement, insertion or deletion. This can be achieved using any suitable technology, including but not limited to recombinant methods. Replacement includes inserting different amino acids to replace natural amino acids. Insertion includes inserting additional amino acid residues into the protein at one or more positions. Deletion includes deleting one or more amino acid residues in the protein. The change of this characteristic can be produced by methods known in the art. For example, oligonucleotide site-directed mutagenesis of genes encoding one or more proteins (e.g., capsid proteins) can result in the production of capsid proteins with the desired changes. In vitro expression of altered proteins in mammalian cells infected by picornaviruses can result in the incorporation of altered proteins into picornavirus virion particles.

[0169] In some embodiments, the picornavirus can be modified to reduce or eliminate the immune response of the picornavirus. Such modified picornaviruses are referred to as "immunoprotective picornaviruses." Suitable modifications may include packaging the picornavirus in liposomes, micelles, or other vehicles to shield the picornavirus from the subject's immune system. Alternatively, the outer capsid of the picornavirus virion particle can be removed or altered because the proteins present in the outer capsid are major determinants of the host's humoral and cellular responses.

[0170] In some embodiments, picornaviruses can be modified by making changes to the viral nucleic acid sequence, including untranslated regions (UTRs), regulatory regions, and the like.

[0171] The picornavirus can be further modified by inserting an exogenous nucleic acid encoding a protein of interest or its components. Exogenous nucleic acids can be inserted into RNA using standard methods known to those skilled in the art (see, for example, Ferran and Skuse (Eds), "Recombinant Virus Vaccines Methods and Protocols", 2017, Springer Protocols; Green and Joseph. (2012), Molecular cloning: a laboratory manual, fourth edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; Ausubel et al. (1987-2016), Current Protocols in Molecular Biology. New York, NY, John Wiley & Sons). Non-limiting examples of exogenous nucleic acids that can be inserted into the picornaviruses of the present invention include those encoding cytokines (e.g., interleukins, interferons), chemokines, immune checkpoint inhibitors (e.g., PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, ICOS, LAG-3, TIM3, GAL9, CD28, OX-40, TIGIT), and the like.

[0172] Additionally or alternatively, picornaviruses can be modified by bioselection for certain traits (also known in the art as directed evolution). For example, picornaviruses can be bioselected for enhanced infectivity, enhanced safety, enhanced lytic ability, enhanced migration between cancer cells, enhanced DAF and / or FcRn binding, enhanced ability to replicate at mammalian body temperature (33°C-39°C, 35°C-39°C, 37°C-39°C), enhanced lytic activity against cancer cells expressing PD-L1, etc. Methods for bioselecting viruses with target traits are known to those skilled in the art (see, for example, Zainutdinov et al. “Directed evolution as a tool for the selection of oncolytic RNAviruses with desired phenotypes”, Oncolytic Virotherapy; Macclesfield Vol. 8, (2019): 9-26; Svyatchenko et al. “Bioselection of coxsackievirus B6 strain variants with altered tropism to human cancer cell lines”, Arch Virol (2017) 162: 3355–3362; Yan et al. “Developing Novel Oncolytic Adenoviruses through Bioselection”, J. Virol, 2003, p. 2640–2650).

[0173] The present invention includes isolated nucleic acid molecules from picornaviruses. In some embodiments, the picornavirus is capable of binding to at least a DAF receptor on the surface of a cancer cell. In some embodiments, the nucleic acid molecule can be derived from a picornavirus and can be a single-stranded RNA or a synthetically produced viral RNA or a complementary DNA from a virus. It will be appreciated that the nucleic acid sequences of the present invention include nucleic acid sequences that have been derived from picornaviruses, including, for example, nucleic acid sequences encoding viral genomes or sequences thereof that are sufficient to allow the production of viruses or to induce lytic infection of cells. For example, the nucleic acid molecule can comprise a single viral RNA or DNA molecule, such as a complementary DNA molecule or a plurality of such molecules encoding different viral sequences.

[0174] It will be understood that in the context of this specification, the term "derived from" therefore includes sequences that may be viral RNA isolated directly from a picornavirus, synthetic viral RNA, or cDNA encoding a viral genome or component thereof corresponding to the isolated sequence. The term also includes synthetic polynucleotide sequences that contain one or more changes in sequence compared to the wild-type or parental sequence, including, for example, changes in the capsid protein.

[0175] Any suitable method for isolating viral RNA may be used, including methods based on extraction using phenol / chloroform, such as those provided in commercial kit form for isolating viral RNA, e.g. LS reagent (GIBCO BRL, Life Technologies Grand Island, NY, USA), using a magnetic bead-based separation method such as Ambion MagMax TM Viral RNA Isolation Kit. Methods for isolating viral RNA are generally described in, for example, Ausubel et al. (1987-2016), Current Protocols in Molecular Biology. New York, NY, John Wiley & Sons; and Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbour Laboratory Press, New York.

[0176] It will be appreciated that, in the context of this specification, the present invention does not require the absence of contaminant material to be considered "isolated," such as nucleic acid sequences of cellular debris, such as viral RNA, whether isolated directly from a virus, synthesized, provided as a plasmid molecule, or produced in vitro, for example, using bacteriophage T7 RNA polymerase, from a cDNA template (e.g., encoding a viral genome or component thereof). Thus, in the context of this specification, RNA will be considered isolated when non-RNA components from the source material, such as cellular proteins, have been partially or completely removed from the RNA. For example, RNA will be considered "isolated" when greater than 50% of the non-RNA material has been removed. Preferably, greater than 60% of the non-RNA material has been removed, more preferably greater than 70%, 80%, or 90% of the non-RNA material has been removed. Typically, RNA will contain less than 10% contaminant material, more preferably less than 5%. Thus, for viral RNA, the RNA will preferably be greater than 95% pure, even more preferably greater than 97% pure, or greater than 99% pure.

[0177] The nucleic acid molecule may comprise a nucleic acid sequence comprising, or consisting of, SEQ ID NO: 41, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 144, or SEQ ID NO: 156. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, considerable sequence variation is possible in these polynucleotide molecules.

[0178] The present invention also provides isolated polynucleotide sequences that are substantially similar to the polynucleotides disclosed herein, such as SEQ ID NO:41, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:72, SEQ ID NO:84, SEQ ID NO:96, SEQ ID NO:108, SEQ ID NO:120, SEQ ID NO:132, SEQ ID NO:144 or SEQ ID NO:156, wherein such sequences comprise or provide a picornavirus having the ability to bind to the DAF receptor and optionally additional cellular receptors and lyse infected cancer cells. The polynucleotide sequence variants have the same qualitative biological activity as the base sequence (e.g., SEQ ID NO:41, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:72, SEQ ID NO:84, SEQ ID NO:96, SEQ ID NO:108, SEQ ID NO:120, SEQ ID NO:132, SEQ ID NO:144, or SEQ ID NO:156) and have at least about 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the base sequence (e.g., SEQ ID NO:41, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:72, SEQ ID NO:84, SEQ ID NO:96, SEQ ID NO:108, SEQ ID NO:120, SEQ ID NO:132, SEQ ID NO:144, or SEQ ID NO:156). As used herein, "sequence identity" refers to the residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window as determined by computer programs known in the art, such as GAP (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) provided in the GCG program package (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-453).

[0179] The picornaviruses, modified forms thereof, and picornavirus RNA / cDNA of the present invention can be prepared using standard techniques well known in the art (see, for example, Chen et al. 2019, "Physical, chemical, and synthetic virology: Reprogramming viruses as controllable nanodevices, Wiley Interdiscip Rev Nanomed Nanobiotechnol. 11(3):e1545. doi: 10.1002 / wnan.1545; Geunther et al 2014, "Synthetic virology: engineering viruses for gene delivery." Wiley interdisciplinary reviews. Nanomedicine and Nanobiotechnology vol. 6, 6: 548-58. doi: 10.1002 / wnan.1287). Non-limiting examples of modifications of viruses and / or their RNA / cDNA include insertion of tissue-specific miRNA and / or encoding immune checkpoint inhibitors, such as nucleic acids that bind to monoclonal antibodies against immune checkpoint targets (e.g., PD-1, PD-L1, CTLA-4, IDO, TIM-3, LAG-3, TIGIT, etc.).

[0180] The picornaviruses of the present invention and / or their modified forms can be administered to a subject in the form of a pharmaceutical composition comprising the virus and a pharmaceutically acceptable carrier. The composition can comprise any suitable concentration, for example, about 10 5 Virus particles / mL to about 10 15 The concentration range is about 10 virus particles / mL, or about 10 6 virus particles / mL, or about 10 7 virus particles / mL, or about 10 8 virus particles / mL, or about 10 9 virus particles / mL, or about 10 10 virus particles / mL, or about 10 11 virus particles / mL, or about 10 12 Virus particles / mL, about 10 13 virus particles / mL, or about 10 14 virus particles / mL or about 10 15 Virus particles / mL of virus.

[0181] A stock solution of the viral composition can be diluted to a suitable volume for administration, for example, to achieve a desired dose of viral particles administered in a desired volume. For example, a solution containing about 10 5 Virus particles to about 10 15 Virus particles, or about 10 6 Virus particles, or about 10 7 Virus particles, or about 10 8 Virus particles, or about 10 9 Virus particles, or about 10 10 Virus particles, or about 10 11 Virus particles, or about 10 12 Virus particles, or about 10 13 Virus particles, or about 10 14 Virus particles, or about 10 15 The volume of virus administered is affected by the mode of administration. For example, virus administered by injection is typically in a smaller volume, such as about 0.5 mL to about 10 mL. As a further example, intravenous administration of virus can typically use about 100 mL to about 500 mL of virus diluted in normal saline, which is infused by an automated pump over about 30 minutes.

[0182] The pharmaceutical compositions provided herein may comprise about 10 8 and about 10 15 virus particles / mL, about 10 8 and 10 12 virus particles / mL or about 10 8 and 10 10 Virus particles / mL for picornaviruses.

[0183] The pharmaceutical compositions provided herein can comprise a picornavirus having a particle to infectivity ratio of less than or equal to about 3000:1, less than or equal to about 300:1, in the range of about 200:1 to about 5:1, in the range of about 50:1 to about 10:1, or less than about 20:1.

[0184] The pharmaceutical compositions provided herein can further comprise host cell DNA levels less than or equal to about 200 ng / mL, within a range of 1 to about 100 ng / mL, within a range of about 1 to about 50 ng / mL, or within a range of about 1 to about 10 ng / mL.

[0185] Pharmaceutical compositions comprising the picornavirus of the present invention and / or its modified form may further include pharmaceutically acceptable diluents, excipients and / or adjuvants. Carriers, diluents, excipients and adjuvants must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not unacceptably deleterious to the recipient.

[0186] The virus can be administered as naked viral RNA encoding the virus, rather than as viral particles, as described in, for example, PCT / AU2006 / 000051, entitled "Methods and compositions for treating tumors," filed January 17, 2006, and published as WO2006 / 074526, the entire contents of which are incorporated herein by reference. In such embodiments, the viral RNA can be administered in the form of liposomes. Liposomes are typically derived from phospholipids or other lipid substances and are formed by monolayer or multilayer hydrated lipid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. Compositions in liposome form may contain stabilizers, preservatives, excipients, etc. Preferred lipids are phospholipids and phosphatidylcholines (lecithins), both of which are natural and synthetic. Methods for forming liposomes are known in the art, and reference is made in particular to Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, NY (1976), p. 33 et seq., the contents of which are incorporated herein by reference.

[0187] Alternatively, viral RNA can be administered in the form of lipid nanoparticles (LNPs). Lipid nanoparticles (LNPs) are generally composed of four main types of lipids: ionizable cationic lipids, which are critical for encapsulating nucleic acids and assisting endosome release; phospholipids, which contribute to the structural integrity of nanoparticles; cholesterol, which improves stability and mobility; and polyethylene glycol (PEG)-lipids, which extend in vivo circulation time by forming steric barriers. However, other nontoxic, physiologically acceptable and metabolizable lipids that can form LNPs can be used, as understood by those skilled in the art. The formation of LNPs generally includes microfluid mixing or ethanol dilution, in which lipids and therapeutic payloads are rapidly mixed in an aqueous environment. However, other methods of forming LNPs are known in the art.

[0188] The routes of administration of the picornaviruses and modified forms thereof and pharmaceutical compositions comprising the same according to the present invention to a given subject include, but are not limited to, intratumoral, intravenous, intravesical, subcutaneous, oral, intracystic, cutaneous, isolated liver perfusion, intrahepatic, ocular and topical administration routes, and any combination thereof.

[0189] Picornavirus of the present invention and / or its modified form can be by any suitable means, for example, by injection to experimenter, use.Injection can for example be systemic, parenteral, or directly in cancer.Considering factors such as the feasibility of the tumor type for example, the size and position of tumor, tumor direct injection, injection in the focus of tumor can be carried out by any suitable means known to technicians.Increase or maximize the injection technique of the distribution of virus in whole tumor and can provide improved therapeutic outcome.For example, in the treatment of melanoma and other solid tumors, multiple lesions can be injected under dose hyperfractionation mode, (2.0mL is injected in the tumor of>2.5cm, 1.0mL is injected in 1.5 to 2.5cm tumor, 0.5mL is injected in 0.5 to 1.5cm tumor) to 4.0mL maximum value from maximum lesion.After the initial injection with picornavirus described herein, any injection focus that diameter is reduced to<0.5cm can be injected with picornavirus and / or its modified form of 0.1mL according to the treatment regimen of regulation, until focus decomposes completely.

[0190] In one embodiment of the present invention, in the treatment of colorectal cancer and other solid tumors of a specified length, single and multiple lesions may be treated using the following dosage regimen:

[0191] 1. For tumors ≥50 mm in length:

[0192] • Inject up to 10 mL of IVX037 for a single lesion.

[0193] If multiple lesions are injected, at least 5.0 mL is dispensed into the largest lesion, and the remaining 5.0 mL is divided among the smaller lesions according to their size.

[0194] 2. For tumors ≥25 mm and <50 mm in length:

[0195] • For a single lesion, inject 3.0 mL of IVX037.

[0196] If multiple lesions are injected, up to an additional 3.0 mL may be injected and dispensed based on size.

[0197] 3. For tumors ≥10 mm and <25 mm in length:

[0198] • For a single lesion, inject 1.0 mL of IVX037.

[0199] • If multiple lesions are injected (up to 4), the dose can be divided among the target lesions, each receiving 1 mL and 25% of the total dose.

[0200] 4. For tumors ≥5 mm and <10 mm in length:

[0201] If the treated lesion regresses to <10 mm, inject 0.5 mL of IVX037 until it disappears.

[0202] Maximizing the number of initially infected cancer cells and regions throughout the tumor will theoretically increase the amount of cancer cells destroyed. It will also increase the amount of viral progeny produced by the tumor, thereby increasing the chance of sustained viremia for seeding distant tumors. Any suitable method for achieving the desired distribution of the administered virus through the tumor can be used and will be apparent to the skilled artisan.

[0203] The picornaviruses of the present invention and / or modified forms thereof can be administered to a subject, tumor, or cancer cell simultaneously or sequentially with other agents.

[0204] For example, they can be administered in combination with immunostimulants. Immunostimulants can be selected from any suitable agent. In the context of the present invention, immunostimulants will be understood as any agent that can stimulate an immune response to tumor / cancer cells when administered to an individual. Immunostimulants can be any agent that interacts with immune checkpoint molecules to block, reduce or offset immune checkpoint molecules or complexes comprising immune checkpoint molecules to reduce the ability of an individual's anti-tumor response based on innate immunity. Therefore, immunostimulants reduce the "handbrake" effect that immune checkpoint molecules have on anti-tumor responses. For example, an immunostimulant can be any agent targeting an immune checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, ICOS, TIM3, GAL9, CD28, OX-40 and TIGIT. It will also be understood that when the immunostimulatory agent targets an immune checkpoint, the term immunostimulatory agent as used herein may also refer to an immune checkpoint inhibitor.

[0205] In some embodiments, the immunostimulatory agent is an antibody, such as a monoclonal antibody. Antibodies for use in the present invention can be prepared by methods well known in the art, including preparing monoclonal antibodies using well-known techniques and screening for high affinity antibodies, or by first identifying monoclonal antibodies with moderately high affinity and then improving affinity using well-known methods (see, e.g., Huse et al., International Rev. Immunol. 10:129-137 (1993); Yelton, et al., J. Immunol. 155:1994-2004 (1995); W, et al., Proc. Natl. Acad. Sci. (USA) 95:6037-6042 (1998); Crameri et al., Nature Medicine 2:100-103 (1996); Stemmer, Proc. Natl. Acad. Sci. (USA) 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994); the portions of these documents describing antibody preparation are incorporated herein by reference). Alternatively, the antibodies may be obtained rather than prepared. Examples of antibodies that target checkpoint inhibitor molecules include nivolumab (BMS-936558, MDX-1106, ONO-4538), a fully human immunoglobulin G4 (IgG4) monoclonal PD-1 antibody that was the first of its kind tested in a phase I trial of 107 patients with metastatic melanoma (see Sosman et al. 2012b); lambrolizumab (MK-3475), a humanized monoclonal IgG4 PD-1 antibody that was studied in a phase I trial including 132 patients with metastatic melanoma (see Iannone et al. 2012); and BMS-936559, a fully human IgG4 PD-L1 antibody that was tested as part of a phase I trial in 55 patients with metastatic melanoma (see Brahmer et al. 2012).

[0206] Additionally or alternatively, the picornavirus of the present invention and / or its modified form can be combined with CAR-T cells and / or natural killer (NK) cells (simultaneously or sequentially) to administer to a subject, a tumor or a cancer cell. The method for producing CAR-T cells is well known to those skilled in the art (see, for example, Zhao et al. "Universal CARs, universal T cells, and universal CAR T cells ", Journal of Hematology & Oncology, volume 11, Article number: 132 (2018);Wang and Rivière, "Clinical manufacturing of CAR Tcells: foundation of a promising therapy ", Molecular Therapy-Oncolytics (2016) 3, 16015;Vormittag et al. "A Guide to Manufacturing CAR-T Cell Therapies ", CurrOpin Biotechnol. 2018 Oct;53: 164-181). By way of non-limiting example, CAR-T cells can be engineered to bind any one or more of the following on the surface of cancer cells: EGFRVIII, Interleukin 13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Muc1, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Muc1, EphA2, CD123, CD19, Claudin 18.2.

[0207] Additionally or alternatively, the picornaviruses of the present invention and / or modified forms thereof can be administered to a subject, tumor, or cancer cell in combination (simultaneously or sequentially) with a chemotherapeutic agent. By way of non-limiting example, suitable chemotherapeutic agents include ABVD, AC chemotherapy, amsacrine, (Amsidine), asparaginase, azacitidine, BEACOPP chemotherapy, BEAM chemotherapy, bendamustine, BEP chemotherapy, bleomycin, busulfan, Cabazitaxel, Capecitabine and docetaxel, Capecitabine, Carboplatin, carboplatin and etoposide chemotherapy, carmustine, CAV chemotherapy, chlorambucil, ChlVPP chemotherapy, CHOP chemotherapy, cisplatin, cisplatin and fluorouracil chemotherapy, 5FU, cisplatin and topotecan chemotherapy, (CT), cisplatin, capecitabine and trastuzumab, (HCX), cladribine, Clofarabine, CMF chemotherapy, CODOX-M chemotherapy, Clistatase, ( Asparaginase or L-asparaginase), CTD chemotherapy, CVP chemotherapy, cyclophosphamide, cytarabine, dacarbazine, (DTIC), actinomycin D, (Cosmegen ), daunorubicin, De Gramont and modified de Gramont chemotherapy, DHAP chemotherapy, docetaxel, Docetaxel and carboplatin chemotherapy, Docetaxel and cisplatin chemotherapy, Doxorubicin, (Adriamycin@), Doxorubicin and Ifosfamide chemotherapy, EC chemotherapy, ECF chemotherapy, E-CMF chemotherapy, (Epi-CMF), ECX chemotherapy, EOX chemotherapy, Epirubicin, Eribulin, ESHAP chemotherapy, etoposide, Etoposide and cisplatin chemotherapy, (EP / PE), FCR chemotherapy, FEC chemotherapy, FEC-T chemotherapy, FLAG-Ida, FLOT chemotherapy, fludarabine, Fluorouracil, (5FU), FOLFIRINOX chemotherapy, GemCarbo chemotherapy, gemcitabine, Gemcitabine and capecitabine (GemCap), Gemcitabine and cisplatin chemotherapy (GemCis or GemCisplat), Tablets, (carmustine), hydroxycarbamide, ( Hydroxyurea), Hyper-CVAD chemotherapy, idarubicin, Ifosfamide, Ifosfamide, carboplatin, etoposide-ICE chemotherapy, irinotecan, Irinotecan with fluorouracil and folinic acid, (5FU) and (FOLFIRI), Leucovorin, (folinic acid), liposomal daunorubicin, Liposomal doxorubicin, Lomustine, melphalan, Mercaptopurine, Mesna, Methotrexate, mitomycin, (mitomycin C ), mitomycin and fluorouracil, (5FU), mitotane, Mitoxantrone, MPT chemotherapy, MPT chemotherapy, MVAC chemotherapy, Nab-paclitaxel, Oxaliplatin, Oxaliplatin with fluorouracil and folinic acid chemotherapy, (5FU) and (FOLFOX or OxMdG), paclitaxel, Paclitaxel and carboplatin chemotherapy, (Taxol / Carbo), PCV chemotherapy, pemetrexed, pemetrexed and carboplatin, pemetrexed and cisplatin chemotherapy, pentostatin, Pertuzumab, trastuzumab and docetaxel, PMitCEBO chemotherapy, procarbazine, raltitrexed, Rasburicase, R-CHOP chemotherapy, R-CVP, R-DHAP chemotherapy, R-ICE chemotherapy, streptozocin, TAC chemotherapy, TC (Taxotere and cyclophosphamide) chemotherapy, temozolomide, Thiotepa, thioguanine, TIP chemotherapy, topotecan, Trabectedin, Trioxan, trifluridine-tipirimidine hydrochloride, Vinblastine, vincristine, Changchunruibin, Vinorelbine and carboplatin chemotherapy, (VP), vinorelbine and cisplatin chemotherapy, (VP), XELOX (or CAPOX), XELOX (or CAPOX).

[0208] The methods of the present invention can be used in combination with surgical treatment of cancer. For example, after tumor resection, the subject can be treated with the methods of the present invention. This is expected to prevent or reduce tumor recurrence.

[0209] Additionally or alternatively, the methods of the present invention can be used in combination with neoadjuvant therapy. For example, neoadjuvant therapy can be followed by treatment of the subject with the methods of the present invention. This is expected to prevent or reduce tumor recurrence.

[0210] Additionally or alternatively, the methods of the present invention can be used in combination with radiation therapy (e.g., X-rays, protons, and / or other particles). For example, radiation therapy can be followed by treatment of a subject using the methods of the present invention. This is expected to prevent or reduce tumor recurrence.

[0211] The method may include a single or multiple doses of any one or more of a picornavirus, an immunostimulant, a CAR-T cell, a natural killer (NK) cell, a chemotherapeutic agent, and / or a radiotherapeutic agent. The method of the present invention includes administering a picornavirus described herein and / or its modified form and / or its RNA and / or cDNA produced from RNA to a subject, a tumor, or a cancer cell. Although there is no specific limitation on the type of cancer that can be treated, alleviated, or prevented according to the methods described herein, in some embodiments, cancer is identified as a cancer that is resistant to treatment with immune checkpoint inhibitors (i.e., a reduced level of response) in a general sense or in the case of a specific subject, tumor, or cancer cell. Non-limiting examples of such cancers include liver cancer, pancreatic cancer, head and neck cancer, gastric cancer, colorectal cancer, gastric cancer, breast cancer, sarcoma, lymphoma, brain cancer, and ovarian cancer. The cancer can be characterized by cancer cells expressing decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

[0212] The present invention also relates to a kit for use in the method of the present invention. In basic form, the kit may include a pharmaceutical composition comprising the picornavirus of the present invention and / or its modified form; a pharmaceutically acceptable carrier; and instructions for use of the composition. The kit may further include any one or more of the following: immune checkpoint inhibitors, CAR-T cells, natural killer (NK) cells, chemotherapeutic agents, radiotherapeutic agents. The composition may be provided in any suitable container, such as a vial, ampoule, or syringe. The composition may be lyophilized, freeze-dried, provided in liquid form, or in a frozen state.

[0213] The kit may include any number of other additional components. By way of non-limiting example, additional components may include (i) one or more anti-viral agents, such as Plecornil; (ii) one or more additional pharmaceutical compositions comprising oncolytic viruses; (iii) one or more additional pharmaceutical compositions comprising oncolytic viral RNA; (iv) one or more additional therapeutic agents that can be used to treat a patient's cancer. The kit may also include a composition contained in a disposable vial, a pre-filled syringe for direct human administration, diluted in a physiological solution for intravenous infusion, or in a concentrated form that can be appropriately diluted with a physiological solution. Such a solution may be, for example, phosphate-buffered saline or a physiological concentration of NaCl2.

[0214] Those skilled in the art will appreciate that various changes and / or modifications may be made to the invention as disclosed in the specific embodiments without departing from the spirit or scope of the invention as broadly described. Therefore, the present embodiments are to be considered in all respects as illustrative and not restrictive.

[0215] Example

[0216] The present invention will now be described with reference to the following examples, which should not be construed as limiting in any way.

[0217] Example 1: Novel Echovirus 12 strain IVX037 with enhanced DAF-binding ability

[0218] The complete genome sequence of a novel echovirus 12 (E12) strain designated IVX037 (in-house designation) with high affinity DAF binding was determined.

[0219] -Materials and methods

[0220] Viral RNA was extracted from IVX037 virus stock (IVX037 RD PLC 15.06.2020) using the QIAamp Viral RNA Mini Kit (SOP LAB-PROC 018) and reverse transcribed and amplified using a one-step reverse transcription polymerase chain reaction (LAB-PROC 027) using virus-specific primers (see Table 3).

[0221] Table 3. List of virus-specific primers used for sequence analysis

[0222]

[0223]

[0224] Amplified DNA was analyzed on agarose gel (LAB PROC 014) and purified using a QIAquick PCR purification kit (LAB-PROC 016) or a QIAquick gel extraction kit (LAB-PROC 015). Complete viral genome sequences were determined using Sanger sequencing and virus-specific primers (LAB-PROC 012) at AGRF, Westmead NSW, Australia. Genomic sequences were analyzed using Sequencher 5.4.6 version and compared to the wild-type E12 prototype strain Travis virus sequence (GenBank accession number X79047.01). Paired sequence alignment was performed using the Emboss needle global alignment tool (EMBL-EBI).

[0225] -result

[0226] In some embodiments, the complete genomic sequence of the isolated IVX 037 strain, excluding the poly(A) tail, is 7423 nucleotides (nt) (e.g., SEQ ID NO:41, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:72, SEQ ID NO:84, SEQ ID NO:96, SEQ ID NO:108, SEQ ID NO:120, SEQ ID NO:132, SEQ ID NO:144, or SEQ ID NO:156). In some embodiments, the open reading frame (e.g., SEQ ID NO:43, SEQ ID NO:81, SEQ ID NO:93, SEQ ID NO:105, SEQ ID NO:117, SEQ ID NO:129, SEQ ID NO:141, SEQ ID NO:153, or SEQ ID NO:165) encodes a polyprotein of 2193 amino acids (e.g., SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:73, SEQ ID NO:85, SEQ ID NO:97, SEQ ID NO:109, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:145, or SEQ ID NO:157) and is preceded by a 741 nucleotide long 5' untranslated region (UTR) (e.g., SEQ ID NO:44, SEQ ID NO:82, SEQ ID NO:94, SEQ ID NO:106, SEQ ID NO:118, SEQ ID NO:130, SEQ ID NO:142, SEQ ID NO:133 ID NO: 154 or SEQ ID NO: 166) followed by a 103 nt long 3'UTR (e.g., SEQ ID NO: 45, SEQ ID NO: 83, SEQ ID NO: 95, SEQ ID NO: 107, SEQ ID NO: 119, SEQ ID NO: 131, SEQ ID NO: 143, SEQ ID NO: 155 or SEQ ID NO: 167) and a poly (A) tail.

[0227] The viral genome of the IVX037 strain of the present invention is a positive-sense single-stranded RNA virus, and due to the characteristics of the genome, the first 20 nucleotides at the 5' end have not been experimentally determined. However, these regions are highly conserved among enteroviruses, so it is very likely that these sequences represent the authentic sequence of IVX037 (see Polacek et al., (2001). Genetic characterization of the Coxsackievirus B2 3'untranslated region. Journal of General Virology 82, 1339; Mahud et al. (2019). Structure of the 5'Untranslated Region of Enteroviral Genomic RNA. Journal of Virology 93, e01288). The average sequence coverage of the exemplary IVX037 genome of the present invention was >5x, and the vast majority of the genome was sequenced with at least one high-quality sequence in the forward direction and one in the reverse direction. The prediction error probabilities for high-quality and medium-quality values ​​were ≤0.01% and ≤1%, respectively.

[0228] In one embodiment, an undefined position was identified in the genome at nucleotide position 3074 where the viral sequence was identified as having a mixture of U (uracil) and A (adenine). This position was sequenced in both directions using two separate PCR amplicons (see Table 4) with identical results (U / A mixture).

[0229] Table 4. Detailed sequence analysis of the ambiguous nt mixture at position 3074

[0230]

[0231]

[0232] 1 Note: Sequence results are presented here using T (thymidine) determined during Sanger sequencing, whereas the RNA sequence of IVX037 is U (uracil).

[0233] 2 The sequence quality score is a measure of the uncertainty of the base call and due to the presence of both T and A signals at this position, the resulting quality score is lower than the surrounding regions.

[0234] Therefore, this uncertainty is unlikely to be a proofreading error introduced during RT-PCR amplification, but rather that the position actually contains a mixture of the two nucleotides (shown as "W" in the genomic sequence of SEQ ID NO: 41). This sequence uncertainty results in a mixture of two amino acids encoded at amino acid 210 of the capsid protein VP1, where U encodes phenylalanine and A encodes tyrosine, respectively (see Table 5).

[0235] During analysis of the IVX037 viral genome of the present invention, a number of positions were identified where the sequence of the IVX037 genome differed from the E12 Travis prototype strain, including numerous silent and coding nucleotide changes in nonstructural proteins (see Table 5). In some embodiments, a change also occurs at nucleotide position 108 of the 5'UTR, and based on the primary and secondary sequence similarities between the IVX037 virus of the present invention and the closely related Coxsackievirus B3 (CVB3), this nucleotide is predicted to be located in a loop of domain II of the 5'UTR, and this change is unlikely to affect the secondary structure of the IVX037 viral protein.

[0236] In some embodiments, a number of coding changes were also identified in the capsid protein responsible for cell binding (Table 5). In some embodiments, a further amino acid change was identified in the capsid protein VP3 at amino acid 206. In some embodiments, in VP1, an amino acid change was identified at amino acid 230, and a mixture of amino acids was encoded at amino acid VP1 210 (see nucleotide position 3074 in Table 5). Interestingly, in some embodiments, two amino acid changes were detected in VP2 (amino acids 142 and 154), and these two amino acids are predicted to be located in the hypervariable puff region of VP2 identified as the DAF-binding region of E12.

[0237] In addition to the complete genome sequence of the E12 prototype strain Travis, three complete genome sequences of three E12 strains (isolated from the feces of three healthy children in China in 2013) are available in GenBank (Hongbu et al., (2018), Molecular characterization of echovirus 12 strains isolated from healthy children in China. Scientific Reports 8, 11716). These sequences are shown in SEQ ID NOs: 47-49 and were used for further comparative sequence analysis of the IVX037 genome sequence of the present invention. Among the changes in the capsid coding region, three changes (nucleotides 1373 [VP2 amino acid 142], 2348 [VP3 206], and 3074 [VP1210]) were identified in some embodiments as unique among the IVX037 sequence, the E12 prototype strain Travis (SEQ ID NO: 46), and the three Chinese isolates (see Table 5). These changes are all coding changes, and of particular interest is amino acid VP2 141 (N->T), which is located in the hypervariable PUFF region of VP2 involved in virus binding to DAF.

[0238] Table 5. Comparison of genomic differences between the exemplary IVX037 sequence of the present invention, E12 Travis, and three E12 strains isolated from three healthy children in China in 2013. Capsid region nucleotide differences that are unique to the IVX037 sequence among the five viral sequences (IVX037, E12 Travis, and the three Chinese isolates) are highlighted in light green.

[0239]

[0240]

[0241] discuss

[0242] The complete genome sequence of the IVX037 virus of the present invention, selected on cells expressing high levels of the attachment receptor DAF, was identified as having numerous changes compared to the E12 Travis prototype strain. Changes in the capsid protein are of particular interest because these changes can affect viral binding to receptors on the cell surface. Interestingly, no unique changes were identified in the nonstructural proteins, whereas numerous changes in the capsid coding regions were identified when the IVX037 sequence was compared to the E12 Travis prototype strain. In some embodiments, two amino acid changes (VP2 141N->T and VP2 153H->Y) located in the DAF-binding region of the E12 VP2 protein were identified.

[0243] Of the two changes, VP2 142T was unique among the E12 sequences analyzed (the E12 prototype strain Travis and three Chinese isolates from healthy children).

[0244] A sequence alignment is provided below showing exemplary IVX037 sequences of the present invention including structural features compared to the E12 prototype strain Travis and three Chinese isolates from healthy children. CLUSTALO (1.2.4) multiple sequence alignment

[0245] Keywords:

[0246] VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C, 3D

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256] Example 3: Oncolytic activity and testing of IVX037 passaged in colorectal and ovarian cell lines

[0257] The exemplary bioselected IVX-037 strain was subjected to five rounds of serial passage in human ovarian cell culture (DOV-13) and human MSS colorectal cell culture (SW480). Briefly, cancer cell monolayers were infected with the IVX-037 strain for 1 hour at 37°C, followed by application of maintenance medium (DMEM) and incubation of the cultures at 37°C for 24-124 hours until detectable CPE was evident. The infected cells and medium were then used to infect monolayer cultures of the same cell type using the same infection method. This viral passage process was repeated for five cycles to obtain the IVX-037 OVO strain from DOV-13 cells and the IVX-037 COLO strain from SW480 cells.

[0258] -Materials and methods

[0259] Tumor cell lines (2 x 10 4 Cells (cells) were propagated in monolayers in 96-well plates and inoculated with 10-fold serial dilutions of stock preparations of E12 Plevero, IVX-037 COLO, and IVX037 OVO. After incubation for 5 days at 37°C / 5% CO2, the cell monolayers were examined microscopically for the presence of cytopathic effect (CPE). The 50% endpoint titer was calculated using the Karber method, and the mean (± SEM) and minimum MOI (TCID50 / cell) for each cell line were calculated.

[0260] Viral sequences were obtained by Sangar sequencing using conventional methods using a walking strategy.

[0261] -result

[0262] exist Figure 4 In Figure A, the exemplary IVX-037COLO strain of the present invention showed significantly enhanced oncolytic activity (up to 10 % oncolytic activity) in four human MSS-colorectal cancer cell lines compared to CVA21 (historical data publicly available). 3 Furthermore, the IVX-037OVO strain of the present invention showed significantly enhanced activity (up to 10 times higher) in four advanced human ovarian cancer cell lines compared to CVA21 (historical data available to the public). 7 times higher).

[0263] exist Figure 4 In Figure B, the exemplary IVX-037COLO strain of the present invention showed significantly enhanced oncolytic activity (~10-10) in four human MSS-colorectal cancer cell lines compared to the prototype E12 travis strain (E12 P1-Vero) that was passaged once in Vero cells. 2At the same time, compared with the prototype E12 travis strain (E12P1-Vero) that was passaged once in Vero cells, the IVX-037OVO strain of the present invention showed significantly enhanced oncolytic activity in four high-grade human ovarian cancer cell lines (~10-10 2 times higher).

[0264] exist Figure 4 In Figure C, the exemplary IVX-037COLO strain showed slightly enhanced oncolytic activity in 2 / 3 of the human MSS-colorectal cancer cell lines compared to the exemplary IVX-037OVO strain. Meanwhile, the IVX-037OVO strain showed slightly enhanced oncolytic activity in 3 / 3 of the human ovarian cancer cell lines compared to the IVX-037COLO strain.

[0265] Nucleotide sequences of viruses passaged in ovarian and colorectal cancer cell lines were obtained, and examples thereof are shown in SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 144, and SEQ ID NO: 156, respectively. Examples of encoded amino acid sequences related to the obtained nucleic acid sequences are shown in SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 97, SEQ ID NO: 109, SEQ ID NO: 121, SEQ ID NO: 133, SEQ ID NO: 145, and SEQ ID NO: 157, respectively.

[0266] Example 4: IVX037 virus strain upregulates DDX58 (RIG-I), CD274 (PD-L1), IFN-g inducible protein 10 (CXCL10), and PD-L1

[0267] -Materials and methods

[0268] (i) In vitro assay

[0269] The following protocol was used for in vitro assays:

[0270] OAW42, DOV13, or WiDr cells were seeded in 5 x 6-well plates in medium containing 2% FBS (approximately 2.5E+05 cells / mL, 3 mL / well) to reach approximately 90-100% confluence on the second day. A 500 mL volume of serum-free DMEM medium was prepared for cell washing and inoculum preparation. A 100 mL volume was dispensed from this medium and supplemented with 0.5% FBS for maintenance culture. 10 mL of buffer RLT was prepared and 100 μL of 14.3 M B-ME-mercaptoethanol was added for the cell lysis process. Buffer RLT+B-ME was stable at RT for 1 month. Cell counts were obtained using 3 wells from 1 plate for the average cell count / well, and 3 untreated control wells were harvested. The amount of E12 virus was determined and added per well based on the number of cells / well and viral titer (if the viral titer is unknown, use 0.5 mL / well of a 1:10 dilution of p5 DOV stock in serum-free medium*). Remove the medium and wash the cells once with serum-free medium. Dilute the virus to 10 TCID in 0.5 mL of serum-free medium (or as described above*). 50 / cell MOI. Set up four identical plates with 3 wells of virus-infected cells and 3 wells of mock-infected cells (serum-free medium only) at each time point. Note: A total of about 7 mL of inoculum is required to infect 12 wells. Infect the cells with 0.5 mL of diluted virus or serum-free medium. Incubate the plate for 1 hour, gently shaking the plate (or placing it on a shaker) every 15 minutes to ensure that the cell monolayer does not dry out. Remove the inoculum and gently wash 3 times with preheated serum-free medium. Re-inoculate the cells with 2-3 ml of preheated medium containing 0.5% FBS (maintenance medium) and incubate at 37°C for 0, 3, 6 or 9 hours. Note: Use the following protocol to harvest time 0 wells immediately after the re-inoculation step. At each harvest time point, the cell monolayer is photographed, and the culture medium (supernatant) is taken out completely and frozen for subsequent virus titration (according to LAB-PROC 006) and the possible analysis of viral RNA load (according to LAB-PROC 018, LAB-PROC 031, LAB-PROC 032). Note: it is not necessary to collect culture medium from the control sample. The culture medium gathered in the crops from each virus infection well should be distributed into 4x 500 μ L of labeling aliquots before freezing. In addition to the aliquots infected by the virus, the remaining maintenance culture medium aliquots of 4x 500 μ L are also distributed and frozen for use as the negative control for RNA extraction. Note: aliquots should be carried out after dissolving (step 11). According to step 5.5.2-5.5.3 of LAB-PROC025, after removing the culture medium, the cell monolayer is directly dissolved with 350 μ L of buffer RLT (containing β-ME-mercaptoethanol), and lysate is collected after scraping with a cell scraper. Vortex or pipette to mix and ensure no cell clumps are visible, then continue homogenizing the sample using a QIA disruptor (add up to 700uL / disruptor). Freeze samples until all samples are harvested. Use labeling: control wells = Dov13 C#Xhr 202007XX, infected wells = Dov13 IVX037#, Xhr, 202007XX. When all samples are harvested and frozen, thaw samples in a 37°C water bath until just thawed and salts dissolved. If any insoluble material is seen, centrifuge at 3,000–5,000xg for 5 minutes and transfer the supernatant to a new tube. Extract RNA from all samples (starting in step 5.8 RNA Extraction and Purification, LAB-PROC 025). Include an extraction negative control (media with 0.5% FBS) and a low-range positive control (dilution 10 -4The total number of samples was 12 virus-infected samples (3 replicates per time point), 12 uninfected samples, and a negative extraction control. Gene expression analysis was performed on the extracted RNA samples, including a no-template control (NTC), a negative RT-PCR control, and a negative extraction control. The following TaqMan™ gene expression assay was used to detect CXCL10, RIG-I, and CD274 mRNA levels relative to the housekeeping gene GUSB. The TaqMan™ gene expression assay is designed to analyze the expression of CXCL10, GUSB, CD274, and RIG-I genes using RT-qPCR. Other pre-designed TaqMan™ gene expression assays can also be used with this assay. TaqMan™ Gene Expression Assays are designed to detect expressed mRNA (assay type '_m1'), and the assays HS01125296_m1 (CD274), HS00171042_m1 (CXCL10), HS99999908_m1 (GUSB), and Hs01061436_m1 (RIG-I) were selected based on their ability to detect human mRNA for these transcripts without cross-reactivity with mouse, and the assay probes span exons to avoid detection of genomic DNA. Both assays are labeled with FAM-MGB and therefore cannot be multiplexed, but rather need to be tested in a single assay. The TaqPath™ 1-Step Multiplex Master Mix contains components for reverse transcription and real-time qPCR, including Fast DNA polymerase, thermostable MMLV enzyme, RNase inhibitors, and buffer components. The master mix also contains the enzyme UNG to prevent carryover between assays and a blend of dUTP and dTTP to enable UNG activity. The master mix contains MustangPurple as a passive reference dye to provide an internal reference for standardization of fluorescence fluctuations caused by volume or concentration changes. The assay is a relative quantitative gene expression assay that determines expression changes in a test sample relative to a reference sample (e.g., a sample vs. an untreated control sample). The results are analyzed using a comparative Ct method that uses the formula ratio=2ΔCt to compare the Ct values ​​of the treated sample and the control (wherein the mass is a standardized value, i.e., the number of cells extracted or the μg value of RNA used as a template for RT-qPCR).

[0271] (ii) In vivo assay

[0272] The following protocol was used for in vivo assays:

[0273] Immunocompromised SCID-Balb / C mice were injected subcutaneously with MSS-colorectal cell WiDr. Once the tumor was palpable (≥25 mm 3Mice were injected intratumorally with either the virus dilution vehicle (as a control) or the IVX-037 strain of the present invention. Two mice from the control and IVX037 treatment groups were sacrificed 24, 48, and 72 hours after treatment, and their tumors were excised and placed in RNA post-treatment buffer and stored at 4°C prior to RNA extraction and gene expression PCR analysis. TaqMan™ gene expression assays were subsequently used to measure CXCL10, RIG-I, and CD274 mRNA levels in the excised tumors relative to the housekeeping gene GUSB.

[0274] -result

[0275] In vitro challenge of the ovarian cancer cell lines DOV-13, OAW42 and the human MSS-colorectal cell line WiDr with the IVX-037OVO strain of the present invention resulted in a significant upregulation (1.5- to 5-fold) of mRNA encoding DDX58, CD274 and CXCL10 at 10 hours post-infection compared to control infection levels ( Figure 5 ).

[0276] In Balb-C SCID mice, a single intratumoral injection (10 8 TCID 50 ) In vivo challenge of human MSS-colorectal cell xenografts with the IVX-037OVO strain of the present invention resulted in significant upregulation of mRNA encoding DDX58, CD274, and CXCL10 at 24, 48, and 72 hours post-infection. The most pronounced upregulation was observed at 24 hours post-infection, particularly for the DDX58 and CXCL10 genes ( Figure 6 and 7 ).

[0277] Example 5: Increased infectivity of IVX037 in CHO cells transfected with human FcRn receptor

[0278] -Materials and methods

[0279] CHO cells were seeded into 24-well plates to a confluence of approximately 70-90%. The next day, commercially available Lipofectamine 2000 was used to clone the cells using GenEZ vector expressing Fcgrt (encoding human FcRn receptor) in the mammalian expression cloning vector pcDNA3.1+ / C-(K)-DYK. TMORF clones were transfected. A pIND plasmid lacking the Fcgrt gene served as a transfection control. For reverse transfection, cells were simultaneously inoculated with lipofectamine-plasmid complexes. 48 hours later, cells were infected with the IVX-037OVO strain of the present invention. 72 hours after infection, supernatants and cells were harvested and assayed for infectivity. Plates were scored for cytopathic effect after 5 days. Infectivity data are expressed as log tissue culture infectious dose 50% / mL (log10 TCID50 / mL).

[0280] -result

[0281] IVX037 infectivity data on CHO cells transfected with human FcRn using forward and reverse transfection methods showed Figure 8 In both cases, increased infectivity was observed in cells expressing the human FcRn receptor.

[0282] like Figure 9 As shown, infectivity data derived from CHO cells transfected with either a human FcRn plasmid or a pIND control plasmid demonstrate significantly higher levels of infectivity in CHO cells transfected with the FcRn plasmid. The inclusion of the control plasmid confirms that transfection with the FcRn plasmid is due to transfection and not other factors. The data presented here provide strong evidence that expression of the human FcRn receptor confers enhanced susceptibility to IVX037 infection in CHO cells. Statistical differences between groups were determined using one-way ANOVA.

[0283] Example 6: Intratumoral oncolytic activity of IVX037 in human colorectal cancer (Caco-2) xenografts

[0284] -Materials and methods

[0285] Immunocompromised SCID-Balb / C mice were subcutaneously inoculated with human colorectal cancer cells (Caco-2) (2e6 cells). Once tumors became palpable (≥25 mm3), mice received a single intratumoral injection of either control vehicle or the IVX-037OVO strain of the present invention (1e8 TCID50 / injection). Tumor volumes were measured, and animals were sacrificed 28 days after treatment.

[0286] -result

[0287] Figure 10 Figure 3. Mean (A) and individual (B) tumor volumes of mice treated with IVX037. Potent antitumor activity was observed in Caco-2 xenografts following a single intratumoral injection of IVX037. Treatment was well tolerated, and no side effects were observed. Statistical differences between treatment groups were determined using a two-way ANOVA.

[0288] Example 7: Intratumoral oncolytic activity of IVX037 in human gastric cancer (NCI-N87) xenografts

[0289] -Materials and methods

[0290] Immunocompromised SCID-Balb / C mice were subcutaneously inoculated with human colorectal cancer cells NCI-N87 (2e6 cells). Once tumors became palpable (≥25 mm3), mice received a single intratumoral injection of either control vehicle or the IVX-037OVO strain of the present invention (1e8 TCID50 / injection). Tumor volumes were measured, and animals were sacrificed 25 days after treatment.

[0291] -result

[0292] The mean (A) and individual (B) tumor volumes of mice treated with IVX037 strain are shown in Figure 11 Potent antitumor activity was observed in NCI-N87 xenografts following a single intratumoral injection of the IVX037 strain. Treatment was well tolerated, and no side effects were observed. Statistical differences between treatment groups were determined using a two-way ANOVA.

[0293] Example 8: Intratumoral oncolytic activity of IVX037 in human ovarian cancer (IGROV-1) xenografts

[0294] -Materials and methods

[0295] Immunocompromised SCID-Balb / C mice were subcutaneously inoculated with human ovarian cancer cells IGROV-1 (2e6 cells). Once tumors were palpable (≥25 mm3), mice were given a single intratumoral injection of control vehicle or the IVX-037OVO strain of the present invention (1e8 TCID50 / injection). Tumor volumes were measured, and animals were sacrificed 20 days after treatment.

[0296] -result

[0297] Individual tumor volumes of mice treated with IVX037 strain are shown in Figure 12 IGROV-1 xenografts were more aggressive, leading to tumor ulceration. Affected animals were euthanized. Data presented here are individual volumes per animal, not mean values. Potent antitumor activity was observed in IGROV-1 xenografts following a single intratumoral injection of the IVX037 strain, and no tumor ulceration was observed in this group. Treatment was well tolerated, and no side effects were observed. Due to the early termination of the control animals, a two-way ANOVA could not be performed.

[0298] Example 9: Oncolytic Activity of Intravenous IVX037 in Human Colorectal Cancer (WiDr) Xenografts

[0299] -Materials and methods

[0300] Immunocompromised SCID-Balb / C mice were subcutaneously inoculated with human colorectal cancer cells WiDr (2e5 cells). Once tumors became palpable (approximately 4 mm3), mice were injected intravenously four times with either a control vehicle or the IVX-037OVO strain of the present invention (1e8 TCID50 / injection) at intervals of 3 to 4 days. Tumor volume was measured, and animals were terminated 22 days after treatment.

[0301] -result

[0302] Figure 13 Shown are the mean (A) and individual (B) tumor volumes of mice treated with IVX037 strain. Potent antitumor activity was observed in WiDr xenografts following four intravenous injections of IVX037 strain. Treatment was well tolerated, and no side effects were observed. Statistical differences between treatment groups were determined using a two-way ANOVA.

[0303] Example 10: Synthesis of IVX-037OVO virus strain

[0304] The IVX-037OVO strain of the present invention belongs to the Enterovirus B species, specifically, the wild-type echovirus 12 (E12) prototype strain Travis. To obtain IVX-037OVO material with a complete manufacturing history and mitigate the risk of exogenous factors, the IVX-037OVO strain was rescued from a pUC-like plasmid containing the complete E12 viral sequence after transient transfection of Vero MCB LN 1595.01 cells. The E12 viral sequence inserted into the pUC-like plasmid was not genetically modified. This plasmid was synthesized at GeneArt, ThermoFisher Scientific, and the E12 progeny virus rescue procedure was performed at ImVirX. An overview of the IVX037 pre-MVSS preparation is summarized below.

[0305]

[0306] Further details of the IVX-037OVO strain synthesis process are summarized in Table 1 below:

[0307] Table 1: IVX-037OVO synthesis process

[0308]

[0309]

[0310] footnote:

[0311] Bold: Samples selected for further processing in the next processing step

[0312] Example 11: IVX037 as a mediator of immune responses and anti-tumor activity

[0313] CXCL10 is a marker of the severity of viral infection and promotes the recruitment of T cells, natural killer cells, macrophages, and dendritic cells. Evidence suggests that CXCL10 is essential for the recruitment of anti-tumor T cells to melanoma tumors. Furthermore, CXCL10 signaling through the CXCR3 receptor promotes lymphocyte migration to dendritic cells, which is required for response to PD-1 blockade in transplantable mouse models. Clinically, there is evidence that high pre-treatment CXCL9 and CXCL10 levels are associated with response to anti-PD-(L)1 therapy in patients with non-small cell lung cancer, and that CXCL9 and CXCL10 increase during the first few months of treatment in patients with melanoma who respond to PD-1 inhibitor therapy.

[0314] The LEGENDplex bead-based multiplex assay TM Human Essential Immune ResponsePanel(13-plex),#740930,LEGENDplex TM , Biolegend Inc., USA) quantified the patient's serum. This assay can detect 13 inflammatory cytokines / chemokines, including IL-4, IL-2, CXCL10 (IP-10), IL-1β, TNF-α, CCL2 (MCP-1), IL-17A, IL-6, IL-10, IFN-γ, IL-12p70, CXCL8 (IL-8). The staining procedure was performed as recommended by the manufacturer's protocol. Flow cytometry was performed using a BD LSRFortessa TM X-20 (Becton Dickinson Biosciences, San Jose, USA). Cytokine concentration was determined using BioLegend's LEGENDplex supplied by the manufacturer. TM The data analysis software was calculated based on the standard curve. Cytokine data were analyzed on days 1, 8, 15, and 29. Preliminary serum biomarker analysis indicated that IVX037 induced potentially beneficial inflammatory cytokines / chemokines, such as early signs of CXCL10 ( Figure 18 ).

[0315] Example 12: Infectivity and Oncolytic Effects of IVX037 in Hepatocellular Carcinoma (HCC) Cell Lines

[0316] A panel of seven hepatocellular carcinoma cell lines with varying degrees of genetic complexity was purchased from ATCC, including SNU-475, C3A [HepG2 / C3A, a derivative of Hep G2], SNU-449, PLC / PRF / 5, SNU-387, SK-HEP-1, and SNU-423. Figure 19 The data in provide an initial overview of the oncolytic effect of IVX037 on these liver cancer lines.

[0317] Example 13: Phase 1a open-label, non-randomized, multicenter clinical trial of intratumoral IVX037 in patients with advanced microsatellite stable (MSS) colorectal, gastroesophageal, or ovarian cancer: trial ongoing

[0318] background

[0319] In human colorectal, gastric, and ovarian cancer cell cultures, IVX037 challenge induced selective in vitro tumor cell lytic infection through specific viral capsid-cell receptor interactions.

[0320] In SCID mice, microsatellite stable (MSS) colorectal cancer ( Figure 14 ), gastric cancer, and ovarian cancer human xenografts showed significant anti-tumor activity by a single intratumoral injection of IVX037.

[0321] In vivo studies

[0322] In vivo human MSS colorectal cancer xenograft studies in mice revealed that intratumoral administration of IVX037 induced elevated levels of g-INF-responsive genes (CXCL10, RIG-I) and upregulated expression of the important immune checkpoint molecule PD-L1 ( Figure 15 ), indicating an inflammatory phenotype within the treated tumor microenvironment (TME).

[0323] The preliminary pharmacodynamic study was approved by the Newcastle University Animal Ethics Committee and used in-house purified IVX037 propagated from an in-house viral seed stock. Female severe combined immunodeficient (SCID) mice (8 / group) bearing human xenografts of WiDr (an immortalized cell line of human colorectal adenocarcinoma cells) were evaluated by intratumoral (IT) administration of 1x10 8 TCID 50 Activity of a single dose of IVX037 (TR 2020-007)32.

[0324] The results highlighted that although the tumor volume of mice in the vehicle (control) group gradually increased within three weeks after treatment, IVX037 administration reduced the tumor volume of mice in the treatment group 2 days after infection and stabilized until day 9, and then slowly increased until day 21 after infection. Overall, the mean tumor volume of mice in the IVX037-treated group was statistically significantly smaller than that in the vehicle group ( Figure 5 A). In addition, there was no significant change in body weight of mice from either treatment group.

[0325] Gene profiling by RT-PCR in IVX037-treated tumors revealed that expression of retinoic acid-inducible gene 1 (RIG-I) and CXC motif chemokine ligand 10 (CXCL10) increased and peaked 1 day after IVX037 infection (approximately 5-fold change), whereas expression of CD274 (the gene encoding PD-L1) increased approximately 1.3-fold change and remained stable for 3 days after infection ( Figure 15 ).

[0326] When the tumor reaches an average size (approximately 50 mm 3 ), mice were treated with virus IVX037 (n = 8 mice) or control formulation buffer (n = 6 mice) on day 0. Mice were sacrificed 1, 2, and 3 days after treatment. Tumors were harvested and RNA was extracted. Expression of RIG-I, CD274, and CXCL10 was measured using RT-PCR and calculated as fold change.

[0327] It has been proposed that the induction of a virally inflamed TME may allow for increased migration of anti-tumor lymphocytes both within the injection lesion and in distant lesions, as well as elevated levels of cellular targets of immune checkpoint therapy.

[0328] Increased serum levels of CXCL10 and CCL22 were associated with response in melanoma patients administered another RNA oncolytic virus, V927, in combination with pembrolizumab, suggesting that viral replication promotes antitumor immunity (Silk AW, et al, 2023 Cancer Immunol Immunother; 72(6): 1405-1415).

[0329] Clinical trial methods

[0330] This is a Phase 1a, first-in-human, open-label, non-randomized, multicenter clinical trial of intratumoral IVX037 in patients with advanced MSS colorectal, gastroesophageal, or ovarian cancer. The full trial protocol can be found in Figure 17 .

[0331] Inclusion criteria: Patients (pts) must have one injectable tumor of the liver / nodal / peritoneal disease.

[0332] • Exclusion criteria: Candidates for liver surgery or locoregional therapy for liver or other lesions. Clinically significant ascites (grade ≥ 2), ongoing systemic treatment with any corticosteroids (> 10 mg per day).

[0333] Intervention: Patients will be sequentially enrolled in three escalating dose cohorts to receive 1 (n=3 patients), 2 (n=3 patients), or up to 7 doses (n=15 patients) of IVX037 at the investigator's discretion in the absence of dose-limiting toxicities (DLTs), with each dose up to 3 x 10 8 TCID 50 , administered intratumorally on days 1, 15, 29, 43, 57, 71, and 85 (as applicable).

[0334] Primary Objective: To determine the feasibility, safety, and tolerability of intratumoral IVX037, including the incidence of dose-limiting toxicities (DLTs).

[0335] Secondary objective: To evaluate the maximum tolerated dose (MTD) of IVX037 administered as 1, 2, or 3 injections / lesion. Tumor response was assessed using RECIST 1.1, with the first response assessment performed on day 50.

[0336] Exploratory Objectives: The effects of IVX037 administration on several biomarkers in peripheral blood and tumor tissue will be evaluated, including expression levels of tumor-infiltrating lymphocytes and cellular targets of immune checkpoint therapy.

[0337] result

[0338] Patient recruitment began in April 2023 and has currently enrolled 8 patients. IVX037 dosing in Cohorts 1 and 2 is complete, and dosing in Cohort 3 is ongoing.

[0339] IVX037 intralesional administration has been generally well tolerated to date, with all patients demonstrating some level of systemic exposure immediately following injection ( Figure 16 A) No dose-limiting toxicity was observed. Mild flu-like reactions (fatigue, chills, rigors, injection site discomfort) were observed after injection.

[0340] IVX037 has been successfully administered to the liver, lymph nodes, and abdominal metastases.

[0341] All patients have now developed serum neutralizing anti-IVX037 antibodies by day 15 after virus administration ( Figure 16 B).

[0342] Preliminary serum biomarker analysis has shown that IVX037 induces potentially beneficial inflammatory cytokines / chemokines, such as early signs of CXCL10 ( Figure 16 C).

[0343] Recruitment is ongoing, and a phase 1b trial in combination with immune checkpoint blockade is planned.

Claims

1. A modified picornavirus comprising changes in any one or more of the capsid proteins VP1, VP2, and VP3 compared to a wild-type strain of the virus; said changes confer enhanced decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn) binding ability compared to a wild-type strain.

2. The modified picornavirus according to claim 1, comprising said changes in each of the VP2 and VP3 capsid proteins.

3. The modified picornavirus according to claim 1, comprising said changes in each of the VP1, VP2 and VP3 capsid proteins.

4. The modified picornavirus according to any one of claims 1 to 3, further comprising a change in any one or more of the non-structural proteins 2A, 3A, 3C, and 3D compared to the wild-type strain of the virus; the change confers enhanced decay accelerating factor (DAF / CD55) binding ability compared to the wild-type strain.

5. The modified picornavirus according to any one of claims 1 to 4, wherein the wild-type strain and the modified picornavirus have the same nucleotide sequence and / or the same amino acid sequence except for the following changes: (i) changes in any one or more of the capsid proteins VP1, VP2, VP3; and optionally (ii) changes in any one or more of the nonstructural proteins 2A, 3A, 3C, 3D.

6. The modified picornavirus according to any one of claims 1 to 5, wherein the modified picornavirus is an enterovirus.

7. The modified picornavirus of claim 6, wherein the enterovirus is selected from the group consisting of an echovirus, a poliovirus, an unclassified enterovirus, a rhinovirus, a double echovirus, a hepatovirus, and a cardiovirus.

8. The modified picornavirus of any one of claims 1-7, wherein the modified picornavirus is not a coxsackievirus.

9. The modified picornavirus of any one of claims 1-8, wherein the modified picornavirus is an echovirus, enterovirus B85, coxsackievirus A9, coxsackievirus A13, coxsackievirus 15, or coxsackievirus 21.

10. The modified picornavirus of any one of claims 1 to 9, wherein the modified picornavirus is echovirus 1, echovirus 3, echovirus 6, echovirus 7, echovirus 9, echovirus 11, echovirus 12 (E12), echovirus 12, echovirus 13, echovirus 14, echovirus 15, echovirus 17, echovirus 25, echovirus 26, echovirus 29, or echovirus 30.

11. The modified picornavirus according to any one of claims 1 to 10, wherein the change in capsid protein VP2 comprises or consists of an asparagine to threonine change at residue 142.

12. The modified picornavirus according to any one of claims 1 to 11, wherein the change in capsid protein VP3 comprises or consists of an alanine to valine change at residue 206.

13. The modified picornavirus according to any one of claims 1 to 12, wherein the changes in the capsid protein VP2 comprise or consist of a histidine to tyrosine change at residue 154 and / or a serine to asparagine change at residue 168.

14. The modified picornavirus according to any one of claims 1 to 13, wherein the change in the capsid protein VP1 comprises or consists of a tyrosine to histidine change at residue 230.

15. The modified picornavirus according to any one of claims 1 to 14, wherein the change in the capsid protein VP1 comprises or consists of a change from phenylalanine to tyrosine at residue 210.

16. The modified picornavirus according to any one of claims 1 to 15, wherein the change in capsid protein VP1 comprises or consists of a glutamine to arginine change at residue 132.

17. The modified picornavirus of claim 16, wherein the changes confer enhanced neonatal Fc receptor (FcRn) binding.

18. The modified picornavirus according to any one of claims 1 to 17, wherein the changes in nonstructural proteins 2A, 3A, 3C, 3D comprise or consist of any one or more of the following: - a phenylalanine to tyrosine change at residue 47 of the nonstructural 2A protein; - an isoleucine to tyrosine change at residue 6 of the nonstructural 3A protein; - a histidine to arginine change at residue 39 of the nonstructural 3C protein; - An isoleucine to leucine change at residue 123 of the unstructured 3D protein.

19. The modified picornavirus of any one of claims 1-18, wherein the wild-type strain is Echovirus 12 strain Travis.

20. The modified picornavirus of any one of claims 1-19, wherein the wild-type strain is Echovirus 12 strain Travis, comprising or consisting of the amino acid sequence defined in SEQ ID NO:

46.

21. The modified picornavirus of any one of claims 1 to 20, further comprising at least one nucleic acid sequence encoding an exogenous protein or a component thereof.

22. The modified picornavirus according to claim 21, wherein the exogenous protein is an immunostimulatory protein or an agent (e.g., an antibody) that can bind to an immune checkpoint molecule or a ligand of the immune checkpoint molecule and inhibit its biological activity.

23. The modified picornavirus of claim 22, wherein the immunostimulatory protein is selected from any one or more of the following: interleukins, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, interferons, including but not limited to IFN-γ, TNF-α, prodrug converting enzymes, biologically active components thereof, and combinations thereof.

24. The modified picornavirus of claim 22, wherein the immune checkpoint molecule is selected from any one or more of the following: PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, LAG-3, ICOS, TIM3, GAL9, CD28, OX-40, and TIGIT, biologically active components thereof, and combinations thereof.

25. The modified picornavirus according to any one of claims 1 to 24, further comprising one or more components for expressing tissue-specific miRNAs capable of inhibiting the replication of the modified picornavirus in a tissue-specific manner.

26. The modified picornavirus of any one of claims 1-25, comprising enhanced oncolytic activity compared to a wild-type strain.

27. The modified picornavirus of claim 26, wherein the enhanced oncolytic activity is against any one or more of ovarian cancer cells, colorectal cancer cells, gastric cancer cells, liver cancer cells, pancreatic cancer cells, head and neck cancer cells, gastric cancer cells, breast cancer cells, sarcoma cells, lymphoma cells, brain cancer cells.

28. The modified picornavirus of claim 27, wherein the cancer cell expresses decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

29. The modified picornavirus of any one of claims 1-28, wherein the picornavirus comprises: (i) a VP1 capsid protein amino acid sequence comprising any one of SEQ ID NOs: 54-57, 74, 86, 98, 110, 122, 134, 146 or 158; (ii) a VP2 capsid protein amino acid sequence comprising any one of SEQ ID NO: 58-61, 75, 87, 99, 111, 123, 135, 147 or 159; (iii) a VP3 capsid protein amino acid sequence comprising any one of SEQ ID NO: 62, 63, 76, 88, 100, 112, 124, 136, 148 or 160; (iv) a nonstructural protein 2A sequence comprising any one of SEQ ID NO: 64, 65, 77, 89, 101, 113, 125, 137, 149, or 161; (v) a nonstructural protein 3A sequence comprising any one of SEQ ID NO: 66, 67, 78, 90, 102, 114, 126, 138, 150, or 162; (vi) a nonstructural protein 3C sequence comprising any one of SEQ ID NO: 68, 69, 79, 91, 103, 115, 127, 139, 151, or 163; (vii) a nonstructural protein 3D sequence comprising any one of SEQ ID NO: 70, 71, 80, 92, 104, 116, 128, 140, 152 or 164.

30. The modified picornavirus of any one of claims 1 to 29, comprising or consisting of an amino acid sequence as defined in SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 73, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 109, SEQ ID NO: 121, SEQ ID NO: 133, SEQ ID NO: 145, or SEQ ID NO: 157, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 73, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 109, SEQ ID NO: 121, SEQ ID NO: 133, SEQ ID NO: 145, or SEQ ID NO:

157.

31. The modified picornavirus according to any one of claims 1 to 30, which is encoded by a nucleotide sequence comprising an RNA or cDNA / DNA sequence defined in SEQ ID NO: 41, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 144 or SEQ ID NO: 156, or a combination thereof with an RNA or cDNA / DNA sequence defined in SEQ ID NO: 41, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 144 or SEQ ID NO:

156. A variant of the RNA or cDNA / DNA sequence defined in NO: 156 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, or consisting of said RNA or cDNA / DNA sequence or said variant.

32. The modified picornavirus of any one of claims 1-31, which is synthesized using recombinant methods.

33. A pharmaceutical composition comprising any one or more of the following and a pharmaceutically acceptable carrier, excipient or diluent: - a modified picornavirus according to any one of claims 1 to 32, - the RNA of the modified picornavirus according to any one of claims 1 to 32, - a complementary DNA (cDNA) encoding all or part of the genome of the modified picornavirus according to any one of claims 1 to 32.

34. The pharmaceutical composition of claim 33, wherein the RNA of the modified picornavirus (e.g., synthetic RNA) and / or complementary DNA (cDNA) encoding all or a portion of the genome of the modified picornavirus is provided within a nanoparticle (e.g., a lipid nanoparticle).

35. The pharmaceutical composition of claim 33 or 34, further comprising CAR-T cells, natural killer (NK) cells, immunostimulatory proteins and / or agents (e.g., antibodies) capable of binding to immune checkpoint molecules or ligands of the immune checkpoint molecules.

36. The pharmaceutical composition of claim 35, wherein the CAR-T cells are engineered to bind to any one or more of the following on the surface of cancer cells: EGFRVIII, interleukin 13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Muc1, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Muc1, EphA2, CD123, CD19, Claudin 18.

2.

37. The pharmaceutical composition of claim 35 or 36, wherein the immunostimulatory protein is selected from any one or more of the following: interleukins, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, interferons, including but not limited to IFN-γ, TNF-α, prodrug converting enzymes, biologically active components thereof, and combinations thereof.

38. The pharmaceutical composition of any one of claims 35-37, wherein the immune checkpoint molecule is selected from any one or more of the following: PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, LAG-3, ICOS, TIM3, GAL9, CD28, OX-40 and TIGIT, biologically active components thereof, and combinations thereof.

39. The pharmaceutical composition according to any one of claims 33-38, wherein the composition comprises about 10 8 to about 10 15 Virus particles / mL, about 10 8 to 10 12 virus particles / mL or about 10 8 to 10 10 Virus particles / mL of picornavirus.

40. The pharmaceutical composition of any one of claims 33-39, wherein the composition comprises a particle to infectivity ratio of less than or equal to about 3000: 1, less than or equal to about 300: 1, in the range of about 200: 1 to about 5: 1, in the range of about 50: 1 to about 10: 1, or less than about 20:

1.

41. The pharmaceutical composition of any one of claims 33-40, wherein the composition comprises a host cell DNA level of less than or equal to about 200 ng / mL, within a range of 1 to about 100 ng / mL, within a range of about 1 to about 50 ng / mL, or within a range of about 1 to about 10 ng / mL.

42. A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of any one or more of: - a modified picornavirus according to any one of claims 1 to 32; - RNA of the modified picornavirus according to any one of claims 1 to 32; - a complementary DNA (cDNA) encoding all or part of the genome of the modified picornavirus according to any one of claims 1 to 32; - A pharmaceutical composition according to any one of claims 33 to 41.

43. The method of claim 42, wherein the modified picornavirus is bioselected for any one or more of: enhanced migration between cancer cells, enhanced binding to DAF and / or FcRn, enhanced replication capacity at mammalian body temperature (33°C–39°C), and enhanced lytic activity against cancer cells expressing any one or more of PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, ICOS, TIM3, LAG-3, GAL9, CD28, and / or OX-40.

44. The method of claim 43, wherein the modified picornavirus is bioselected in vivo using tumor xenografts.

45. The method of claim 44, wherein the tumor is from the subject.

46. ​​The method of any one of claims 42-45, wherein the therapeutically effective amount is administered to the subject by parenteral, intravenous, intravesical, subcutaneous, oral, intratumoral, ophthalmic, topical, or systemic administration.

47. The method of any one of claims 42-46, wherein the therapeutically effective amount is co-administered with CAR-T cells and / or natural killer (NK) cells.

48. The method of claim 47, wherein the CAR-T cells are engineered to bind to any one or more of the following on the surface of cancer cells: EGFRVIII, interleukin 13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Muc1, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Muc1, EphA2, CD123, CD19, Claudin 18.

2.

49. The method of claim 47 or 48, wherein the CAR-T cells and / or natural killer (NK) cells are co-administered before or after administering the therapeutically effective amount to the subject.

50. The method of any one of claims 42-49, wherein the therapeutically effective amount is co-administered with an immunostimulatory protein and / or an agent (e.g., an antibody) that can bind to an immune checkpoint molecule or a ligand of the immune checkpoint molecule.

51. The method of claim 50, wherein the immunostimulatory protein is selected from any one or more of the following: interleukins, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, interferons, including but not limited to IFN-γ, TNF-α, prodrug converting enzymes, biologically active components thereof, and combinations thereof.

52. The method of claim 50 or 51, wherein the immune checkpoint molecule is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, ICOS, LAG-3, TIM3, GAL9, CD28, OX-40, and TIGIT.

53. The method of any one of claims 50-52, wherein the agent is a monoclonal antibody.

54. The method of any one of claims 50-53, wherein the immunostimulatory protein and / or agent is co-administered before or after administering the therapeutically effective amount to the subject.

55. The method of any one of claims 42-54, wherein the cancer is classified as a cancer resistant to immune checkpoint therapy.

56. The method of any one of claims 42-55, wherein the cancer is ovarian cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, head and neck cancer, stomach cancer, breast cancer, sarcoma, lymphoma, or brain cancer.

57. The method of claim 56, wherein the cancer comprises cancer cells expressing decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

58. Use of any one or more of the following in the preparation of a medicament for treating cancer in a subject: - a modified picornavirus according to any one of claims 1 to 32; - RNA of the picornavirus according to any one of claims 1 to 32; - a complementary DNA (cDNA) encoding all or part of the genome of the picornavirus according to any one of claims 1 to 32; - A pharmaceutical composition according to any one of claims 33 to 41.

59. The method of claim 58, wherein the drug further comprises CAR-T cells, natural killer (NK) cells, immunostimulatory proteins and / or agents (e.g., antibodies) capable of binding to immune checkpoint molecules or ligands of the immune checkpoint molecules.

60. The modified picornavirus according to any one of claims 1 to 32, the RNA of the picornavirus according to any one of claims 1 to 32, the complementary DNA (cDNA) encoding all or part of the genome of the picornavirus according to any one of claims 1 to 32, and / or the pharmaceutical composition according to any one of claims 33 to 41, for use in treating cancer.

61. The modified picornavirus, RNA and / or cDNA according to claim 60, for use in the treatment of cancer in combination with CAR-T cells and / or natural killer (NK) cells and / or immunostimulants.

62. The use according to claim 59 or the modified picornavirus, RNA and / or cDNA according to claim 61, wherein the CAR-T cell is engineered to bind to any one or more of the following on the surface of cancer cells: EGFRVIII, interleukin 13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Muc1, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Muc1, EphA2, CD123, CD19, Claudin 18.

2.

63. The use according to claim 59 or 62 or the modified picornavirus, RNA and / or cDNA according to claim 60 or 61, wherein the immunostimulatory protein is selected from any one or more of the following: interleukins, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, interferons, including but not limited to IFN-γ, TNF-α, prodrug converting enzymes, biologically active components thereof, and combinations thereof.

64. The use of claim 63 or the modified picornavirus, RNA and / or cDNA of claim 84, wherein the immune checkpoint molecule is selected from the group consisting of: PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, LAG-3, B7RP1, ICOS, TIM3, GAL9, CD28, OX-40 and TIGIT.

65. The use of any one of claims 58, 59, or 62-64, or the modified picornavirus, RNA, and / or cDNA of any one of claims 60-64, wherein the modified picornavirus is bioselected for any one or more of: enhanced migration between cancer cells, enhanced binding to DAF and / or FcRn, enhanced replication capacity at mammalian body temperature (37°C-39°C), enhanced lytic activity against cancer cells expressing any one or more of PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4, B7RP1, LAG-3, ICOS, TIM3, GAL9, CD28, and / or OX-40.

66. The use according to any one of claims 58, 59 or 62-65 or the modified picornavirus, RNA and / or cDNA according to any one of claims 60-65, wherein the modified picornavirus is bioselected in vivo using tumor xenografts.

67. The use according to claim 66 or the modified picornavirus, RNA and / or cDNA according to claim 66, wherein the tumor is from the subject.

68. The use of any one of claims 58, 59, or 62-67, or the modified picornavirus, RNA, and / or cDNA of any one of claims 60-67, wherein the cancer is ovarian cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, head and neck cancer, stomach cancer, breast cancer, sarcoma, lymphoma, or brain cancer.

69. The use of claim 68 or the modified picornavirus, RNA and / or cDNA of claim 68, wherein the cancer comprises cancer cells expressing decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

70. The use of any one of claims 58, 59 or 62-69, or the modified picornavirus, RNA and / or cDNA of any one of claims 60-69, wherein the modified picornavirus comprises an amino acid sequence as defined in SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 73, SEQ ID NO: 85, SEQ ID NO: 97, SEQ ID NO: 109, SEQ ID NO: 121, SEQ ID NO: 133, SEQ ID NO: 145 or SEQ ID NO: 157, or a combination thereof. NO:157 has a variant with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, or consists of said amino acid sequence or said variant.

71. The method of claim 58, 59, or 62-70, or the method of claim 60-7091, wherein the modified picornavirus is encoded by a nucleotide sequence comprising an RNA or cDNA / DNA sequence as defined in SEQ ID NO: 41, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 72, SEQ ID NO: 84, SEQ ID NO: 96, SEQ ID NO: 108, SEQ ID NO: 120, SEQ ID NO: 132, SEQ ID NO: 144, or SEQ ID NO: 156, or a combination thereof. A variant of the RNA or cDNA / DNA sequence defined in NO: 156 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity, or consisting of said RNA or cDNA / DNA sequence or said variant.

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