Oncolytic viruses as therapeutic agents for treating cancer

Abstract

The present invention relates to compositions and methods for treating cancer utilizing oncolytic viruses, components thereof, and / or derivatives thereof. In particular, the present invention relates to modified picornaviruses that contain alterations in one or more of their capsid proteins that confer enhanced binding ability to cancer cells.

Description

[Technical Field]

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

[0002] Viral oncolytic therapy has emerged as a promising treatment for several human and animal cancers.

[0003] Picornaviridae is one of the largest families of viruses named using the Greek word "pico" (very small) and "RNA" for its ribonucleic acid genome. This family includes several clinically important human and animal pathogens, including poliovirus, rhinovirus, and hepatitis A. The Picornaviridae family has been classified into nine genera based on physical virion characteristics, RNA sequence similarity, and viral RNA genome organization (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. pp. 17-24). They are small, non-enveloped, icosahedral viruses whose capsids contain 60 copies of each of four viral proteins, VP1, VP2, VP3, and VP4, forming an icosahedral shell (approximately 300 Å in diameter) that houses a single-stranded, positive-sense RNA genome. A prominent feature of the capsid surface is a depression around a five-fold axis of symmetry (the "canyon"), which is the receptor-binding site for many picornaviruses, including echoviruses that utilize β integrins. Receptor molecules that bind at the canyon have been identified as members of the immunoglobulin superfamily, and upon binding, they remove factors within a pocket just below the canyon surface. When receptors bind within the canyon, the floor of the canyon, corresponding to the roof of the pocket, is depressed. Similarly, binding of antiviral compounds or lipids to the pocket causes the roof of the pocket to expand, corresponding to the floor of the canyon. Thus, the pocket factors and canyon-bound receptors compete with each other for virus binding. The absence of hydrophobic pocket factors destabilizes the virus, initiating a transition to an altered morphology that is a precursor to virion uncoating. Examples of cellular receptors that bind to the canyon include immunoglobulin superfamily receptors (eg, VCAM-1, ICAM-1, PVR, and CAR).

[0004] Some receptors bind to picornaviruses without using the canyon. For example, a subgroup of human rhinoviruses (HRVs) binds 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 conjunction with another receptor. DAF is a member of the complement activation protein family that binds to and promotes the decay of both classical and alternative pathway C3 and C5 convertases, the central amplification enzymes of the complement cascade. DAF is expressed on the surface of virtually all cells and protects host cells from the immune system by rapidly dissociating any convertases that assemble, thereby halting complement attack directed against self-cells. The functional region of DAF consists of four short consensus repeats (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 DAF at different sites on their receptor surfaces.

[0005] Oncolytic virotherapy is based on the ability of viruses to infect and kill tumor cells without destroying normal tissue. While some viruses have a natural preference for tumor cells, most require at least some modification of their receptor tropism to specifically invade and replicate in cancerous cells. Oncolytic picornaviruses have 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 possess the virus-cell entry receptor intercellular adhesion molecule-1 (ICAM-1). The efficacy of CVA21 against melanoma cells has been demonstrated in various preclinical xenograft models using immunodeficient mice and against other forms of cancer. Several other cellular receptor targets exist that provide potential avenues for the design of effective oncolytic picornaviruses.

[0006] The recent emergence of immune checkpoint inhibitor therapy has transformed cancer treatment across a wide range of tumor types. Effective and durable clinical responses have been observed in difficult-to-treat cancer histologies. However, despite these promising long-term responses, many patients fail to respond to immune checkpoint blockade and exhibit primary resistance. In addition, many patients who initially respond to treatment relapse following acquired resistance. Both primary and acquired resistance are the result of complex and constantly evolving interactions between the immune system and cancer cells (see, e.g., Fares et al., American Society of Clinical Oncology Educational Book 39, May 17, 2019, pp. 147-164).

[0007] There remains a need for improved compositions and methods for the treatment, alleviation, or prevention of cancer, including cancer types that are poorly responsive to immune checkpoint inhibitor therapy. Summary of the Invention

[0008] We have generated modified Picornaviridae family viruses that have enhanced ability to bind to decay-accelerating factor (DAF / CD55) on the surface of cancer cells compared to their wild-type counterparts, which have been demonstrated to be effective oncolytic agents against cancer cells.

[0009] Although not limited to a particular form of cancer, the compositions and methods described herein may be 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 may also 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, etc.

[0011] Although not limited thereto, the present invention relates to at least the following embodiments 1 to 71. Embodiment 1. A modified picornavirus, comprising alterations in any one or more of the capsid proteins: VP1, VP2, VP3, compared to a wild-type strain of the virus, that confer enhanced binding ability to decay-accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn), compared to a wild-type strain. Embodiment 2. The modified picornavirus of embodiment 1, comprising said alteration(s) in each of the VP2 and VP3 capsid proteins. Embodiment 3. The modified picornavirus of embodiment 1, comprising said alteration(s) in each of the VP1, VP2, and VP3 capsid proteins. Embodiment 4. The modified picornavirus of any one of embodiments 1-3, further comprising an alteration(s) in any one or more of the following non-structural proteins compared to a wild-type strain of the virus that confers enhanced binding ability to decay-accelerating factor (DAF / CD55) compared to the wild-type strain. Embodiment 5. The wild-type strain and the modified picornavirus are (i) a change(s) in any one or more of the capsid proteins VP1, VP2, VP3, and optionally, (ii) Nonstructural proteins: A modified picornavirus according to any one of embodiments 1 to 4, having the same nucleotide sequence and / or the same amino acid sequence, except for a change(s) in any one or more of 2A, 3A, 3C, and 3D. Embodiment 6. The modified picornavirus of any one of embodiments 1 to 5, wherein the modified picornavirus is an enterovirus. Embodiment 7. The modified picornavirus of embodiment 6, wherein the enterovirus is selected from the group consisting of echovirus, poliovirus, unclassified enterovirus, rhinovirus, paraechovirus, hepatovirus, and cardiovirus. Embodiment 8. The modified picornavirus of any one of embodiments 1 to 7, wherein the modified picornavirus is not a coxsackievirus. Embodiment 9. The modified picornavirus of any one of embodiments 1 to 8, wherein the modified picornavirus is an echovirus, enterovirus B85, coxsackievirus A9, coxsackievirus A13, coxsackievirus 15, or coxsackievirus 21. Embodiment 10. The modified picornavirus of any one of embodiments 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. Embodiment 11. The modified picornavirus of any one of embodiments 1 to 10, wherein the change(s) in capsid protein VP2 comprise or consist of an asparagine to threonine change at residue 142. Embodiment 12. The modified picornavirus of any one of embodiments 1 to 11, wherein the change(s) in the capsid protein VP3 comprise or consist of an alanine to valine change at residue 206. Embodiment 13. The modified picornavirus of any one of embodiments 1 to 12, wherein the change(s) in 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. Embodiment 14. The modified picornavirus of any one of embodiments 1 to 13, wherein the change(s) in the capsid protein VP1 comprise or consist of a change from tyrosine to histidine at residue 230. Embodiment 15. The modified picornavirus of any one of embodiments 1 to 14, wherein the change(s) in the capsid protein VP1 comprise or consist of a change from phenylalanine to tyrosine at residue 210. Embodiment 16. The modified picornavirus of any one of embodiments 1 to 15, wherein the change(s) in the capsid protein VP1 comprise or consist of a change from glutamine to arginine at residue 132. Embodiment 17. The modified picornavirus of embodiment 16, wherein the alteration confers enhanced binding to the neonatal Fc receptor (FcRn). Embodiment 18. Nonstructural proteins: The change(s) in 2A, 3A, 3C, 3D are: - a phenylalanine to tyrosine change at residue 47 of the unstructured 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 nonstructural 3D protein. Embodiment 19. The modified picornavirus of any one of embodiments 1 to 18, wherein the wild-type strain is Echovirus 12 strain Travis. Embodiment 20. The modified picornavirus of any one of embodiments 1 to 19, wherein the wild-type strain is Echovirus 12 strain Travis, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46. Embodiment 21. The modified picornavirus of any one of embodiments 1 to 20, further comprising at least one nucleic acid sequence encoding an exogenous protein or component thereof. Embodiment 22. The modified picornavirus of embodiment 21, wherein the exogenous protein is an immunostimulatory protein or an agent (e.g., an antibody) capable of binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule and inhibiting its biological activity. Embodiment 23. The modified picornavirus of embodiment 22, wherein the immunostimulatory protein is selected from any one or more of an interleukin, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, an interferon, including but not limited to IFN-γ, TNF-α, a prodrug converting enzyme, biologically active component(s) thereof, or a combination thereof. Embodiment 24. The modified picornavirus of embodiment 22, wherein the immune checkpoint molecule is selected from 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, OX-40, and TIGIT, biologically active component(s) thereof, or combinations thereof. Embodiment 25. The modified picornavirus of any one of embodiments 1 to 24, further comprising one or more components for expression of tissue-specific miRNAs capable of inhibiting replication of the modified picornavirus in a tissue-specific manner. Embodiment 26. A modified picornavirus according to any one of embodiments 1 to 25, comprising enhanced oncolytic activity compared to a wild-type strain. Embodiment 27. The modified picornavirus of embodiment 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, stomach cancer cells, breast cancer cells, sarcoma cells, lymphoma cells, and brain cancer cells. Embodiment 28. The modified picornavirus of embodiment 27, wherein the cancer cells express decay-accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn). Embodiment 29. The picornavirus is (i) a VP1 capsid protein amino acid sequence comprising any one of SEQ ID NOs: 54 to 57, 74, 86, 98, 110, 122, 134, 146, or 158; (ii) 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; (iii) 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; (iv) a nonstructural protein 2A sequence comprising any one of SEQ ID NOs: 64, 65, 77, 89, 101, 113, 125, 137, 149, or 161; (v) a nonstructural protein 3A sequence comprising any one of SEQ ID NOs: 66, 67, 78, 90, 102, 114, 126, 138, 150, or 162; (vi) a nonstructural protein 3C sequence comprising any one of SEQ ID NOs: 68, 69, 79, 91, 103, 115, 127, 139, 151, or 163; (vii) A modified picornavirus according to any one of embodiments 1 to 28, comprising a nonstructural protein 3D sequence comprising any one of SEQ ID NOs: 70, 71, 80, 92, 104, 116, 128, 140, 152, or 164. Embodiment 30. The modified picornavirus of any one of embodiments 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. Embodiment 31. The modified picornavirus of any one of embodiments 1 to 30, encoded by a nucleotide sequence comprising or consisting of the 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 variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the 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. Embodiment 32. A modified picornavirus according to any one of embodiments 1 to 31, which is synthesized using recombinant means. Embodiment 33. - a modified picornavirus according to any one of embodiments 1 to 32; - RNA of a modified picornavirus according to any one of embodiments 1 to 32, - one or more complementary DNAs (cDNAs) encoding all or part of the genome of a modified picornavirus according to any one of embodiments 1 to 32; and a pharmaceutically acceptable carrier, excipient, or diluent. Embodiment 34. The pharmaceutical composition of embodiment 33, wherein the RNA (e.g., synthetic RNA) of the modified picornavirus and / or complementary DNA (cDNA) encoding all or part of the genome of the modified picornavirus is provided within a nanoparticle (e.g., a lipid nanoparticle). Embodiment 35. The pharmaceutical composition of embodiment 33 or 34, further comprising an agent (e.g., an antibody) capable of binding to CAR-T cells, natural killer (NK) cells, immunostimulatory proteins, and / or immune checkpoint molecules or ligands of immune checkpoint molecules. Embodiment 36. The pharmaceutical composition of embodiment 35, wherein the CAR-T cells are engineered to bind to any one or more of EGFRVIII, Interleukin-13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Mucl, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Mucl, EphA2, CD123, CD19, Claudin 18.2 on the surface of cancerous cells. Embodiment 37. The pharmaceutical composition of embodiment 35 or 36, wherein the immunostimulatory protein is selected from any one or more of an interleukin, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, an interferon, including but not limited to IFN-γ, TNF-α, a prodrug converting enzyme, biologically active component(s) thereof, or a combination thereof. Embodiment 38. The pharmaceutical composition of any one of embodiments 35 to 37, wherein the immune checkpoint molecule is selected from 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, OX-40, and TIGIT, biologically active component(s) thereof, or combinations thereof. Embodiment 39. The composition comprises about 108 ~about 10 15 Virus particles / mL, approximately 10 8 ~10 12 virus particles / mL, or approximately 10 8 ~10 10 39. The pharmaceutical composition of any one of embodiments 33 to 38, comprising a picornavirus in viral particles / mL. Embodiment 40. The pharmaceutical composition of any one of embodiments 33 to 39, wherein the composition comprises a particle to infectivity ratio of about 3000:1 or less, about 300:1 or less, 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. Embodiment 41. The pharmaceutical composition of any one of embodiments 33 to 40, wherein the composition comprises a level of host cell DNA of less than about 200 ng / mL, in the range of about 1 to about 100 ng / mL, in the range of about 1 to about 50 ng / mL, or in the range of about 1 to about 10 ng / mL. Embodiment 42. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of - a modified picornavirus according to any one of embodiments 1 to 32; - RNA of a modified picornavirus according to any one of embodiments 1 to 32, a complementary DNA (cDNA) encoding all or part of the genome of a modified picornavirus according to any one of embodiments 1 to 32, - A method comprising administering any one or more of the pharmaceutical compositions according to any one of embodiments 33 to 41. Embodiment 43. The method of embodiment 42, wherein the modified picornavirus is bioselected for any one or more of: enhanced movement among cancer cells; enhanced DAF and / or FcRn binding; enhanced replication capacity at mammalian body temperature (33°C to 39°C); enhanced lytic activity of 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. Embodiment 44 The method of embodiment 43, wherein the modified picornavirus is bioselected in vivo using tumor xenographs. Embodiment 45. The method of embodiment 44, wherein the tumor is from a subject. Embodiment 46. The method of any one of embodiments 42-45, wherein the therapeutically effective amount is administered to the subject by parenteral, intravenous, intravesical, subcutaneous, oral, intratumoral, ocular, topical, or systemic administration. Embodiment 47. The method of any one of embodiments 42 to 46, wherein the therapeutically effective amount is co-administered with CAR-T cells and / or natural killer (NK) cells. Embodiment 48. The method of embodiment 47, wherein the CAR-T cells are engineered to bind to any one or more of EGFRVIII, Interleukin-13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Mucl, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Mucl, EphA2, CD123, CD19, Claudin 18.2 on the surface of cancerous cells. Embodiment 49. The method of embodiment 47 or 48, wherein the CAR-T cells and / or natural killer (NK) cells are co-administered before or after administration of a therapeutically effective amount to the subject. Embodiment 50. The method of any one of embodiments 42 to 49, wherein the therapeutically effective amount is co-administered with an immunostimulatory protein and / or an agent (e.g., an antibody) capable of binding to an immune checkpoint molecule or a ligand of an immune checkpoint molecule. Embodiment 51. The method of embodiment 50, wherein the immunostimulatory protein is selected from any one or more of an interleukin, including but not limited to, interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, an interferon, including but not limited to IFN-γ, TNF-α, a prodrug converting enzyme, biologically active component(s) thereof, or a combination thereof. Embodiment 52. The method of embodiment 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. Embodiment 53. The method of any one of embodiments 50 to 52, wherein the agent is a monoclonal antibody. Embodiment 54. The method of 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. Embodiment 55. The method of any one of embodiments 42 to 54, wherein the cancer is classified as a cancer resistant to immune checkpoint therapy. Embodiment 56. The method of any one of embodiments 42 to 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. Embodiment 57. The method of embodiment 56, wherein the cancer comprises cancerous cells that express decay-accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn). Embodiment 58. In the manufacture of a medicament for treating cancer in a subject, - a modified picornavirus according to any one of embodiments 1 to 32; Picornaviral RNA according to any one of embodiments 1 to 32, a complementary DNA (cDNA) encoding all or part of the genome of a picornavirus according to any one of embodiments 1 to 32, - any one or more of the pharmaceutical compositions according to any one of embodiments 33 to 41, use. Embodiment 59. The use of embodiment 58, wherein the medicament further comprises an agent (e.g., an antibody) capable of binding to CAR-T cells, natural killer (NK) cells, immunostimulatory proteins, and / or immune checkpoint molecules or ligands of immune checkpoint molecules. Embodiment 60. A modified picornavirus according to any one of embodiments 1 to 32, RNA of a picornavirus according to any one of embodiments 1 to 32, a complementary DNA (cDNA) encoding all or part of the genome of a picornavirus according to any one of embodiments 1 to 32, and / or a pharmaceutical composition according to any one of embodiments 33 to 41, for use in the treatment of cancer. Embodiment 61. The modified picornavirus, RNA, and / or cDNA of embodiment 60 for use in treating cancer in combination with CAR-T cells, and / or natural killer (NK) cells, and / or immunostimulatory agents. Embodiment 62. The use of embodiment 59, or the modified picornavirus, RNA, and / or cDNA of embodiment 61, wherein the CAR-T cells are engineered to bind to any one or more of EGFRVIII, Interleukin-13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Mucl, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Mucl, EphA2, CD123, CD19, Claudin18.2 on the surface of cancerous cells. Embodiment 63. The use of embodiment 59 or 62, or the modified picornavirus, RNA, and / or cDNA of embodiment 60 or 61, wherein the immunostimulatory protein is selected from any one or more of 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 component(s) thereof, and combinations thereof. Embodiment 64. The use of embodiment 63, or the modified picornavirus, RNA, and / or cDNA of embodiment 84, wherein the immune checkpoint molecule is selected from the group consisting of PD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134 L, CD137, CD137 L, CD80, CD86, B7-H3, B7-H4, LAG-3, B7RP1, ICOS, TIM3, GAL9, CD28, OX-40, and TIGIT. Embodiment 65. The 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 movement among cancer cells; enhanced DAF and / or FcRn binding; enhanced replication capacity at mammalian body temperature (37°C to 39°C); enhanced lytic activity of 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. Embodiment 66. The use of any one of embodiments 58, 59, or 62 to 65, or the modified picornavirus, RNA, and / or cDNA of any one of embodiments 60 to 65, wherein the modified picornavirus is bioselected in vivo using tumor xenography. Embodiment 67. The use of embodiment 66, or the modified picornavirus, RNA, and / or cDNA of embodiment 66, wherein the tumor is from a subject. Embodiment 68. The use of any one of embodiments 58, 59, or 62 to 67, or the modified picornavirus, RNA, and / or cDNA of any one of embodiments 60 to 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. Embodiment 69. The use of embodiment 68, or the modified picornavirus, RNA, and / or cDNA of embodiment 68, wherein the cancer comprises cancerous cells expressing decay-accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn). Embodiment 70. The use according to any one of embodiments 58, 59, or 62 to 69, or the modified picornavirus, RNA, and / or cDNA according to any one of embodiments 60 to 69, wherein the modified picornavirus comprises or consists 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. Embodiment 71. The use according to any one of embodiments 58, 59, or 62 to 70, or the modified picornavirus, RNA, and / or cDNA according to any one of embodiments 60 to 70, wherein the modified picornavirus is encoded by a nucleotide sequence comprising or consisting of 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 variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the 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.

[0012] definition Below are some definitions that may be helpful in understanding the description of the present invention. These are not intended to limit the scope of the present invention to those terms alone, but rather as general definitions presented for a better understanding of the following description.

[0013] As used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "rhinovirus" includes a plurality of rhinoviruses and / or modified forms thereof. Thus, within the context of this specification, the singular also encompasses the plural unless the specific context clearly dictates otherwise. For example, if the invention is stated to include a method of treating cancer by administering an oncolytic virus or oncolytic viral RNA, this will be understood to encompass the administration of one or more such viruses or viral RNAs. Similarly, unless the context requires otherwise or unless specifically stated otherwise, integers, steps, or elements of the invention recited herein as singular integers, steps, or elements clearly encompass both the singular and plural forms of the recited integer, step, or element.

[0014] As used herein, the addition of "(s)" to a given term will be understood to convey singular or plural options. For example, "changes" will be understood to include a single change or multiple changes.

[0015] In the context of this specification, when a numerical range is provided, it will be understood to encompass the stated endpoints of the range and all values ​​between those endpoints, including any sub-ranges within those endpoints.

[0016] As used herein, the term "comprising" means "comprising" in a non-exhaustive sense. Variations of the word "comprising," such as "comprise" and "comprises," have correspondingly varied meanings. Thus, for example, a composition "comprising" a picornavirus may consist solely of the picornavirus, or it may include one or more additional components (e.g., a pharmaceutically acceptable excipient, carrier, or diluent).

[0017] As used herein, the term "therapeutically effective amount" includes, by its meaning, a non-toxic but sufficient amount of a given agent(s) of the present invention (e.g., an oncolytic picornavirus or viral RNA from an oncolytic picornavirus) to provide the desired therapeutic effect. The exact amount of agent(s) required will vary from subject to 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 particular agent being administered, and the mode of administration. Thus, an exact "effective amount" cannot be specified. However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0018] 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 a wild-type or parent sequence that contains, for example, a mutation in the capsid protein(s).

[0019] As used herein, a "wild-type" strain, virus, sequence, or organism is understood to be a strain, virus, sequence, or organism that occurs in nature or that has been isolated from a naturally occurring source, and also includes artificially synthesized but otherwise identical forms to those occurring in nature. In some embodiments, they may represent the form most frequently observed in a natural population. The wild-type strains, viruses, sequences, or organisms referred to herein can serve as the base strain, virus, sequence, or organism into which the change(s) are introduced. Thus, they can be used as a reference when comparing the modified strains, viruses, sequences, or organisms described herein.

[0020] The term "polynucleotide," as used herein, refers to a single- or double-stranded polymer of deoxyribonucleotide, ribonucleotide bases, or known analogues or naturally occurring nucleotides, or mixtures thereof.

[0021] As used herein, the term "treatment" and related terms such as "treating," "treated," and "treat" refer to any and all uses of improving or alleviating a disease state or symptom, preventing the establishment of a disease, or otherwise preventing, hindering, slowing, or reversing the progression of a disease or other undesirable condition in any way. For the avoidance of doubt, it should be noted that, as used herein, "treatment" and related terms do not require a complete cure or remission of the disease being treated.

[0022] As used herein, the term "subject" or "patient" includes individuals of any species of social, economic, or research importance, including, but not limited to, humans and members of the genera ovine, bovine, equine, porcine, feline, canine, primates, and rodents. A subject or patient may be a mammal, such as a human.

[0023] As used herein, the term "kit" refers to any delivery system for delivering materials. Such delivery systems include systems that allow for the storage, transport, or delivery of reaction reagents (e.g., labels in appropriate containers, reference samples, support materials, etc.) and / or support materials (e.g., buffers, written instructions for conducting the assay, etc.) from one location to another. For example, a kit may include one or more containers, such as a box, that contain the relevant reaction reagents and / or support materials. The term "kit" includes both fragmented kits and combined kits. A "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a subportion of the overall kit components. The containers may be delivered to the intended recipient together or separately. Any delivery system that includes two or more separate containers, each containing a subportion of the overall kit components, is included within the meaning of the term "fragmented kit." A "combined kit" refers to a delivery system that includes all of the components of a reaction assay in a single container (e.g., in a single box that houses each of the desired components).

[0024] The discussion in this specification of any prior art document, or statements in this specification that are derived from or based on those documents, is not an admission that the document or the statements from which it is derived are part of the general general knowledge in the relevant art.

[0025] For purposes of description, all documents referred to herein are incorporated by reference in their entirety unless otherwise stated. [Brief explanation of the drawings]

[0026] Non-limiting embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1] Figure 1 shows novel capsid changes in the DAF binding footprint that enhance the oncolytic activity of the inventive IVX037 strain 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%). [Figure 2]1 shows the three-dimensional structure (front view) of modified echovirus 12 (E12) strain IVX037 according to one embodiment of the present invention. [Figure 3] 1 shows the three-dimensional structure (side view) of modified E12 strain IVX037 according to one embodiment of the present invention. [Figure 4] 1 provides a series of graphs (A-C) comparing the oncolytic activity of a modified E12 strain according to one embodiment of the present invention with other existing E12 strains. [Figure 5] 1 shows that strain IVX037, according to one embodiment of the present invention, induced upregulation of DDX58 (RIG-I), CD274 (PD-L1), and IFN-γ-inducible protein 10 (CXCL10), as well as PD-L1, in in vitro cultures of human MSS-colorectal and ovarian epithelial adenocarcinoma cells 10 hours post-infection. [Figure 6] 1 shows the in vivo antitumor and oncolytic activity of intratumoral IVX037 line according to one embodiment of the present invention in human MSS-colorectal cancer (WiDr) xenografts. [Figure 7] 1 shows that line IVX037, according to one embodiment of the present invention, induced upregulation of DDX58 (RIG-I), CD274 (PD-L1), and IFN-γ-inducible protein 10 (CXCL10) as well as PD-L1 in human MSS-colorectal (WiDr) xenografts. [Figure 8] 1 shows infectivity data of the IVX-037 OVO strain according to one embodiment of the present invention on CHO cells transfected with human FcRn using forward (A) and reverse (B) transfection methods. [Figure 9] 1 shows infectivity data of the IVX-037 OVO strain according to one embodiment of the present invention on CHO cells transfected with either a human FcRn plasmid or a pIND control plasmid (*p<0.05, **p<0.01, ***p<0.001, ***p<0.0001). [Figure 10] Mean (A) and individual (B) tumor volumes of mice treated with the IVX037 OVO strain of the present invention are shown (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 11] 1 shows the mean (A) and individual (B) tumor volumes of mice treated with IVX-037 OVO strain according to one embodiment of the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 12] 4 shows the individual tumor volumes of mice treated with the IVX037 strain of the present invention. [Figure 13] 1 shows the mean (A) and individual (B) tumor volumes of mice treated with IVX-037 OVO strain according to one embodiment of the present invention (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 14] IVX037 infects and replicates in human colorectal cancer xenografts, resulting in tumor growth inhibition. (A) A single injection of IVX037 (1x108 TCID50) was administered intratumorally (it) to WiDr tumor xenografts. (B) Increasing doses of IVX037 (1x104, 1x106, 1x108 TCID50) were injected intratumorally to WiDr tumor xenografts. (C) Mice bearing WiDr tumor xenografts were treated with four intravenous (iv) injections of IVX037 spaced 3-4 days apart. (D) A single injection of IVX037 (1x108 TCID50) was administered intratumorally to Caco-2 tumor xenografts. Tumor volumes are expressed as mean ± SD (mm3). ****P<0.0001. [Figure 15] Expression of RIG-I, CD274, and CXCL10 1, 2, and 3 days after treatment. SCID mice were inoculated with WiDr cells. When tumors reached an average size (approximately 50 mm), 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 collected and RNA was extracted. Expression of RIG-I, CD274, and CXCL10 was measured using RT-PCR and calculated as fold change. [Figure 16](A) Patient serum levels of IVX037 viral RNA. IVX037 virus levels were determined using semi-quantitative RT-qPCR. (B) Patient serum levels of neutralizing antibodies (nAb) after IVX037 administration. (C) Patient serum CXCL10 levels. CXCL10 levels were determined using a multiplex flow cytometry assay. Statistical analysis utilized a paired sample "t-test" technique. [Figure 17] The Phase 1a / b study schema is shown. [Figure 18] Serum levels of CXCL10 in patients under study are shown. P values ​​were calculated by T-test using Graphpad Prism. CXCL10 was expressed as pg / mL. [Figure 19] Dose-response curves for IVX037 are shown for a panel of sensitive liver cancer cell lines. Cell death induced by dose-dependent IVX037 infection is graphed as a percentage of cell viability. Normalized dose-response curves (variable slope) were used as best-fit curves and analyzed using GraphPad Prism. Cell viability data were generated using an XTT cell viability assay. IVX037 dose is defined as MOI, expressed as TCID50 / cell. Micrographs on display demonstrate the cytotoxic effects of IVX037 on these cell lines. DETAILED DESCRIPTION OF THE INVENTION

[0027] The invention will now be described in more detail with reference to the following examples, which are included by way of illustration only.

[0028] Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention is to be understood to include all such variations and modifications. The invention also includes all and any and all combinations of the steps, features, compositions, and compounds referred to or shown herein, individually or collectively, or any two or more of such steps or features.

[0029] The present invention relates to viruses of the Picornaviridae family and modified forms thereof that bind to decay-accelerating factor (DAF) receptors on the surface of cancer cells, facilitating lytic infection of cancer cells by the viruses and, as a result, being effective oncolytic agents in the treatment, mitigation, and / or prevention of cancer.

[0030] The oncolytic picornaviruses of the present invention can be modified to have an enhanced ability to bind to DAF compared to unmodified or wild-type picornavirus strains. The increased ability to bind to DAF can facilitate more effective binding to cancer cells, thereby increasing the effectiveness of the modified picornavirus strain in treating, alleviating, and / or preventing cancer.

[0031] Additionally or alternatively, the oncolytic picornaviruses of the present invention can be modified by other means(s) to enhance their effectiveness in treating, mitigating, and / or preventing cancer. As a non-limiting example, the virus can be subjected to one or various bioselection procedures to increase its ability to target and / or lyse cancer cells. Additionally or alternatively, and also as a non-limiting example, the virus can undergo recombinant modification or incorporate an exogenous nucleic acid encoding a protein that increases its ability to target and / or lyse cancer cells.

[0032] The oncolytic picornaviruses of the present invention and modified forms thereof may 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, and chemotherapeutic agents.

[0033] Although not limited to a particular form of cancer, oncolytic picornaviruses and modified forms thereof may be effective in treating, alleviating, and / or preventing cancer types that are poorly responsive to immune checkpoint therapy, such as cancers with an objective clinical response rate of less than 25%, non-limiting examples of which include liver cancer, pancreatic cancer, head and neck cancer, stomach cancer, colorectal cancer, gastric cancer, breast cancer, sarcoma, lymphoma, brain cancer, and ovarian cancer. The cancer may be characterized by cancerous cells that express decay-accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

[0034] The picornavirus of the present invention can be any picornavirus, including known and classified picornaviruses and unclassified picornaviruses. Picornaviruses can be selected from prototype and clinically isolated strains. Representative types of human picornaviruses include enteroviruses, coxsackieviruses, echoviruses, polioviruses, unclassified enteroviruses, rhinoviruses, paraechoviruses, hepatoviruses, and cardioviruses. In some embodiments, the picornavirus can include enteroviruses, including coxsackieviruses, echoviruses, polioviruses, and unclassified enteroviruses, or viruses from other genus(s) of picornaviruses, which can include rhinoviruses, paraechoviruses, hepatoviruses, cardioviruses, aphthoviruses, erboviruses, coboviruses, and teschoviruses. Modified forms of these viruses are also within the scope of the present invention.

[0035] In certain embodiments, picornaviruses and modified forms thereof can be picornaviruses capable of binding to decay-accelerating factor (DAF, also known as CD55) receptors on the surface of cancer cells, such as, for example, enteroviruses (e.g., coxsackieviruses or echoviruses). Non-limiting examples include enteroviruses 68, 70, coxsackie B virus serotypes 1, 3, and 5, and echovirus serotypes 6, 7, 12, 20, 21, and coxsackievirus A21. Picornaviruses and modified forms thereof can use the DAF receptor to mediate viral attachment to and / or viral entry into cancerous cells.

[0036] Additionally or alternatively, picornaviruses and modified forms thereof may bind to one or more other receptor type(s) on the surface of cancer cells, such as, for example, intercellular adhesion molecule 1 (ICAM-1), intercellular adhesion molecule 1 (ICAM-5), integrin α2 β1, integrin αV β3, integrin αV β6, neonatal Fc receptor (FcRn), coxsackievirus and adenovirus receptor (CAR / CXADR), sialic acid (e.g., alpha 2,3 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).

[0037] The picornaviruses used in the compositions and methods of the present invention may be in their naturally occurring form or in a modified form.

[0038] A picornavirus is "naturally occurring" if it has been isolated from a source in nature and has not been intentionally modified by humans in the laboratory. For example, a picornavirus may be obtained from a "field source," such as from a human patient.

[0039] A picornavirus may be "modified" by altering one or more characteristics compared to a naturally occurring picornavirus.

[0040] For example, the picornavirus may be a recombinant picornavirus derived from two or more picornaviruses with different pathogenic phenotypes so as to contain different antigenic determinants, thereby reducing or preventing an immune response by mammals previously exposed to the picornavirus subtypes. Such recombinant virions can be produced by co-infection of mammalian cells with picornaviruses of different subtypes, such that coat proteins of the different subtypes are rescued and incorporated into the resulting virion capsid.

[0041] Additionally or alternatively, picornaviruses can be modified by introducing a change(s) into a structural protein, such as one or more of VP1, VP2, VP3, and / or VP4. The structural protein(s) can be exposed on the outer surface of the capsid (e.g., one or more of VP1, VP2, VP3). Non-limiting examples of changes in VP1, VP2, and / or VP3 protein(s) include those shown in Table 1 below. [Table 1]

[0042] 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.

[0043] Additionally or alternatively, the picornavirus can be modified by making a change(s) in a nonstructural viral protein such as, for example, one or more of 2A, 2B, 2C, 2BC, 3A, 3B, 3AB, 3C, 3D, and 3CD. [Table 2]

[0044] Without limitation, the picornavirus can 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.

[0045] In connection with any amino acid or nucleotide sequence alterations described herein, including the amino acid changes set forth 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 among different picornavirus strains. Thus, as used herein, a reference to an amino acid change at a given residue position in a picornavirus protein sequence "A" will be understood to encompass the same amino acid change at the equivalent position in the same picornavirus protein sequence "B," which differs in length from the sequence of "A" but still represents the same picornavirus protein. Equivalent amino acid positions in 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.

[0046] Picornavirus proteins can be altered by amino acid substitution, insertion, or deletion. This can be achieved using any suitable technique, including but not limited to recombinant methods. Substitution includes inserting a different amino acid in place of a natural amino acid. Insertion includes inserting an additional amino acid residue into the protein at one or more positions. Deletion includes deleting one or more amino acid residues within the protein. Alterations of this nature can be produced by methods known in the art. For example, oligonucleotide site-directed mutagenesis of the gene(s) encoding one or more of the protein(s) (e.g., capsid protein(s)) can result in the production of the desired altered capsid protein(s). Expression of the altered protein in picornavirus-infected mammalian cells in vitro can result in the incorporation of the modified protein into picornavirus virion particles.

[0047] In some embodiments, picornaviruses may be modified to reduce or eliminate immune responses to the picornavirus. Such modified picornaviruses are referred to as "immunoprotected picornaviruses." Suitable modifications may include packaging the picornavirus in a liposome, micelle, or other vehicle to mask the picornavirus from the subject's immune system. Alternatively, the outer capsid of the picornavirus virion particle may be removed or altered, as proteins present in the outer capsid are primary determinants of the host humoral and cellular responses.

[0048] In some embodiments, picornaviruses can be modified by making changes to viral nucleic acid sequences, including untranslated regions (UTRs), regulatory regions, and the like.

[0049] Picornaviruses can be further modified by the insertion of exogenous nucleic acids encoding the desired protein(s) or components thereof. Exogenous nucleic acids can be inserted into the 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 picornaviruses of the 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.

[0050] Additionally or alternatively, picornaviruses can be engineered for certain traits via bioselection (also known in the art as directed evolution). For example, picornaviruses can be bioselected for enhanced infectivity, enhanced safety, enhanced lytic potential, enhanced migration among cancer cells, enhanced DAF and / or FcRn binding, enhanced replication ability at mammalian body temperatures (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 well known to those skilled in the art (see, for example, Zainutdinov et al., "Directed evolution as a tool for the selection of oncolytic RNA viruses 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, pp. 2640-2650).

[0051] The present invention includes nucleic acid molecules isolated from picornaviruses. In some embodiments, the picornaviruses are capable of at least binding to DAF receptors on the surface of cancer cells. In some embodiments, the nucleic acid molecules may be derived from picornaviruses and may be single-stranded RNA, or viral RNA or complementary DNA synthetically produced from the virus. It should be understood that the nucleic acid sequences of the present invention include, for example, nucleic acid sequences encoding the viral genome, or nucleic acid sequences derived from picornaviruses that contain sufficient sequence to enable virus production or induce lytic infection in cells. For example, the nucleic acid molecule may comprise a single viral RNA or DNA molecule, such as a complementary DNA molecule, or multiple such molecules encoding different viral sequences.

[0052] In the context of this specification, the term "derived from" should therefore be understood to include that the sequence may be a picornavirus, a synthetic viral RNA, or a viral RNA isolated directly from a cDNA encoding the viral genome or a component thereof corresponding to the isolated sequence. The term also includes synthetic polynucleotide sequences that contain one or more changes in the sequence compared to a wild-type or parent sequence, including, for example, alterations in the capsid protein.

[0053] Any suitable method for isolating viral RNA may be used, including, for example, a method based on the use of phenol / chloroform extraction, such as that provided in a commercially available kit for isolating viral RNA, such as Trizol® LS Reagent (GIBCO BRL, Life Technologies, Grand Island, NY, USA), or an isolation method using magnetic bead-based isolation, such as the Ambion MagMax™ viral RNA isolation kit. Methods for isolating viral RNA are generally described, for example, in 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 Harbor Laboratory Press, New York.

[0054] It should be understood that the present invention does not require that a nucleic acid sequence, such as viral RNA, be free of contaminants such as cellular debris to be considered "isolated" in the context of this specification, whether isolated directly from a virus, synthesized, presented as a plasmid molecule, or generated in vitro from a cDNA template (e.g., encoding a viral genome or a component thereof) using bacteriophage T7 RNA polymerase. Thus, in the context of this specification, RNA is 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 is considered "isolated" if more than 50% of the non-RNA material has been removed. Preferably, more than 60% of the non-RNA material has been removed, and more preferably, more than 70%, 80%, or 90% of the non-RNA material has been removed. Typically, the RNA contains less than 10% contaminants, more typically less than 5%. Thus, the RNA is preferably more than 95% pure with respect to viral RNA, and even more preferably, more than 97% pure or even more than 99% pure.

[0055] 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 recognize that, given the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules.

[0056] The present invention also provides isolated polynucleotide sequences substantially similar to the polynucleotides disclosed herein, 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, which sequences comprise or provide picornaviruses that have the ability to bind to a DAF receptor and optionally additional cellular receptor(s) and lytically infect cancerous cells. Polynucleotide sequence variants share qualitative biological activity with 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 residues in two sequences that are the same when aligned for maximum correspondence over a particular comparison window 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, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-453).

[0057] The picornaviruses, modified forms thereof, and picornavirus RNA / cDNA of the present invention can be produced using standard techniques well known in the art (e.g., 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 to the viruses and / or their RNA / cDNA include insertion of tissue-specific miRNA and / or nucleic acids encoding immune checkpoint inhibitors, such as monoclonal antibodies that bind to immune checkpoint targets (e.g., PD-1, PD-L1, CTLA-4, IDO, TIM-3, LAG-3, TIGIT, etc.).

[0058] The picornavirus and / or modified forms thereof of the present invention can be administered to a subject in the form of a pharmaceutical composition comprising the virus and a pharmaceutically acceptable carrier. 5 Virus particles / mL ~ approx. 10 15 virus particles / mL, or approximately 10 6 virus particles / mL, or approximately 10 7 virus particles / mL, or approximately 10 8 virus particles / mL, or approximately 10 9 virus particles / mL, or approximately 10 10 virus particles / mL, or approximately 10 11 virus particles / mL, or approximately 10 12 Virus particles / mL, approximately 10 13virus particles / mL, or approximately 10 14 virus particles / mL, or approximately 10 15 The virus may be present at any suitable concentration, such as in the range of virus particles / mL.

[0059] The virus composition stock may be diluted to an appropriate volume suitable for administration, for example, to achieve a desired dose of virus particles administered in a desired volume. For example, a subject may receive approximately 10 5 virus particles ~ approximately 10 15 of virus particles, or about 10 6 of virus particles, or about 10 7 of virus particles, or about 10 8 of virus particles, or about 10 9 of virus particles, or about 10 10 of virus particles, or about 10 11 of virus particles, or about 10 12 of virus particles, or about 10 13 of virus particles, or about 10 14 of virus particles, or about 10 15 The virus may be administered in a dose containing about 100 to 500 mL of virus particles. The volume in which the virus is administered is affected by the mode of administration. For example, administration of the virus by injection typically involves smaller volumes, e.g., about 0.5 mL to about 10 mL. As a further example, intravenous administration of the virus may typically involve diluting about 100 mL to about 500 mL of virus in normal saline and infusing it over approximately 30 minutes using an automated pump.

[0060] The pharmaceutical compositions provided herein contain picornaviruses in an amount of about 10 8 ~about 10 15 of virus particles / mL, approximately 10 8 ~10 12 virus particles / mL, or approximately 10 8 ~10 10 The virus particles may contain up to 10 ...

[0061] The pharmaceutical compositions provided herein may comprise a picornavirus at a particle-to-infectious ratio of about 3000:1 or less, about 300:1 or less, 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.

[0062] The pharmaceutical compositions provided herein may further comprise a level of host cell DNA of about 200 ng / mL or less, in the range of about 1 to about 100 ng / mL, in the range of about 1 to about 50 ng / mL, or in the range of about 1 to about 10 ng / mL.

[0063] Pharmaceutical compositions comprising the picornavirus and / or modified forms thereof of the present invention may further comprise a pharmaceutically acceptable diluent, excipient, and / or adjuvant. The carrier, diluent, excipient, and adjuvant must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not unacceptably deleterious to the recipient subject.

[0064] The virus may be administered as naked viral RNA encoding the virus, rather than as a viral particle, as described, for example, in PCT Application No. PCT / AU2006 / 000051, entitled "Methods and Composition for the Treatment of Neoplasms," 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 may be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances and are formed by mono- or multi-lamellar hydrated liquid crystals dispersed in an aqueous medium. Any non-toxic, physiologically acceptable, and metabolizable lipid capable of forming liposomes may be used. The liposome-formed composition may contain stabilizers, preservatives, excipients, and the like. Preferred lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods for forming liposomes are known in the art, and specific references thereto are made 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.

[0065] Alternatively, viral RNA can be administered in the form of lipid nanoparticles (LNPs). Lipid nanoparticles (LNPs) are typically composed of four main types of lipids: ionizable cationic lipids, which are essential for encapsulating nucleic acids and aiding endosomal release; phospholipids, which contribute to the structural integrity of the nanoparticle; cholesterol, which enhances stability and fluidity; and polyethylene glycol (PEG) lipids, which extend circulation time in vivo by forming a steric barrier. However, other non-toxic, physiologically acceptable, and metabolizable lipids capable of forming LNPs can be used, as will be understood by those skilled in the art. Formation of LNPs generally involves microfluidic mixing or ethanol dilution, in which lipids and therapeutic payloads are rapidly mixed in an aqueous environment. However, other methods for forming LNPs are known in the art.

[0066] Routes of administration of the picornaviruses and modified forms thereof according to the present invention, and pharmaceutical compositions comprising them, to a given subject include, but are not limited to, intratumoral, intravenous, intravesical, subcutaneous, oral, intravesicular, cutaneous, isolated liver perfusion, intrahepatic, ocular and topical administration routes, and any combination thereof.

[0067] The picornaviruses and / or modified forms thereof of the present invention can be administered to a subject by any suitable means, such as injection. Injection may be, for example, systemic, parenteral, or by direct injection into the cancer. Intralesional injection of tumors can be performed by any suitable means known to those skilled in the art, taking into account factors such as the type of tumor being treated, the size and location of the tumor, and the tumor's accessibility to direct injection. Injection techniques that increase or maximize distribution of the virus throughout the tumor may provide improved therapeutic outcomes. For example, in the treatment of melanoma and other solid tumors, multiple lesions may be injected in a multi-fractionated dose pattern, starting with the largest lesion(s) and continuing up to a maximum of 4.0 mL (2.0 mL for tumors larger than 2.5 cm, 1.0 mL for tumors between 1.5 and 2.5 cm, and 0.5 mL for tumors between 0.5 and 1.5 cm). After the initial injection of a picornavirus described herein, any injected lesion that shrinks to less than 0.5 cm in diameter may be injected with 0.1 mL of picornavirus and / or modified forms thereof according to the treatment schedule described until the lesion has completely resolved.

[0068] In one embodiment of the present invention, in the treatment of colorectal cancer and other solid tumors of a particular length, single and multiple lesions may be treated using the following dosing regimen. 1. For tumors 50mm or longer: A maximum of 10 mL of IVX 037 is injected into a single lesion. If injecting multiple lesions, the largest lesion should be allotted at least 5.0 mL, with the remaining 5.0 mL divided among smaller lesions based on size. 2. For tumors 25mm or more but less than 50mm in length: A single lesion will receive 3.0 mL of IVX037. If injecting multiple lesions, up to 3.0 mL may be injected additionally, distributed based on size. 3. For tumors 10mm or more but less than 25mm in length: A single lesion will receive 1.0 mL of IVX037. If multiple lesions are injected (maximum 4), the dose may be distributed among the target lesions, each receiving 1 mL and 25% of the total dose. 4. For tumors ≥ 5mm but < 10mm in length: If the treated lesion regresses to less than 10 mm, the volume of IVX037 injected is 0.5 mL until it disappears.

[0069] Theoretically, maximizing the number of cancer cells and regions throughout the tumor that are initially infected increases the amount of cancer cells destroyed. It also increases the amount of viral progeny produced by the tumor, thus increasing the likelihood of ongoing viremia due to distant tumor dissemination. Any suitable means for achieving the desired distribution of the administered virus throughout the tumor may be used, as will be apparent to those skilled in the art.

[0070] The picornaviruses and / or modified forms thereof of the present invention may be administered to a subject, tumor, or cancer cell along with other agents, either simultaneously or sequentially.

[0071] For example, they may be administered in combination with immunostimulatory agent(s). The immunostimulatory agent(s) may be selected from any suitable agent. In the context of the present invention, an immunostimulatory agent will be understood as any agent that can stimulate an immune response against tumor / cancer cells when administered to an individual. An immunostimulatory agent may be any agent that interacts with an immune checkpoint molecule to block, reduce, or counteract the ability of that immune checkpoint molecule or a complex containing that immune checkpoint molecule in reducing an individual's innate immune-based anti-tumor response. Thus, the immunostimulatory agent reduces the "handbrake" effect that immune checkpoint molecules have on anti-tumor responses. For example, the immunostimulatory agent can be any agent that targets an immune checkpoint molecule 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, TIM3, GAL9, CD28, OX-40, and TIGIT. It should also be understood that the term immunostimulatory agent, as used herein, can also be referred to as an immune checkpoint inhibitor when the immunostimulatory agent targets an immune checkpoint.

[0072] In some embodiments, the immunostimulatory agent is an antibody, e.g., a monoclonal antibody. Preparation of antibodies for use in the present invention may be carried out 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 a monoclonal antibody with reasonably high affinity and then improving the affinity using well-known methods (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 sections of these documents describing the preparation of antibodies are incorporated herein by reference. Alternatively, antibodies may be obtained rather than prepared. Examples of antibodies targeting checkpoint inhibitor molecules include those targeting 10 patients with metastatic melanoma. 7 These include nivolumab (BMS-936558, MDX-1106, ONO-4538), a fully human immunoglobulin G4 (IgG4) monoclonal PD-1 antibody that is the first of its class to be tested in a Phase I trial in human patients (see Sosman et al. 2012b); lambrolizumab (MK-3475), a humanized monoclonal IgG4 PD-1 antibody that has been studied in a Phase I trial involving 132 patients with metastatic melanoma (see Iannone et al. 2012); and BMS-936559, a fully human IgG4 PD-L1 antibody that has been tested in 55 patients with metastatic melanoma as part of a Phase I trial (see Brahmer et al. 2012).

[0073] Additionally or alternatively, the picornaviruses and / or modified forms thereof of the present invention can be administered to a subject, tumor, or cancer cells in combination (simultaneously or sequentially) with CAR-T cells and / or natural killer (NK) cells. Methods for generating CAR-T cells are 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 Riviere, "Clinical manufacturing of CAR T cells: foundation of a promising therapy," Molecular Therapy-Oncolytics (2016) 3, 16015; Vormittag et al., "A Guide to Manufacturing CAR-T Cell Therapies," Curr Opin Biotechnol. 2018 Oct; 53: 164-181). As non-limiting examples, CAR-T cells can be engineered to bind to any one or more of EGFRVIII, Interleukin-13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Mucl, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Mucl, EphA2, CD123, CD19, Claudin 18.2 on the surface of cancerous cells.

[0074] Additionally or alternatively, the picornaviruses and / or modified forms thereof of the present invention may be administered to a subject, tumor, or cancer cell in combination (concurrently or sequentially) with chemotherapeutic agent(s). By way of non-limiting example, suitable chemotherapeutic agents include ABVD, AC chemotherapy, amsacrine, (Amcidine), asparaginase, azacitidine, (Vidaza®), BEACOPP chemotherapy, BEAM chemotherapy, bendamustine, (Levact®), BEP chemotherapy, bleomycin, busulfan, (Busilvex®, Myleran®), cabazitaxel, (Jevtana®), capecetabine and docetaxel, capecitabine, (Xeloda®), carboplatin, carboplatin and etoposide chemotherapy, carmustine, (BiCNU®), CAV chemotherapy, chlorambucil, (Leu keran®), ChlVPP chemotherapy, CHOP chemotherapy, cisplatin, cisplatin and fluorouracil chemotherapy, 5FU, cisplatin and topotecan chemotherapy, (CT), cisplatin, capecitabine and trastuzumab, (HCX), cladribine, (Leustat®, LITAK®), clofarabine, (Evoltra®), CMF chemotherapy, CODOX-M chemotherapy, crisantaspase, (Erwinase®, asparaginase or L-asparaginase), CTD chemotherapy, CVP chemotherapy, cyclophosphamide, cytarabine, dacarbazine, (DTIC), dactinomycin, (Cosmegen Lyovac®), daunorubicin, De Gramont and modified deGramont chemotherapy, DHAP chemotherapy, docetaxel, (Taxotere®), docetaxel and carboplatin chemotherapy, (Taxotere®), docetaxel and cisplatin chemotherapy, (Taxotere®), doxorubicin, (Adriamycin®), doxorubicin and ifosfamide chemotherapy, EC chemotherapy, ECF chemotherapy, E-CMF chemotherapy, (Epi-CMF), ECX chemotherapy, EOX chemotherapy, epirubicin, (Pharmorubicin® (trademark), eribulin, (Halaven®), ESHAP chemotherapy, etoposide, (Etopophos®, Vepesid®), etoposide and cisplatin chemotherapy, (EP / PE), FCR chemotherapy, FEC chemotherapy, FEC-T chemotherapy, FLAG-Ida, FLOT chemotherapy, fludarabine, (Fludara®), fluorouracil, (5FU), FOLFIRINOX chemotherapy, GemCarbo chemotherapy, gemcitabine, (Gemzar®), gemcitabine Gemcitabine and capecitabine (GemCap), gemcitabine and cisplatin chemotherapy, (GemCis or GemCisplat), Gliadel® wafers, (carmustine), hydroxycarbamide, (Hydrea®, hydroxyurea), Hyper-CVAD chemotherapy, idarubicin, (Zavedos®), ifosfamide, (Mitoxana®), ifosfamide, carboplatin, etoposide-ICE chemotherapy, irinotecan, (Campto®), Irinotecan, including fluorouracil and folinic acid, (5FU) and (FOLFIRI), leucovorin, (folinic acid), liposomal daunorubicin, (DaunoXome®), liposomal doxorubicin, (Caelyx®, Myocet®), lomustine, melphalan, (Alkeran®), mercaptopurine, (Puri-Nethol®), mesna, (Uromitexan®), methotrexate, mitomycin, (Mitomycin CKyowa®), mitomycin and fluorouracil, (5FU), mitotane, (Lysodren®), mitoxantrone, MPT chemotherapy, MPT chemotherapy, MVAC chemotherapy, Nab-paclitaxel, (Abraxane®), oxaliplatin, (Eloxatin®), oxaliplatin including fluorouracil and folinic acid chemotherapy, (5FU) and (FOLFOX or OxMdG), paclitaxel Cel, (Taxol®), paclitaxel and carboplatin chemotherapy, (Taxol / Carbo), PCV chemotherapy, pemetrexed, pemetrexed and carboplatin, pemetrexed and cisplatin chemotherapy, pentostatin, (Nipent®), pertuzumab, trastuzumab, and docetaxel, PMitCEBO chemotherapy, procarbazine, raltitrexed, (Tomudex®), rasuburicase, (Fa Sturtec®), R-CHOP chemotherapy, R-CVP, R-DHAP chemotherapy, R-ICE chemotherapy, streptozocin, (Zanosar®), TAC chemotherapy, TC (Taxotere and cyclophosphamide) chemotherapy, temozolomide, (Temodal®), thiotepa, thioguanine, (Lanvis®), TIP chemotherapy, topotecan, (Hycamtin®), trabectedin, (Yonde lis®), treosulfan, trifluridine-tipiracil hydrochloride, (Lonsurf®), vinblastine, (Velbe®), vincristine, (Oncovin®), vinorelbine, (Navelbine®), vinorelbine and carboplatin chemotherapy, (VP), vinorelbine and cisplatin chemotherapy, (VP), XELOX (or CAPOX), XELOX (or CAPOX).

[0075] The methods of the present invention can be used in conjunction with surgical treatment of cancer. For example, following tumor resection, a subject can be treated using the methods of the present invention. This is expected to prevent or reduce tumor recurrence.

[0076] Additionally or alternatively, the methods of the invention can be used in combination with neoadjuvant therapy. For example, neoadjuvant therapy can be followed by treatment of a subject using the methods of the invention. This is expected to prevent or reduce tumor recurrence.

[0077] 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.

[0078] The methods may include single or multiple administrations of any one or more of a picornavirus, an immunostimulatory agent, CAR-T cells, natural killer (NK) cells, a chemotherapeutic agent, and / or a radiotherapeutic agent. The methods of the present invention include administering to a subject, tumor, or cancer cell a picornavirus and / or a modified form thereof described herein, and / or their RNA and / or cDNA produced from the RNA. There is no particular limit to the type of cancer that can be treated, ameliorated, or prevented according to the methods described herein, although in some embodiments, the cancer is recognized to have resistance (i.e., a reduced level of response) to treatment with immune checkpoint inhibitors, either generally 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, stomach cancer, colorectal cancer, gastric cancer, breast cancer, sarcoma, lymphoma, brain cancer, and ovarian cancer. Cancers can be characterized by cancerous cells that express decay-accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

[0079] The present invention also relates to kits for use in the methods of the present invention. In a basic form, the kit may include a pharmaceutical composition containing the picornavirus of the present invention and / or a modified form thereof, a pharmaceutically acceptable carrier, and instructions for use of the composition. The kit may further include any one or more of an immune checkpoint inhibitor, CAR-T cells, natural killer (NK) cells, a chemotherapeutic agent, or a radiotherapeutic agent. The composition may be provided in any suitable container, such as a vial, an ampoule, or a syringe. The composition may be provided in lyophilized, freeze-dried, liquid, or frozen form.

[0080] The kit may include any number of other additional components. By way of non-limiting example, the additional components may include: (i) one or more antiviral agents, such as Plecornil; (ii) one or more additional pharmaceutical compositions comprising an oncolytic virus; (iii) one or more additional pharmaceutical compositions comprising oncolytic viral RNA; or (iv) one or more additional therapeutic agents useful for treating cancer in a patient. The kit may also include compositions contained in single-use vials, pre-filled syringes for direct human administration, diluted with physiological solution for intravenous infusion, or in a concentrated form that allows for suitable dilution with physiological solution. Such solutions may be, for example, phosphate-buffered saline or physiologically concentrated NaCl.

[0081] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention disclosed in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. [Example]

[0082] The present invention will now be described with reference to the following examples, which should not be construed as limiting in any way.

[0083] Example 1: Novel echovirus strain IVX037 with enhanced DAF binding ability The complete genome sequence of a novel echovirus 12 (E12) strain, designated IVX037 (internal name), with high affinity DAF binding was determined.

[0084] Materials and Methods 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 one-step reverse transcription polymerase chain reaction (LAB-PROC 027) using virus-specific primers (see Table 3). [Table 3]

[0085] Amplified DNA was analyzed on an 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). The complete viral genome sequence was determined at AGRF (Westmead, NSW, Australia) using Sanger sequencing and virus-specific primers (LAB-PROC 012). The genome sequence was analyzed using Sequencia version 5.4.6 and compared to the wild-type E12 prototype strain Travis virus sequence (GenBank accession number X79047.01). Pairwise sequence alignments were performed using the Embossed Needle Global Alignment tool (EMBL-EBI).

[0086] -result In some embodiments, the complete genome sequence of the isolated strain IVX037 was 7423 nucleotides (nt), excluding the poly(A) tail (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), with a 741 nucleotide long 5' untranslated region. The nucleotide sequence begins with 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.

[0087] The viral genome of the IVX037 strain of the present invention is a positive-sense single-stranded RNA virus, and due to the nature of the genome, the first 20 nucleotides of the 5'-end have not been experimentally determined. However, these regions are well conserved among enteroviruses, and therefore, it is highly likely that these sequences represent the true 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 our exemplary IVX037 genome is greater than 5x, with the majority of the genome having at least one sequence determined in the forward direction and one in the reverse direction with high quality (high and medium quality values ​​predict a probability of error of 0.01% and 1%, respectively).

[0088] In one embodiment, one ambiguous position was identified in the genome at nucleotide position 3074, where the viral sequence was identified to have a mixture of U (uracil) and A (adenine). This position was sequenced in both directions using two separate PCR amplicons (see Table 4) with the same result (a mixture of U / A). [Table 4]

[0089] Therefore, this ambiguity is unlikely to be a proofreading error introduced during RT-PCR amplification, but this position actually contains a mixture of both of these nucleotides (shown as "W" in the genome sequence of SEQ ID NO: 41). This sequence ambiguity results in a mixture of two amino acids encoded at amino acid 210 in the capsid protein VP1, where U encodes phenylalanine and A tyrosine, respectively (see Table 5).

[0090] During analysis of the IVX037 viral genome of the present invention, several positions were identified where the sequence of the IVX037 genome differs from the E12 Travis prototype strain, including several both silent and coding nucleotide changes in nonstructural proteins (see Table 5). In some embodiments, a change is also found at nucleotide position 108 of the 5'UTR; based on primary and secondary sequence similarity between the IVX037 virus of the present invention and the closely related Coxsackievirus B3 (CVB3), this nucleotide is predicted to be located in the loop of domain II of the 5'UTR, and this change is unlikely to affect the secondary structure of the IVX037 viral protein.

[0091] In some embodiments, several coding changes were also identified in the capsid protein responsible for cell binding (Table 5). In some embodiments, an additional amino acid change was identified in capsid protein VP3 at amino acid 206. In some embodiments, one amino acid change was identified in VP1 at amino acid 230, and a mixture of amino acids is encoded by 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 both of these amino acids are predicted to map to the hypervariable puff region of VP2, identified as the DAF binding region of E12.

[0092] In addition to the complete genome sequence of the E12 prototype strain Travis, there are three complete genome sequences of three E12 strains (isolated from the stool of three healthy children in China in 2013) available in GenBank (Hongbu et al., (2018), Molecular characterization of echovirus 12 strain isolated from healthy children in China. Scientific Reports 8, 11716). These sequences are set forth in SEQ ID NOS: 47-49 and were used for further comparative sequence analysis of the present IVX037 genome sequence. Among the changes in the capsid coding region, three changes (nucleotides 1373 [VP2 amino acid 142], 2348 [VP3 206], and 3074 [VP1 210]) were identified as unique, in some embodiments, between the IVX037 sequence, the E12 prototype strain Travis (SEQ ID NOS: 46), and the three Chinese isolates (see Table 5). All of these changes are coding changes, with amino acid VP2 141 (N->T) located in the hypervariable puff region of VP2 involved in virus binding to DAF being of particular interest. [Table 5-1] [Table 5-2]

[0093] Consideration 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 to have several changes compared to the E12 Travis prototype strain. Changes in the capsid protein are particularly interesting because they may affect viral binding to receptors on the cell surface. Interestingly, when the IVX037 sequence was compared to the E12 Travis prototype strain, several changes were identified in the capsid-coding region, but no unique changes were identified in the nonstructural proteins. In some embodiments, two amino acid changes (VP2 141 N->T and VP2 153 H->Y) located in the DAF-binding region of the E12 VP2 protein were identified.

[0094] Of these two changes, VP2 142 T is unique among the E12 sequences analyzed (E12 prototype strain Travis and three Chinese isolates from healthy children).

[0095] A sequence alignment showing the exemplary IVX037 sequence of the invention, including structural features, compared to the E12 prototype strain Travis and three Chinese isolates from healthy children is shown below. CLUSTAL O(1.2.4) multiple sequence alignment key: VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C, 3D [Sequence Table 1-1] TIFF2026506348000008.tif115170 [Sequence Table 1-2] TIFF2026506348000009.tif242170 [Sequence Table 1-3] TIFF2026506348000010.tif252170 [Sequence Tables 1-4] TIFF2026506348000011.tif252170 [Sequence Tables 1-5] TIFF2026506348000012.tif249170 [Sequence Tables 1-6] TIFF2026506348000013.tif250170 [Sequence Tables 1-7] TIFF2026506348000014.tif245170 [Sequence Tables 1-8] TIFF2026506348000015.tif221170 [Sequence Tables 1-9] TIFF2026506348000016.tif240170 [Sequence Table 1-10] TIFF2026506348000017.tif127170

[0096] Example 3: Oncolytic activity and testing of IVX037 in colorectal and ovarian cell lines 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, and then maintenance medium (DMEM) was applied. The cultures were incubated 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 methodology. This virus passaging process was repeated five cycles to obtain the IVX-037 OVO strain from DOV-13 cells and the IVX-037 COLO strain from SW480 cells.

[0097] Materials and Methods Tumor cell lines (2 × 10 4 Cells) were grown in monolayers in 96-well plates and inoculated with 10-fold serial dilutions of stock preparations of E12 P1 Vero, IVX-037 COLO, and IVX037 OVO strains. After 5 days of incubation at 37°C / 5% CO2, cell monolayers were examined microscopically for the presence of cytopathic effect (CPE). The 50 percent endpoint titer was calculated using the method of Karber, and the mean (±SEM) was calculated with the minimum MOI (TCID50 / cell) for each cell line.

[0098] Viral sequences were obtained by Sangar sequencing using a walking strategy using conventional methodologies.

[0099] -result In Figure 4A, the exemplary IVX-037 COLO line of the present invention exhibited significantly enhanced oncolytic activity (up to 10 ) in four human MSS-colorectal cancer cell lines compared to that of CVA21 (publicly available historical data). 3 Additionally, the present invention's IVX-037 OVO line exhibits significantly enhanced activity (up to 10 times higher) in four aggressive human ovarian cancer cell lines compared to CVA21 (publicly available historical data). 7 This indicates that the

[0100] In Figure 4B, the exemplary IVX-037 COLO line of the present invention exhibited significantly enhanced oncolytic activity (approximately 10-10) in four human MSS-colorectal cancer cell lines compared to that exhibited by the prototype E12 Travis line, which was passaged in Vero cells (E12 P1-Vero). 2 On the other hand, the IVX-037 OVO strain of the present invention exhibited significantly enhanced oncolytic activity (approximately 10-10 times higher) in four highly malignant human ovarian cancer cell lines compared to the prototype E12 Travis strain, which was passaged once in Vero cells (E12 P1-Vero). 2 This indicates that the

[0101] In Figure 4C, the exemplary IVX-037 COLO line exhibits significantly enhanced oncolytic activity in two of three human MSS-colorectal cancer cell lines compared to the exemplary IVX-037 OVO line, while the IVX-037 OVO line exhibits moderately enhanced oncolytic activity in three of three human ovarian cancer cell lines compared to the IVX-037 COLO line.

[0102] Nucleotide sequences of viruses passaged in ovarian and colorectal cancer cell lines were obtained, examples of which 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 associated with the resulting 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.

[0103] Example 4: IVX037 virus strain upregulates DDX58 (RIG-I), CD274 (PD-L1), and IFN-g-inducible protein 10 (CXCL10), and PD-L1 Materials and Methods (i) In vitro assay The following protocol was followed in performing the in vitro assay: Seed OAW42, DOV13, or WiDr cells in 5 x 6-well plates in medium containing 2% FBS (approximately 2.5E+05 cells / mL, 3 mL / well) to achieve approximately 90-100% confluency the next day. Prepare 500 mL of serum-free DMEM medium for cell washing and inoculum preparation. From this medium, allocate 100 mL of maintenance medium supplemented with 0.5% FBS. For the cell lysis process, prepare 10 mL of Buffer RLT by adding 100 µL of 14.3 M B-ME-mercaptoethanol. Buffer RLT+B-ME is stable for one month at room temperature. For the average cell number per well, obtain cell counts using three wells of one plate and three untreated control wells. Determine the amount of E12 virus to add per well based on the number of cells / well and the virus titer (if the virus titer is unknown, use 0.5 mL / well of p5 DOV stock diluted 1:10 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 above*). 50Achieve an MOI of 100 / cell. Set up four identical plates with three virus-infected wells and three mock-infected wells (serum-free medium only) per time point. NB: A total of approximately 7 mL of inoculum is required to infect 12 wells. Infect cells with 0.5 mL of diluted virus or serum-free medium. Incubate the plate for 1 hour, gently rocking the plate (or placing it on a rocker) every 15 minutes to prevent the cell monolayer from drying out. Remove the inoculum and gently wash three times with prewarmed serum-free medium. Re-feed the cells with 2-3 mL of prewarmed medium containing 0.5% FBS (maintenance medium) and incubate at 37°C for 0, 3, 6, or 9 hours. NB: Harvest the time 0 wells immediately after the re-feeding step using the following protocol: At each collection time point, photographs of the cell monolayer are taken, the medium (supernatant) is completely removed, and frozen for later virus titration according to LAB-PROC006, and for potential analysis of viral RNA load according to LAB-PROC018, LAB-PROC031, and LAB-PROC032. NB: Medium collected from control samples is not necessary. The medium collected from each virus-infected well should be aliquoted into 4 x 500 µL labeled aliquots before freezing. In addition to the virus-infected aliquots, 4 x 500 µL aliquots of residual maintenance medium are aliquoted and frozen for use as negative controls in RNA extraction. NB: Aliquoting should be performed after lysis (step 11). Immediately after medium removal, lyse the cell monolayer with 350 µL of Buffer RLT (containing beta-ME-mercaptoethanol) and collect the lysate after scraping with a cell scraper according to steps 5.5.2-5.5.3 of LAB-PROC 025. Mix by vortexing or pipetting and ensure no cell clumps are visible before homogenizing the sample using a QIA shredder (add a maximum of 700uL / shredder). Freeze the samples until all samples have been collected. Use label nomenclature: control wells = Dov13 C# Xhr 202007 XX, infected wells = Dov13 IVX037#, Xhr, 202007XX. Once all samples have been collected and frozen, thaw the samples in a 37°C water bath until they are completely thawed and the salts have dissolved.If any insoluble material is visible, centrifuge at 3,000-5,000 x g for 5 min and transfer the supernatant to a new tube. Extract RNA from all samples (start with step 5.8 RNA Extraction and Purification LAB-PROC 025). Extract a negative control (medium containing 0.5% FBS) and a low-range positive control (diluted control stock 10). -4) extraction. Total number of samples: 12 virus-infected samples (3 replicates per time point), 12 non-infected samples, and a negative extraction control. Gene expression analysis was performed on the extracted RNA samples, including a no template control (NTC), an RT-PCR negative control, and an extracted negative control. The following TaqMan™ Gene Expression Assays were used to detect mRNA levels of CXCL10, RIG-I, and CD274 relative to the level of the housekeeping gene GUSB. The TaqMan™ Gene Expression Assays are designed to analyze the expression of CXCL10, GUSB, CD274, and RIG-I gene expression using RT-qPCR. Other predesigned TaqMan™ Gene Expression Assays may also be used in this assay. TaqMan™ Gene Expression Assays are designed to detect expressed mRNA (assay type '_m1'). 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, but without cross-reactivity with mouse. The assay probes span exons to avoid detecting genomic DNA. Both assays are labeled with FAM-MGB and therefore cannot be multiplexed but should be tested in singleplex assays. The TaqPath™ 1-Step Multiplex Master Mix contains components for both reverse transcription and real-time qPCR, including fast DNA polymerase, thermostable MMLV enzyme, RNase inhibitor, 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 Mustang Purple as a passive reference dye to provide an internal standard for normalizing fluorescence fluctuations due to changes in volume or concentration. The assay is a relative quantitative gene expression assay that determines changes in expression in a test sample relative to a reference sample (e.g., sample vs. untreated control sample). Results are calculated by dividing the Ct values ​​of treated samples by the formula ratio = 2.ΔCt (Mass is analyzed using the comparative Ct method compared to a control using a normalization group, i.e., number of cells extracted or μg of RNA used as RT-qPCR template).

[0104] (ii) In vivo assay In performing the in vivo assay, the following protocol was followed: Immunodeficient SCID-Balb / C mice were injected sc with MSS-colorectal cells WiDr. When tumors became physically palpable (25 mm 3 (above), mice received an it injection of virus-diluted vehicle as a control or the IVX-037 strain of the present invention. Two mice from the control and IVX037-treated groups were sacrificed 24, 48, and 72 hours after treatment, and tumors were excised, placed in RNA buffer, and stored at 4°C prior to RNA extraction and gene expression PCR analysis. The following TaqMan™ gene expression assays were used to detect the levels of CXCL10, RIG-I, and CD274 mRNA relative to the level of the housekeeping gene GUSB in the excised tumors.

[0105] -result In vitro challenge of the ovarian cancer cell lines DOV-13, OAW42, and the human MSS colorectal cell line WiDr with our IVX-037 OVO line resulted in a significant upregulation (1.5- to 5-fold) of mRNA encoding DDX58, CD274, and CXCL10 at 10 h postinfection compared to control infection levels (Figure 5 ).

[0106] A single intratumoral injection of the IVX-037 OVO strain of the present invention (10 8 TCID 50 In vivo challenge of human MSS-colorectal cell xenografts in Balb-C SCID mice with IgG4-mediated HIV-1 infection resulted in a significant upregulation of mRNA encoding DDX58, CD274, and CXCL10 at 24, 48, and 72 hours postinfection. The most significant upregulation was observed at 24 hours postinfection, particularly for the DXD-58 and CXCL10 genes (Figures 6 and 7).

[0107] Example 5: Increased infectivity of IVX037 in CHO cells transfected with the human FcRn receptor Materials and Methods CHO cells were seeded into 24-well plates and allowed to reach approximately 70-90% confluence. The following day, transfection was performed using commercially available Lipofectamine 2000 with a GenEZ™ ORF clone expressing Fcgrt (encoding the human FcRn receptor) in the mammalian expression cloning vector pcDNA3.1+ / C-(K)-DYK. A pIND plasmid lacking the Fcgrt gene was used as a transfection control. For reverse transfection, cells were seeded simultaneously with the Lipofectamine-plasmid complex. 48 hours later, cells were infected with the IVX-037 OVO strain of the present invention. After 72 hours of infection, supernatants and cells were collected, and an infectivity assay was performed. Plates were scored 5 days later for cytopathic effects. Infectivity data were expressed as 50% log tissue culture infectious dose per mL (log10 TCID50 / mL).

[0108] -result IVX037 infectivity data on CHO cells transfected with human FcRn using forward and reverse transfection methods are shown in Figure 8. In both cases, increased infectivity was observed in cells expressing the human FcRn receptor.

[0109] As shown in Figure 9, infectivity data from CHO cells transfected with either the human FcRn plasmid or the pIND control plasmid showed significantly higher levels of infectivity in CHO cells transfected with the FcRn plasmid. The inclusion of the control plasmid confirmed that transfection with the FcRn plasmid was due to transfection and not other causes. The data here provide strong evidence that expression of the human FcRn receptor resulted in improved susceptibility to IVX037 infection in CHO cells. Statistical differences between groups were determined using one-way ANOVA.

[0110] Example 6: Oncolytic activity of intratumoral IVX037 in human colorectal cancer (Caco-2) xenografts Materials and Methods Immunocompromised SCID-Balb / C mice were subcutaneously inoculated with human colorectal cancer cells, Caco-2 (2e6 cells). When tumors became physically palpable (25 mm or larger), mice received a single intratumoral injection of control vehicle or the inventive IVX-037 OVO strain (1e8 TCID50 / injection). Tumor volumes were measured, and animals were terminated 28 days after treatment.

[0111] -result Figure 10 shows the mean (A) and individual (B) tumor volumes in mice treated with IVX037. Strong antitumor activity was observed in Caco-2 xenografts after a single intratumoral injection of the IVX037 strain. Treatment was well tolerated, and no adverse effects were observed. Statistical differences between treatment groups were determined using two-way ANOVA.

[0112] Example 7: Oncolytic activity of intratumoral IVX037 in human gastric cancer (NCI-N87) xenografts Materials and Methods Immunocompromised SCID-Balb / C mice were subcutaneously inoculated with human colorectal cancer cells NCI-N87 (2e6 cells). When tumors became physically palpable (≥25 mm), mice received a single intratumoral injection of control vehicle or the inventive IVX-037 OVO strain (1e8 TCID50 / injection). Tumor volumes were measured, and animals were terminated 25 days after treatment.

[0113] -result The mean (A) and individual (B) tumor volumes of mice treated with the IVX037 strain are shown in Figure 11. Strong antitumor activity was observed in NCI-N87 xenografts after a single intratumoral injection of the IVX037 strain. Treatment was well tolerated, and no adverse effects were observed. Statistical differences between treatment groups were determined using two-way ANOVA.

[0114] Example 8: Oncolytic activity of intratumoral IVX037 in human ovarian cancer (IGROV-1) xenografts Materials and Methods Immunocompromised SCID-Balb / C mice were subcutaneously inoculated with human ovarian cancer cells IGROV-1 (2e6 cells). When tumors became physically palpable (≥25 mm), mice received a single intratumoral injection of control vehicle or the inventive IVX-037 OVO strain (1e8 TCID50 / injection). Tumor volumes were measured, and animals were terminated 20 days after treatment.

[0115] -result Individual tumor volumes in mice treated with IVX037 strain are shown in Figure 12. IGROV-1 xenografts that developed tumor ulceration were more aggressive. Affected animals were euthanized. The data presented here are individual volumes for each animal, not average values. Strong antitumor activity was observed in IGROV-1 xenografts after a single intratumoral injection of IVX037 strain; no tumor ulceration was observed in this group. Treatment was well tolerated, and no adverse effects were observed. Premature termination of control animals prevented two-way ANOVA from being performed.

[0116] Example 9: Oncolytic activity of intravenous IVX037 in human colorectal cancer (WiDr) xenografts Materials and Methods Immunocompromised SCID-Balb / C mice were subcutaneously inoculated with human colorectal cancer cells, WiDr (2e5 cells). Once tumors were physically palpable (approximately 4 mm), mice received four intravenous injections of control vehicle or the inventive IVX-037 OVO strain (1e8 TCID50 / injection) at 3-4 day intervals. Tumor volumes were measured, and animals were terminated after 22 days of treatment.

[0117] -result Figure 13 shows the mean (A) and individual (B) tumor volumes in mice treated with the IVX037 strain. After four intravenous injections of the IVX037 strain, strong antitumor activity was observed in WiDr xenografts. Treatment was well tolerated, and no adverse effects were observed. Statistical differences between treatment groups were determined using two-way ANOVA.

[0118] Example 10: Synthesis of IVX-037 OVO virus strain The present invention's IVX-037 OVO strain belongs to the Enterovirus species B, specifically wild-type echovirus 12 (E12), the prototype strain Travis. To obtain IVX-037 OVO material with a complete manufacturing history and mitigate potential drug risks, the IVX-037 OVO strain was rescued from a pUC-like plasmid containing the complete E12 viral sequence after transient transfection in Vero MCB LN 1595.01 cells. The E12 viral sequence inserted into the pUC-like plasmid was not genetically modified. The plasmid was synthesized at GeneArt, ThermoFisher Scientific, and the E12 progeny virus rescue procedure was performed at ImmVirX. An overview of IVX037 pre-MVSS production is outlined below. [ka]

[0119] Further details of the IVX-037 OVO strain synthesis process are outlined in Table 1 below. [Table 6-1] [Table 6-2]

[0120] Example 11: IVX037 as a mediator in immune responses and antitumor activity CXCL10 is a marker of the severity of viral infection and facilitates the recruitment of T cells, natural killer cells, macrophages, and dendritic cells. Evidence suggests that CXCL10 is required for the recruitment of antitumor T cells to melanoma tumors. Furthermore, CXCL10 signaling through the CXCR3 receptor promotes lymphocyte migration to dendritic cells, which was necessary for the response to PD-1 blockade in a transplantable mouse model. Clinically, high pretreatment CXCL9 and CXCL10 levels correlate with response to anti-PD-1 therapy in patients with non-small cell lung cancer, and there is evidence that CXCL9 and CXCL10 levels increase during the first few months of treatment in patients with melanoma who respond to PD-1 inhibitor therapy.

[0121] Patient serum was quantified using a multiplex bead-based assay (LEGENDplex™ Human Essential Immune Response Panel (13-plex), #740930, LEGENDplex™, BioLegend Inc., USA). 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, and CXCL8 (IL-8). Staining procedures were performed as suggested by the manufacturer's protocol. Flow cytometry was performed using a BD LSRFortessa™ X-20 (Becton Dickinson Biosciences, San Jose, USA). Cytokine concentrations were calculated based on a standard curve using BioLegend's LEGENDplex™ data analysis software provided by the manufacturer. Cytokine data were analyzed on days 1, 8, 15, and 29. Preliminary serum biomarker analysis shows early signs of IVX037 induction of potentially beneficial inflammatory cytokines / chemokines such as CXCL10 (Figure 18).

[0122] Example 12: Infectivity and oncolytic activity of IVX037 in hepatocellular carcinoma (HCC) cell lines A panel of seven liver cancer cell lines with varying degrees of genetic complexity was purchased from ATCC: SNU-475, C3A [HepG2 / C3A, a derivative of Hep G2], SNU-449, PLC / PRF / 5, SNU-387, SK-HEP-1, and SNU-423. The data in Figure 19 provide a preliminary summary of the oncolytic activity of IVX037 against these liver cancer lines.

[0123] 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: Clinical Trial background IVX037 challenge can induce selective in vitro tumor cell lytic infection in human colorectal, gastric, and ovarian cancer cell cultures via specific viral capsid-cell receptor interactions. A single intratumoral injection of IVX037 in microsatellite-stable (MSS) colorectal (Figure 14), gastric, and ovarian cancer human xenografts in SCID mice demonstrated significant antitumor activity.

[0124] In vivo studies In vivo human MSS colorectal cancer xenograft studies in mice revealed that intratumoral administration of IVX037 induced elevated levels of g-INF response genes (CXCL10, RIG-I) and upregulated the expression of PD-L1 (Figure 15), a key immune checkpoint molecule, indicating an inflammatory phenotype within the treated tumor microenvironment (TME).

[0125] The primary pharmacodynamic study was approved by the Newcastle Animal Ethics Committee and utilized in-house purified IVX037 grown from an in-house virus seed stock. Female severe combined immunodeficient (SCID) mice (8 per group) bearing WiDr (an immortalized cell line of human colorectal adenocarcinoma cells) human xenografts were administered 1 × 10 8 TCID 50 evaluated the activity of a single dose of IVX037 administered intratumorally (IT) in a randomized controlled trial (TR 2020-007)32.

[0126] The results highlighted that while tumor volume in mice in the vehicle (control) group gradually increased over the three weeks following treatment, IVX037 administration reduced tumor volume in mice in the treatment group two days post-infection, stabilized by day 9, and then slowly increased by day 21 post-infection. Overall, the mean tumor volume of mice in the IVX037-treated group was statistically significantly smaller compared to mice in the vehicle group over the three weeks following treatment (Figure 5A). Furthermore, there was no significant change in the body weight of mice from either treatment group.

[0127] Gene profiling analysis by RT-PCR on IVX037-treated tumors revealed that expression of retinoic acid-inducible gene I (RIG-I) and C-X-C motif chemokine ligand 10 (CXCL10) increased, peaking 1 day after IVX037 infection (approximately 5-fold change), and expression of CD274, the gene encoding PD-L1, increased approximately 1.3-fold change and remained constant over 3 days post-infection (Figure 15).

[0128] The tumor was of average size (approximately 50 mm 3 When tumor size reached 1000 mg / kg / day (n = 8 mice), mice were treated with virus IVX 037 (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 collected and RNA was extracted. Expression of RIG-I, CD274, and CXCL10 was measured using RT-PCR and calculated as fold change.

[0129] It is suggested that the induction of a viral inflammatory TME could potentially allow increased migration of antitumor lymphocytes in both injected and distant lesions, as well as increased levels of cellular targets for immune checkpoint therapy.

[0130] Increased serum levels of CXCL10 and CCL22 in melanoma patients treated with pembrolizumab in combination with another RNA oncolytic virus, V927, were associated with responses suggesting that viral replication contributes to antitumor immunity (Silk AW, et al, 2023 Cancer Immunol Immunother;72(6):1405-1415).

[0131] Clinical trial method 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 general study schema can be seen in Figure 17. Inclusion criteria: Patients (pts) must have one of the following injectable tumors: liver / nodal / peritoneal disease. Exclusion criteria: Candidates for liver surgery or locoregional therapy of liver or other lesions. Clinically significant ascites (grade 2 or higher), continuous systemic treatment with any corticosteroid (more than 10 mg daily). Intervention: Patients will be enrolled sequentially into 3 dose-escalation cohorts, with up to 3 x 10 for Cohort 3 administered intratumorally on days 1, 15, 29, 43, 57, 71, and 85, if applicable. 8 TCID 50 Patients will receive one (n=3 pts), two (n=3 pts), or up to seven doses of IVX037, as tolerated at the investigator's discretion in the absence of DLTs (n=15). Primary Objective: To determine the feasibility, safety, and tolerability of intratumoral IVX037, including the incidence of dose-limiting toxicities (DLTs). Secondary objective: To evaluate the maximum tolerated dose (MTD) of IVX037 administered as one, two, or three injections per lesion. Tumor response will be assessed using RECIST 1.1, with the first response assessment occurring at day 50. Exploratory Objective: Several biomarker effects of IVX037 administration in peripheral blood and tumor tissue addressing tumor-infiltrating lymphocytes and cellular target expression levels for immune checkpoint therapy will be evaluated.

[0132] result Patient recruitment began in April 2023, with eight patients currently enrolled. Dosing of IVX037 in cohorts 1 and 2 has been completed, and dosing in cohort 3 is ongoing. To date, intralesional IVX037 administration has been generally well tolerated, with all patients experiencing some degree of systemic exposure immediately following injection (Figure 16A), and no dose-limiting toxicities have been observed. Mild flu-like reactions (fatigue, chills, rigors, and injection site discomfort) have been observed following injection. ·IVX037 has been successfully administered to liver, lymph nodes, and abdominal metastases. Currently, all patients have developed serum neutralizing anti-IVX037 antibodies by day 15 after virus administration (Figure 16B). Preliminary serum biomarker analysis shows early signs of IVX037 induction of potentially beneficial inflammatory cytokines / chemokines such as CXCL10 (Figure 16C). Recruitment is ongoing, with a Phase 1b trial planned in combination with immune checkpoint blockade.

Claims

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

2. 2. The modified picornavirus of claim 1, comprising said alteration(s) in each of said VP2 and VP3 capsid proteins.

3. 2. The modified picornavirus of claim 1, comprising said alteration(s) in each of said VP1, VP2, and VP3 capsid proteins.

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

5. The wild-type strain and the modified picornavirus are (i) the alteration(s) in any one or more of capsid proteins VP1, VP2, VP3, and optionally, (ii) The modified picornavirus of any one of claims 1 to 4, having the same nucleotide sequence and / or the same amino acid sequence except for the change(s) 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. 7. The modified picornavirus of claim 6, wherein the enterovirus is selected from the group consisting of echovirus, poliovirus, unclassified enterovirus, rhinovirus, paraechovirus, hepatovirus, and cardiovirus.

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

9. The modified picornavirus according to any one of claims 1 to 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. 11. The modified picornavirus according to any one of claims 1 to 10, wherein said change(s) in capsid protein VP2 comprise or consist of an asparagine to threonine change at residue 142.

12. 12. The modified picornavirus according to any one of claims 1 to 11, wherein said change(s) in capsid protein VP3 comprise or consist of an alanine to valine change at residue 206.

13. 13. The modified picornavirus of any one of claims 1 to 12, wherein the change(s) in 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. 14. The modified picornavirus according to any one of claims 1 to 13, wherein said change(s) in capsid protein VP1 comprise or consist of a change from tyrosine to histidine at residue 230.

15. 15. The modified picornavirus according to any one of claims 1 to 14, wherein said change(s) in capsid protein VP1 comprise or consist of a change from phenylalanine to tyrosine at residue 210.

16. 16. The modified picornavirus according to any one of claims 1 to 15, wherein said change(s) in capsid protein VP1 comprise or consist of a change from glutamine to arginine at residue 132.

17. 17. The modified picornavirus of claim 16, wherein the alteration confers enhanced binding to the neonatal Fc receptor (FcRn).

18. The change(s) in the nonstructural proteins: 2A, 3A, 3C, 3D 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 nonstructural 3D protein.

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

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

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

23. 23. The modified picornavirus of claim 22, wherein the immunostimulatory protein is selected from any one or more of an interleukin, including but not limited to, interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, an interferon, including but not limited to IFN-γ, TNF-α, a prodrug converting enzyme, biologically active component(s) thereof, or a combination thereof.

24. 23. The modified picornavirus of claim 22, wherein the immune checkpoint molecule is selected from 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, OX-40, and TIGIT, biologically active component(s) thereof, or combinations thereof.

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

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

27. 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, stomach cancer cells, breast cancer cells, sarcoma cells, lymphoma cells, and brain cancer cells.

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

29. The picornavirus is (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 NOs: 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 NOs: 62, 63, 76, 88, 100, 112, 124, 136, 148, or 160; (iv) a nonstructural protein 2A sequence comprising any one of SEQ ID NOs: 64, 65, 77, 89, 101, 113, 125, 137, 149, or 161; (v) a nonstructural protein 3A sequence comprising any one of SEQ ID NOs: 66, 67, 78, 90, 102, 114, 126, 138, 150, or 162; (vi) a nonstructural protein 3C sequence comprising any one of SEQ ID NOs: 68, 69, 79, 91, 103, 115, 127, 139, 151, or 163; (vii) a modified picornavirus according to any one of claims 1 to 28, comprising a nonstructural protein 3D sequence comprising any one of SEQ ID NOs: 70, 71, 80, 92, 104, 116, 128, 140, 152, or 164.

30. 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. 31. The modified picornavirus of any one of claims 1 to 30, encoded by a nucleotide sequence comprising or consisting of 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 variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the 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.

32. 32. The modified picornavirus of any one of claims 1 to 31, synthesized using recombinant means.

33. - a modified picornavirus according to any one of claims 1 to 32, - modified picornavirus RNA according to any one of claims 1 to 32, - one or more complementary DNAs (cDNAs) encoding all or part of the genome of a modified picornavirus according to any one of claims 1 to 32, and a pharmaceutically acceptable carrier, excipient, or diluent.

34. The pharmaceutical composition of claim 33, wherein the RNA (e.g., synthetic RNA) of the modified picornavirus and / or complementary DNA (cDNA) encoding all or part 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 an agent (e.g., an antibody) capable of binding to a CAR-T cell, a natural killer (NK) cell, an immunostimulatory protein, and / or an immune checkpoint molecule or a ligand of said immune checkpoint molecule.

36. 36. The pharmaceutical composition of claim 35, wherein the CAR-T cells are engineered to bind to any one or more of EGFR VIII, Interleukin-13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Mucl, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Mucl, EphA2, CD123, CD19, Claudin 18.2 on the surface of cancerous cells.

37. 37. The pharmaceutical composition of claim 35 or 36, wherein the immunostimulatory protein is selected from any one or more of an interleukin, including but not limited to, interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, an interferon, including but not limited to IFN-γ, TNF-α, a prodrug converting enzyme, biologically active component(s) thereof, or a combination thereof.

38. 38. The pharmaceutical composition of any one of claims 35 to 37, wherein the immune checkpoint molecule is selected from 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, OX-40, and TIGIT, biologically active component(s) thereof, or combinations thereof.

39. The composition is ８ ~about 10 １５ Virus particles / mL, approximately 10 ８ ~10 １２ viral particles / mL, or approximately 10 ８ ~10 １０ 39. The pharmaceutical composition of any one of claims 33 to 38, comprising a picornavirus in viral particles / mL.

40. 40. The pharmaceutical composition of any one of claims 33-39, wherein the composition comprises a particle to infectivity ratio of about 3000:1 or less, about 300:1 or less, 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. 41. The pharmaceutical composition of any one of claims 33-40, wherein the composition comprises a level of host cell DNA of about 200 ng / mL or less, in the range of about 1 to about 100 ng / mL, in the range of about 1 to about 50 ng / mL, or in the range of about 1 to about 10 ng / mL.

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

43. 43. The method of claim 42, wherein the modified picornavirus is bioselected for any one or more of: enhanced migration among cancer cells; enhanced DAF and / or FcRn binding; enhanced replication capacity at mammalian body temperature (33°C to 39°C); enhanced lytic activity of 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. 44. The method of claim 43, wherein the modified picornavirus is bioselected in vivo using tumor xenographs.

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

46. 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, ocular, topical, or systemic administration.

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

48. 48. The method of claim 47, wherein the CAR-T cells are engineered to bind to any one or more of EGFR VIII, Interleukin-13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Mucl, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Mucl, EphA2, CD123, CD19, Claudin 18.2 on the surface of cancerous cells.

49. 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 administration of the therapeutically effective amount to the subject.

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

51. 51. The method of claim 50, wherein the immunostimulatory protein is selected from any one or more of an interleukin, including but not limited to, interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, an interferon, including but not limited to IFN-γ, TNF-α, a prodrug converting enzyme, biologically active component(s) thereof, or a combination thereof.

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

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

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

56. 56. The method of any one of claims 42 to 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. 57. The method of claim 56, wherein the cancer comprises cancerous cells that express decay-accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

58. In the manufacture of a pharmaceutical product for treating cancer in a subject, - a modified picornavirus according to any one of claims 1 to 32, - Picornavirus RNA according to any one of claims 1 to 32, - a complementary DNA (cDNA) encoding all or part of the genome of a picornavirus according to any one of claims 1 to 32, - any one or more of the pharmaceutical compositions according to any one of claims 33 to 41, use.

59. 59. The use of claim 58, wherein the medicament further comprises an agent (e.g., an antibody) capable of binding to a CAR-T cell, a natural killer (NK) cell, an immunostimulatory protein, and / or an immune checkpoint molecule or a ligand of said immune checkpoint molecule.

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

61. 61. The modified picornavirus, RNA, and / or cDNA of claim 60 for use in treating cancer in combination with CAR-T cells, and / or natural killer (NK) cells, and / or immunostimulatory agents.

62. 62. The use of claim 59, or the modified picornavirus, RNA, and / or cDNA of claim 61, wherein the CAR-T cells are engineered to bind to any one or more of EGFR VIII, Interleukin 13Ra2, FAP, GD2, EpCam, CD133, CD70, Her2, CEA, GAP, CD5, CD38, Mucl, GPC3, BCMA, Meso, PSCA, CD33, PSMA, ROR1, Mucl, EphA2, CD123, CD19, Claudin 18.2 on the surface of cancerous cells.

63. 63. The use of claim 59 or 62, or the modified picornavirus, RNA, and / or cDNA of claim 60 or 61, wherein the immunostimulatory protein is selected from any one or more of an interleukin, including but not limited to interleukin-2 (IL-2), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-21 (IL-21), RANTES, GM-CSF, an interferon, including but not limited to IFN-γ, TNF-α, a prodrug converting enzyme, biologically active component(s) thereof, or a combination thereof.

64. 85. 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. 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 movement among cancer cells; enhanced DAF and / or FcRn binding; enhanced replication capacity at mammalian body temperature (37°C to 39°C); enhanced lytic activity of 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. 66. The use of any one of claims 58, 59 or 62 to 65, or the modified picornavirus, RNA and / or cDNA of any one of claims 60 to 65, wherein the modified picornavirus is bioselected in vivo using a tumor xenograph.

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

68. 68. The use of any one of claims 58, 59 or 62 to 67, or the modified picornavirus, RNA and / or cDNA of any one of claims 60 to 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. 69. The use of claim 68, or the modified picornavirus, RNA, and / or cDNA of claim 68, wherein the cancer comprises cancerous cells expressing decay accelerating factor (DAF / CD55) and / or neonatal Fc receptor (FcRn).

70. 70. The use of any one of claims 58, 59 or 62 to 69, or the modified picornavirus, RNA and / or cDNA of any one of claims 60 to 69, wherein the modified picornavirus comprises or consists 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.

71. 96, 97%, 98% or 99% sequence identity to the 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 variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the 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.

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