Methods of treating solid or lymphatic tumors by combination therapy

Abstract

To provide methods of treating solid or lymphatic tumors by combination therapy.SOLUTION: The present invention provides methods for treating an individual having solid or lymphatic tumor, comprising locally administering to the site of the tumor an oncolytic virus, and systemically administering an immunomodulator (including a combination of immunomodulators). The methods may further comprise local administration to the site of the tumor a second immunomodulator (including a combination of immunomodulators). Also provided are compositions and kits for the cancer therapy methods.SELECTED DRAWING: None

Description

Cross - Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 306,470, filed on March 10, 2016, the content of which is hereby incorporated by reference in its entirety. Submission of Sequence Listing in ASCII Text File

[0002] The content of the following submission in ASCII text file is hereby incorporated by reference in its entirety: Sequence Listing in computer - readable form (CRF) (file name: 744442000340SEQLIST.txt, recording date: March 9, 2017, size: 3KB).

Technical Field

[0003] The present invention relates to cancer immunotherapy comprising administration of oncolytic viruses and one or more immunomodulatory agents.

Background Art

[0004] The human immune system of innate and adaptive immunity is an extremely complex system that has not yet been successfully utilized in the fight against cancer. One explanation is that since cancer generally develops in the second half of life, the development of an immunological response against cancer is not essential in the process of evolution according to the theory of survival of the fittest. Perhaps various aspects of the human immune system are not particularly designed for such purposes, which is equivalent to killing cells regarded as "self". Even after extensive removal of the primary tumor, it is still a problem to prevent the formation of metastases caused by either the growth from micrometastases that already existed at the time of surgery or the formation of new metastases by tumor cells or tumor stem cells that were not completely removed or re - attached after surgery. In short, in late - stage cancer, surgery and / or radiotherapy can only treat macroscopic lesions, and most patients relapse and are in a state where they cannot receive further treatment.

[0005] Recently, the FDA has approved two immunotherapeutic drugs for prostate cancer and melanoma. The first drug, PROVENGE®, utilizes a GM-CSF fusion molecule with prostate antigen to activate mononuclear cells or antigen-presenting cells in advanced cancer patients in vitro, which can increase the overall survival of these patients. The second drug is an anti-CTLA-4 monoclonal antibody, which has been shown to have a profound enhancing effect on the development of T effector cells. The oncolytic virus CG0070 has also been shown to induce long-term complete responses among bladder cancer patients after six consecutive weekly intravesical treatments (see Burke JM, et al. Journal of Urology Dec, 188(6)2391-7, 2012).

[0006] Current cancer immunotherapy methods face various fundamental challenges. For example, tumor-specific immune T lymphocytes in cancer patients usually occur at a low frequency systemically, even when they are present. The possible reasons are the generally weak antigenicity and specific immunogenicity of tumor antigens of common cancers, and the presence of overwhelming amounts of suppressor activity by cytokines and regulatory cells such as Tregs and tumor-associated macrophages. Furthermore, the traditional idea of ​​using non-specific components to promote immune responses against specific components has proven to be largely unsuccessful, since the human body is inherently limited in its ability to generate highly specific immunological responses against its own cells. After all, most cancer cells are not immunogenic enough to be differentiated from normal cells. Even if such immune responses from non-specific immunological components occur, they are still temporary.

[0007] For at least the reasons discussed above, in vitro preformulated therapeutic cancer vaccines using available tumor antigens and adjuvants have been tried without significant success for several decades, and it is clear that improved methods of cancer immunotherapy are needed. The disclosures of all publications, patents, patent applications, and published patent applications referenced herein are hereby incorporated by reference in their entirety.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

[0009] The present application provides a method, a composition (including a pharmaceutical composition), and a kit for treating a solid tumor or a lymphatic tumor in an individual, including local administration of a tumor-lysing virus to the site of the tumor and systemic administration of an immunomodulatory agent (including a combination of immunomodulatory agents). The method, composition, and kit may further include local administration of an immunomodulatory agent (including a combination of immunomodulatory agents), inactivated tumor cells, pretreatment, and / or pre-treatment.

[0010] Accordingly, one aspect of the present application provides a method for treating a solid tumor or a lymphatic tumor in an individual, the method including: a) locally administering an effective amount of a tumor-lysing virus to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the tumor-lysing virus includes a viral vector including a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the tumor-lysing virus preferentially proliferates in cancer cells, such as cancer cells with a deletion in the Rb pathway. In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter, and for example, the human E2F-1 promoter includes the nucleotide sequence set forth in SEQ ID NO: 1.

[0011] In some embodiments according to any of the above methods, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL-12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LTαβ. In some embodiments, the immune-related molecule is GM-CSF.

[0012] In some embodiments according to any one of the methods provided above, the oncolytic virus is selected from the group consisting of adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, measles virus, Seneca Valley virus, coxsackievirus, reovirus, vesicular stomatitis virus, Maraba and rhabdovirus, and parvovirus. In some embodiments, the oncolytic virus is an oncolytic adenovirus. In some embodiments, the viral gene essential for virus replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E1 promoter or the E3 promoter.

[0013] In some embodiments according to any one of the methods provided above, the oncolytic virus is adenovirus serotype 5, the endogenous E1a promoter of the native adenovirus is replaced with the human E2F-1 promoter, and the endogenous E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding human GM-CSF. In some embodiments, the oncolytic virus is CG0070.

[0014] In some embodiments according to any one of the methods provided above, the oncolytic virus is about 1×10 8 ~ about 1×10 14It is administered at a dosage of viral particles. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the oncolytic virus is administered over a period of about one week to about six weeks.

[0015] In some embodiments according to any one of the methods provided above, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor.

[0016] In some embodiments according to any one of the methods provided above, the oncolytic virus and the immunomodulatory agent are administered continuously. In some embodiments, the oncolytic virus is administered prior to the administration of the immunomodulatory agent. In some embodiments, the oncolytic virus is administered after the administration of the immunomodulatory agent. In some embodiments, the oncolytic virus and the immunomodulatory agent are administered simultaneously.

[0017] In some embodiments according to any one of the methods provided above, the immunomodulatory agent is a regulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an inhibitor of PD-L1. In some embodiments, the inhibitor of PD-L1 is an anti-PD-L1 antibody such as atezolizumab. In some embodiments, the immunomodulatory agent is an immune stimulant selected from the group consisting of activators of OX40, 4-1BB, and CD40. In some embodiments, the immune stimulant is an activator of OX40, such as an agonistic antibody of OX40. In some embodiments, the immunomodulatory agent is administered intravenously.

[0018] In some embodiments according to any one of the methods provided above, the method further comprises locally administering a second immunomodulatory agent (including combinations of immunomodulatory agents) to the site of the tumor (such as directly into the tumor or into the tissue having the tumor). In some embodiments, the second immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the second immunomodulatory agent is an immune stimulant selected from the group consisting of activators of OX40, 4-1BB, and CD40. In some embodiments, the second immunomodulatory agent is administered directly into the tumor. In some embodiments, the immunomodulatory agent is administered prior to or after the administration of the second immunomodulatory agent.

[0019] In some embodiments according to any one of the methods provided above that further comprise locally administering a second immunomodulatory agent to the site of the tumor, the method further comprises administering a third immunomodulatory agent (systemically or locally to the site of the tumor). In some embodiments, the third immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the third immunomodulatory agent is an immune stimulant selected from the group consisting of activators of OX40, 4-1BB, and CD40. In some embodiments, the second immunomodulatory agent and the third immunomodulatory agent are administered simultaneously (such as in the same composition). In some embodiments, the second immunomodulatory agent and the third immunomodulatory agent are administered sequentially.

[0020] In some embodiments according to any one of the methods provided above, the method further comprises locally administering a pretreatment composition to the site of the tumor prior to the administration of the oncolytic virus. In some embodiments, the pretreatment composition comprises a transduction enhancer such as N-dodecyl-β-D-maltoside (DDM).

[0021] In some embodiments according to any one of the methods provided above, the individual undergoes pre-treatment prior to administration of the oncolytic virus and the immunomodulatory agent. In some embodiments, the pre-treatment is radiotherapy. In some embodiments, the pre-treatment includes administration of a therapeutic agent, such as an agent that increases the concentration of cytokines involved in the immunogenic pathway and / or an agent that causes dysfunction or damage to the structural components of the tumor. In some embodiments, the therapeutic agent is selected from the group consisting of anti-VEGF antibodies, hyaluronidase, CCL21, and N-dodecyl-β-maltoside. In some embodiments, the pre-treatment is provided at a dosage insufficient to treat the tumor.

[0022] In some embodiments according to any one of the methods provided above, the method further includes locally administering an effective amount of inactivated tumor cells to the site of the tumor. In some embodiments, the inactivated tumor cells are autologous. In some embodiments, the inactivated tumor cells are allogeneic. In some embodiments, the inactivated tumor cells are derived from a tumor cell line. In some embodiments, the inactivated tumor cells are inactivated by irradiation. In some embodiments, the oncolytic virus and the inactivated tumor cells are administered simultaneously, such as in a single composition. In some embodiments, the oncolytic virus and the inactivated tumor cells are mixed immediately prior to administration.

[0023] In some embodiments according to any one of the methods provided above, the solid tumor or lymphatic tumor is a bladder cancer, such as a muscle-invasive bladder cancer or a non-muscle-invasive bladder cancer. In some embodiments, the oncolytic virus is administered intravesically.

[0024] In some embodiments according to any one of the methods provided above, the individual has a high expression of one or more biomarkers within the tumor. In some embodiments, the one or more biomarkers are selected from PD-1, PD-L1, and PD-L2. In some embodiments, the one or more biomarkers are selected from CD80, CD83, CD86, and HLA class II antigens within tumor-derived mature dendritic cells. In some embodiments, the one or more biomarkers are selected from CXCL9, CXCL10, CXCL11, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1, HES4, MTIB, MTIE, MTIG, MTIH, GADD45A, LAMP3, and miR-155.

[0025] In some embodiments according to any one of the methods provided above, the individual is a human individual.

[0026] Another aspect of the present application provides a kit for treating solid tumors or lymphatic tumors in an individual, the kit comprising: a) a oncolytic virus; b) an immunomodulatory agent; and c) a device for locally administering the oncolytic virus to the site of the tumor, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule, and the immunomodulatory agent is formulated for systemic administration. In some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LTαβ. In some embodiments, the oncolytic virus is an oncolytic adenovirus such as adenovirus serotype 5, wherein the endogenous E1a promoter of the native adenovirus is replaced with a human E2F-1 promoter, and the endogenous E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding human GM-CSF. In some embodiments, the oncolytic virus is CG0070.

[0027] In some embodiments according to any of the kits provided above, the immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an inhibitor of PD-L1, such as an anti-PD-L1 antibody, for example, atezolizumab.

[0028] In some embodiments according to any of the kits provided above, the immunomodulatory agent is an immunostimulant selected from the group consisting of activators of OX40, 4-1BB, and CD40. In some embodiments, the immunomodulatory agent is an agonist antibody of OX40.

[0029] In some embodiments according to any of the kits provided above, the kit further comprises a second immunomodulatory agent (including a combination of immunomodulatory agents) formulated for local administration to the site of the tumor. In some embodiments, the kit further comprises a third immunomodulatory agent (for example, for systemic administration or local administration to the site of the tumor).

[0030] In some embodiments according to any of the kits provided above, the kit further comprises a pretreatment composition containing a transduction enhancer such as N-dodecyl-β-D-maltoside (DDM).

[0031] In some embodiments according to any of the kits provided above, the kit further comprises an immune-related molecule selected from the group consisting of GM-CSF, IL-2, IL12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, LTαβ, STING activator, PRRago, TLR stimulant, and RLR stimulant.

[0032] In some embodiments according to any of the kits provided above, the kit further comprises a plurality of inactivated tumor cells. In some embodiments, the kit further comprises instructions for mixing the oncolytic virus and the inactivated tumor cells prior to administration. In some embodiments, the device for local administration is used for simultaneous administration of the plurality of inactivated tumor cells and the oncolytic virus.

[0033] In some embodiments according to any of the kits provided above, the device for local administration is for directly administering the oncolytic virus into the tumor.

[0034] In some embodiments according to any of the kits provided above, the device for local administration is for administering the oncolytic virus to the tissue having the tumor.

[0035] Another aspect of the present application provides a method for treating a solid tumor or a lymphatic tumor in an individual, the method comprising: a) systemically administering an effective amount of an oncolytic virus (such as intravenously) to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including an antibody that recognizes CTLA-4, a combination of immunomodulatory agents, etc.) (such as intravenously), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule. The embodiments described above applicable to local administration of the oncolytic virus are also applicable to methods comprising systemic administration of the oncolytic virus.

[0036] These and other aspects and advantages of the present invention will become apparent from the following detailed description and the appended "claims". It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. In certain embodiments, for example, the following are provided: (Item 1) A method for treating a solid tumor or a lymphatic tumor in an individual, comprising: a) locally administering an effective amount of an oncolytic virus to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent, wherein the oncolytic virus comprises a tumor cell-specific promoter operably linked to a viral gene essential for the replication of the virus, and a heterologous gene encoding an immune-related molecule, and the method comprises a viral vector. (Item 2) The method according to item 1, wherein the oncolytic virus preferentially proliferates in cancer cells. (Item 3) The method according to item 2, wherein the cancer cells have a deletion in the Rb pathway. (Item 4) The method according to item 3, wherein the tumor-specific promoter is an E2F-1 promoter. (Item 5) The method according to any one of items 1 to 4, wherein the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL-12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LTαβ. (Item 6) The method according to any one of items 1 to 5, wherein the heterologous gene is operably linked to a viral promoter. (Item 7) The method according to any one of items 1 to 6, wherein the oncolytic virus is selected from the group consisting of adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, measles virus, Seneca Valley virus, coxsackievirus, reovirus, vesicular stomatitis virus, Maraba and rhabdovirus, and parvovirus. (Item 8) The method according to item 7, wherein the oncolytic virus is an oncolytic adenovirus. (Item 9) The method according to item 8, wherein the viral gene essential for the replication of the virus is selected from the group consisting of E1A, E1B, and E4. (Item 10) The method according to any one of items 1 to 9, wherein the oncolytic virus is adenovirus serotype 5, the endogenous E1a promoter of the natural adenovirus is replaced with the human E2F-1 promoter, and the endogenous E3 19kD coding region of the natural adenovirus is replaced with a nucleic acid encoding human GM-CSF. (Item 11) The method according to item 10, wherein the oncolytic virus is CG0070. (Item 12) The oncolytic virus is administered at a dose of about 1×10 8 ~ about 1×10 14 virus particles, the method according to any one of items 1 to 11. (Item 13) The method according to any one of items 1 to 12, wherein the oncolytic virus is administered once a week. (Item 14) The method according to any one of items 1 to 13, wherein the oncolytic virus is administered over a period of about 1 week to about 6 weeks. (Item 15) The method according to any one of items 1 to 14, wherein the oncolytic virus is administered directly into the tumor. (Item 16) The method according to any one of items 1 to 14, wherein the oncolytic virus is administered to the tissue having the tumor. (Item 17) The method according to any one of items 1 to 16, wherein the immunomodulatory agent is administered intravenously. (Item 18) The method according to any one of items 1 to 17, wherein the oncolytic virus and the immunomodulatory agent are administered continuously. (Item 19) The method according to any one of items 1 to 17, wherein the oncolytic virus and the immunomodulatory agent are administered simultaneously. (Item 20) The method according to any one of items 1 to 19, wherein the immunomodulatory agent is a regulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. (Item 21) The method according to any one of items 1 to 19, wherein the immunomodulatory agent is an immune stimulant. (Item 22) The method according to item 21, wherein the immune stimulant is an activator of a molecule selected from the group consisting of OX40, 4-1BB, and CD40. (Item 23) The method according to any one of items 1 to 22, further comprising locally administering a second immunomodulatory agent to the site of the tumor. (Item 24) The method according to item 23, further comprising administering a third immunomodulatory agent. (Item 25) The method according to any one of items 1 to 24, further comprising locally administering a pretreatment composition to the site of the tumor prior to the administration of the oncolytic virus. (Item 26) The method according to item 25, wherein the pretreatment composition contains a transduction enhancer. (Item 27) The method according to any one of items 1 to 26, wherein the individual undergoes a prior treatment prior to the administration of the oncolytic virus and the immunomodulatory agent. (Item 28) The method according to item 27, wherein the prior treatment is radiotherapy. (Item 29) The method according to item 27, wherein the prior treatment includes the administration of a therapeutic agent. (Item 30) The method according to item 29, wherein the therapeutic agent is an agent that increases the concentration of a cytokine involved in the immunogenic pathway. (Item 31) The method according to item 29, wherein the therapeutic agent is an agent that causes dysfunction or damage to the structural components of the tumor. (Item 32) The method according to any one of Items 29 to 31, wherein the therapeutic agent is selected from the group consisting of an anti-VEGF antibody, hyaluronidase, CCL21, and N-dodecyl-β-maltoside. (Item 33) The method according to any one of Items 27 to 32, wherein the pretreatment is provided at a dose insufficient to treat the tumor. (Item 34) The method according to any one of Items 1 to 33, further comprising locally administering an effective amount of inactivated tumor cells to the site of the tumor. (Item 35) The method according to any one of Items 1 to 34, wherein the solid tumor or lymphatic tumor is bladder cancer. (Item 36) The method according to Item 35, wherein the oncolytic virus is administered into the bladder. (Item 37) The method according to any one of Items 1 to 36, wherein the individual has high expression of one or more biomarkers selected from PD-1, PD-L1, and PD-L2 in the tumor. (Item 38) The method according to any one of Items 1 to 37, wherein the individual has high expression of one or more biomarkers selected from CD80, CD83, CD86, and HLA class II antigen in tumor-derived mature dendritic cells. (Item 39) The method according to any one of Items 1 to 38, wherein the individual has high expression of one or more biomarkers selected from the group consisting of CXCL9, CXCL10, CXCL11, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1, HES4, MTIB, MTIE, MTIG, MTIH, GADD45A, LAMP3, and miR-155. (Item 40) A kit for treating solid tumors or lymphatic tumors in an individual, comprising: a) an oncolytic virus; b) an immunomodulatory agent; and c) a device for locally administering the oncolytic virus to the site of the tumor, wherein the oncolytic virus comprises a tumor cell-specific promoter operably linked to a viral gene essential for replication of the virus, and a heterologous gene encoding an immune-related molecule, and the oncolytic virus is contained in a viral vector, and the immunomodulatory agent is formulated for systemic administration.

Brief Description of the Drawings

[0037]

Figure 1

Modes for Carrying Out the Invention

[0038] The present invention provides a method and composition for treating solid tumors or lymphatic tumors in an individual by locally administering an effective amount of an oncolytic virus (such as CG0070) to the site of the tumor and systemically administering an effective amount of an immunomodulatory agent (including combinations of immunomodulatory agents such as immunostimulants and / or immune checkpoint inhibitors). The method and composition may further include local administration of an immunomodulatory agent (including combinations of immunomodulatory agents). For example, one exemplary tumor suitable for the methods described herein is bladder cancer, where the immunomodulatory agent can be administered intravenously while the oncolytic virus can be administered intravesically.

[0039] The present invention provides a live and real-time "in vivo" cancer vaccine system generated inside the human body by combining local (such as intratumoral) delivery of oncolytic viruses with systemic (such as intravenous) delivery of immunomodulatory agents. A prominent feature of the present invention is the oncolytic virus, which has both a tumor cell-specific promoter operably linked to a viral gene essential for replication and a heterologous gene encoding an immune-related molecule such as GM-CSF. Thereby, local administration of the oncolytic virus enables both virus-mediated tumor-specific infection and simultaneous local delivery of immune-related molecules to the tumor site. Combined with systemic delivery of an immunomodulatory agent (including combinations of immunomodulatory agents), and optionally further combined with local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents), the cancer vaccine system may provide therapeutic components to the tumor and the human body in appropriate effective amounts, at appropriate times, and in an appropriate order to induce an enhanced immune response against the tumor.

[0040] Therefore, the combinations described herein are thought to be capable of fully exploiting the oncolytic and immunogenic responses within an individual and enhancing the therapeutic effect of cancer immunotherapy. It will be understood by those skilled in the art that in the combination therapy methods described herein, it is necessary to administer one agent or composition in combination with another agent. The dosage, dosing schedule, route of administration, and order of administration of each agent (such as the oncolytic virus and each immunomodulatory agent) of the combination therapies provided herein can be independently optimized to yield optimal treatment results. The method may also be further combined with pretreatment such as local administration of inactivated tumor cells and / or local radiotherapy or local administration of cytokines, chemokines, or other beneficial therapeutic agents to increase the success rate of treatment.

[0041] In one aspect, a method of treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) intravesically administering an effective amount of an oncolytic virus; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule.

[0042] In some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus to the site of the tumor; b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents); and c) locally administering an effective amount of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) intravesically administering an effective amount of an oncolytic virus; b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents); and c) intravesically administering an effective amount of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule.

[0043] Furthermore, compositions (such as pharmaceutical compositions), kits, and products useful in the methods described herein are also provided. In one aspect, a kit for treating a solid tumor or a lymphatic tumor in an individual is provided, the kit comprising: a) an oncolytic virus; b) an immunomodulatory agent (including combinations of immunomodulatory agents); and c) an apparatus for locally administering the oncolytic virus to the site of the tumor, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. Definitions

[0044] As used herein, "treatment" or "treating" is a method for obtaining a beneficial or desired result, including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, slowing or decelerating the progression of the disease, inducing remission of the disease state, achieving remission (partial or complete) of the disease, reducing the dosage of one or more other medications required to treat the disease, slowing the progression of the disease, improving the quality of life, and / or extending the survival period. "Treatment" also encompasses a reduction in the pathological consequences of cancer. The methods of the present invention contemplate any one or more of these aspects of treatment.

[0045] "Adjuvant setting" refers to a clinical setting in which an individual has a history of cancer and in which treatment has typically (but not necessarily) been effective for that individual, and includes, but is not limited to, surgical treatment (e.g., surgical resection), radiation therapy, and chemotherapy. Treatment and administration in an "adjuvant setting" refer to the mechanism of subsequent treatment.

[0046] "Neo - adjuvant setting" refers to the clinical setting in which the method is carried out before primary / radical treatment. The neo - adjuvant setting as used herein also refers to any "tumor site preparation" treatment modality that is used in a continuous manner in combination with a therapeutic component (e.g., oncolytic virus and immunomodulatory agent, or oncolytic virus, immunomodulatory agent and inactivated tumor cells) as described in the present invention.

[0047] As used herein, the term "effective amount" refers to the amount of a compound or composition sufficient to treat a specified disorder, condition or disease, such as alleviating, ameliorating, reducing, and / or delaying one or more of the symptoms. In the context of cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce the rate of tumor growth (such as inhibiting tumor growth) or prevent or delay other unwanted cell growth in cancer. In some embodiments, the effective amount is an amount sufficient to delay the progression of cancer. In some embodiments, the effective amount is an amount sufficient to prevent or delay recurrence. In some embodiments, the effective amount is an amount sufficient to reduce the recurrence rate in an individual. The effective amount can be administered in one or more administrations. An effective amount of a drug or composition may (i) reduce the number of cancer cells, (ii) shrink the tumor size, (iii) inhibit, delay, slow down, and preferably stop the infiltration of cancer cells into peripheral organs to some extent, (iv) inhibit (i.e., delay to some extent and preferably stop) tumor metastasis, (v) inhibit tumor growth, (vi) prevent the occurrence and / or recurrence of tumors, (vii) delay the occurrence and / or recurrence of tumors, (viii) reduce the recurrence rate of tumors, and / or (ix) alleviate to some extent one or more of the symptoms associated with cancer. As understood in the art, an "effective amount" can be in one or more administrations, i.e., a single administration or multiple administrations may be required to achieve the desired treatment endpoint.

[0048] "In combination with" or "combined with" refers to administering the administration of the oncolytic virus described in this specification in addition to the administration of other agents (such as immunomodulatory agents, inactivated tumor cells, etc.) under the same treatment plan for the same individual, that is, adding the administration of one treatment modality to the administration of another treatment modality. Thus, "in combination with" or "combined with" refers to administering the administration of one treatment modality before, during, or after the delivery of another treatment modality to the individual.

[0049] As used herein, the term "co - administration" means that the first treatment and the second treatment as combination therapies are administered simultaneously. When the first and second treatments are co - administered, the first and second treatments may be contained in the same composition (e.g., a composition containing both the first and second treatments), or may be contained in separate compositions (e.g., the first treatment is contained in one composition and the second treatment is contained in another composition).

[0050] As used herein, the term "sequential administration" or "in sequence" means that the first treatment and the second treatment as combination therapies are administered with a time difference of more than about 1 minute, such as more than any of about 5, 10, 15, 20, 30, 40, 50, 60 minutes, or more. In some cases, the term "sequential administration" means that the first treatment and the second treatment as combination therapies are administered with a time difference of more than about 1 day, such as more than any of about 1 day - 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks, or more. Either the first treatment or the second treatment may be administered first. The first and second treatments may be contained in separate compositions, which may be included in the same or different packages or kits.

[0051] The term "administered immediately before" means that the first treatment is administered within about 15 minutes, such as within any of about 10, 5, or 1 minute before the administration of the second treatment. The term "administered immediately after" means that the first treatment is administered within about 15 minutes, such as within any of about 15, 10, or 1 minute after the administration of the second treatment.

[0052] As used herein, the terms "specific," "specificity," or "selective" or "selectivity" when used to describe a compound as an inhibitor mean that the compound preferentially interacts (e.g., binds, modifies, inhibits) with a particular target (e.g., a protein and an enzyme) over a non-target.

[0053] As used herein, the terms "transduction" and "gene transfer" include all methods well known in the art of introducing DNA into a cell using an infectious agent (such as a virus) or other means for the expression of a protein or molecule of interest. In addition to viruses or virus-like substances, chemical-based gene transfer methods such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine), electroporation, cell squeezing, sonoporation, optical gene transfer, impalefection, protein fusion, plasmid delivery, or non-chemical methods such as transposons, gene guns, magnectofection or magnet-assisted gene transfer, particle-based methods such as using a particle gun, and hybrid methods such as nucleofection exist.

[0054] As used herein, the term "tumor site preparation" describes a single treatment modality or a combination of two or more treatment modalities used in a sequential manner in conjunction with a therapeutic component (e.g., an oncolytic virus and an immunomodulatory agent, or an oncolytic virus, an immunomodulatory agent, and an inactivated tumor cell), which or these treatment modalities are applied directly or indirectly (e.g., via IV therapy) to a tumor site (such as cancer cells or tissues containing cancer cells). Exemplary treatment modalities for tumor site preparation include, but are not limited to, administration of immune-related molecules, irradiation, and administration of therapeutic agents. All tumor site preparations described herein may include administration of a single molecule or agent, or a combination of two or more molecules and / or agents.

[0055] It will be understood that embodiments of the invention described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0056] Reference to "about" a value or parameter described herein includes (and describes) variations that are directed to the value or parameter itself. For example, a reference to "about X" includes a description of "X."

[0057] As used herein, a value or parameter with negation (not), such as "not Reference to data generally means and describes a value or parameter "other" than that value or parameter being affirmed. For example, "the method is not used to treat cancer of type X" means "the method is used to treat cancers of types other than X."

[0058] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0059] As used in this specification and the appended claims, the singular forms "a," "or," and "the" are intended to include the plural of the referent unless the context clearly dictates a singular referent. Methods for Treating Solid Tumors or Lymphoid Tumors

[0060] In one aspect, the present invention provides a method for treating a solid tumor or a lymphatic tumor (such as bladder cancer) in an individual (such as a human). The method includes: a) locally administering an effective amount of an oncolytic virus to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents). The oncolytic virus includes a viral vector containing a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the oncolytic virus is selected from the group consisting of adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, measles virus, Seneca Valley virus, coxsackievirus, reovirus, vesicular stomatitis virus, Maraba and rhabdovirus, and parvovirus. In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation, or genetic modification). In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method includes systemic administration of a combination of immunomodulatory agents including one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further includes local administration of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor.

[0061] Another aspect of the present application provides a method for treating a solid tumor or a lymphatic tumor in an individual, the method comprising: a) systemically administering (such as intravenously) an effective amount of an oncolytic virus to the site of the tumor; and b) systemically administering (such as intravenously) an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule. Embodiments described herein as applicable to local administration of an oncolytic virus are also applicable to methods comprising systemic administration of an oncolytic virus.

[0062] Exemplary viruses suitable for use as oncolytic viruses in the present invention include adenoviruses such as H101 (ONCOCRINE®), CG-TG-102 (Ad5 / 3-D24-GM-CSF), and CG0070; herpes simplex viruses such as Talimogene laherparapvec (T-VEC®) and HSV-1716 (SEPREHVIR®); reoviruses such as REOLYSIN®; vaccinia viruses such as JX-594; Seneca Valley viruses such as NTX-010 and SVV-001; Newcastle disease viruses such as NDV-NS1 and GL-ONC1; polioviruses such as PVS-RIPO; measles viruses such as MV-NIS; coxsackieviruses such as CAVATAK™; vesicular stomatitis virus; Maraba and rhabdoviruses; parvoviruses and mumps viruses, but are not limited thereto. In some embodiments, the oncolytic virus is an oncolytic adenovirus. In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus is only a part, or multiple parts, of a wild-type oncolytic virus that can cause an infection, inflammation, or infection-like effect. In some embodiments, the virus is proliferative. In some embodiments, the virus preferentially proliferates within tumor cells. In some embodiments, the oncolytic virus preferentially proliferates within cancer cells lacking a deletion in the Rb pathway.

[0063] In some embodiments, a method of treating a solid tumor or a lymphatic tumor (such as bladder cancer) in an individual (such as a human) is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the oncolytic virus is proliferative. In some embodiments, the oncolytic virus preferentially proliferates in cancer cells, such as Rb pathway-deleted cancer cells. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor.

[0064] In some embodiments, a method of treating a solid tumor or a lymphatic tumor (such as bladder cancer) in an individual (such as a human) is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter. For example, the E2F-1 promoter comprises the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents, including one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor.

[0065] In some embodiments, the methods described herein further comprise locally administering an immune-related molecule (such as a cytokine, chemokine, or PRRago, i.e., a pathogen recognition receptor agonist) to the site of the tumor. In some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL-12, interferon (such as type 1, type 2, or type 3 interferon, e.g., interferon γ), CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LTαβ. In some embodiments, the immune-related molecule is selected from the group consisting of STING (i.e., stimulator of interferon genes) activators (such as CDN, i.e., cyclic dinucleotide), PRRago (such as CpG, imiquimod, or poly I:C), TLR stimulants (such as GS-9620, AED-1419, CYT-003-QbG10, AVE-0675, or PF-7909), and RLR stimulants (such as RIG-I, Mda5, or LGP2 stimulants). In some embodiments, the immune-related molecule attracts dendritic cells, T cells, B cells, and / or follicular helper T cells. In some embodiments, the immune-related molecule is administered separately from the oncolytic virus (e.g., in a separate composition or as a separate entity within the same composition). In some embodiments, the immune-related molecule is administered by transduction to the site of the tumor. Exemplary transduction methods well known in the art include, but are not limited to, the use of calcium phosphate, dendrimers, liposomes, cationic polymers, electroporation, cell squeezing, sonoporation, optical gene transfer, protoplast fusion, impalefection, hydrodynamic delivery, gene gun, magnetofection, viral gene transfer, and nucleofection. In some embodiments, the immune-related molecule is expressed by an oncolytic virus. For example, the oncolytic virus may contain a nucleic acid encoding the immune-related molecule, and the nucleic acid may be present within the viral vector or on a separate vector.

[0066] The present invention is based in part on unpublished results obtained from clinical trials by the inventors. Without being bound by any theory or hypothesis, the viral oncolytic virus, CG0070, which is specifically designed to replicate only within cancer cells, is thought to provide an "appropriate amount" of GM-CSF at the tumor site "in real time" during the period of cancer cell death. Delivery of GM-CSF by the oncolytic virus during the period of cancer cell death "at" the tumor site is considered essential for both antigen-presenting cells to mature existing antigens, neoantigens, and tolerance-breaking antigens (TBAs), and to cross-present these to activated T cells from this cell death mixture. High doses of GM-CSF can lead to a non-focused immune system and may induce an immediate increase in local and systemic suppressors, while low doses of GM-CSF may be insufficient for the inflammatory process and activation of related immune cells. Therefore, an appropriate amount of GM-CSF is required at the tumor site in this treatment scenario. The delicate balance at the tumor site involving an appropriate amount of GM-CSF and the "live" cancer cell death mixture at the site is thought to attract an adaptive immune response specific to cancer cells. Thus, an oncolytic virus that is cancer-specific and oncolytic, in combination with an appropriate amount of GM-CSF or other suitable immune-related molecules, either expressed by the oncolytic virus in response to any oncolytic virus delivered to the tumor site during the period of cell death, infection, or inflammation, or secreted by the body's defenses, is considered an ideal choice for effective cancer immunotherapy.

[0067] In some embodiments, the immune-related molecule enhances the immune response within an individual. Immune-related molecules include, but are not limited to, cytokines, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony-stimulating factors (CSFs), erythropoietin, thrombopoietin, tumor necrosis factor α (TNF), TNF-β, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interferon α, interferon β, interferon γ, interferon λ, a stem cell growth factor called "S1 factor", human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), liver growth factor, prostaglandins, fibroblast growth factors, prolactin, placental lactogen, OB protein, Mullerian duct inhibitory factor, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, NGF-β, platelet-derived growth factor, TGF-α, TGF-β, insulin-like growth factor I, insulin-like growth factor II, macrophage-CSF (M-CSF), IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin, endostatin, lymphotoxin, thalidomide, lenalidomide, or pomalidomide.

[0068] The immune-related molecule can belong to any one of the molecular modalities well-known in the art, including but not limited to aptamers, mRNAs, siRNAs, microRNAs, shRNAs, peptides, antibodies, anticalins, spherical nucleic acids, TALENs, zinc finger nucleases, CRISPR / Cas9, and small molecules.

[0069] Immune-related molecules can be used alone or in combination. For example, any number (such as 1, 2, 3, 4, 5, 6, or more) of immune-related molecules can be used simultaneously or sequentially.

[0070] The oncolytic virus of the present invention comprises a viral vector containing a nucleic acid sequence encoding at least one (e.g., 1, 2, 3, 4, 5, or more) immune-related molecule. In some embodiments, the oncolytic virus comprises a viral vector containing a heterologous gene encoding an immune-related molecule. In some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL-12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LTαβ. In some embodiments, the immune-related molecule is GM-CSF. In some embodiments, the heterologous gene is operably linked to a viral promoter such as the E1 promoter or the E3 promoter.

[0071] Accordingly, in some embodiments, a method of treating a solid tumor or a lymphatic tumor (such as bladder cancer) in an individual (such as a human) is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or a chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for the replication of the oncolytic virus is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor.

[0072] In some embodiments, the oncolytic virus is adenovirus serotype 5. In some embodiments, the endogenous E1a promoter of the native adenovirus is replaced with the human E2F-1 promoter, and the E3 19kD coding region of the native adenovirus is a nucleic acid sequence encoding human GM-CSF. In some embodiments, a polyadenylation signal (PA) is inserted 5' to the E2F-1 promoter. In some embodiments, the nucleic acid encoding human GM-CSF is operably linked to the E3 promoter. In some embodiments, the vector backbone of adenovirus serotype 5 further comprises the E2, E4, late protein regions or inverted terminal repeats (ITRs) identical to the wild-type adenovirus serotype 5 genome. In some embodiments, the oncolytic virus has a genomic structure as shown in FIG. 1. In some embodiments, the oncolytic virus is conditionally replicating. In some embodiments, the oncolytic virus preferentially grows within cancer cells. In some embodiments, the cancer cells are Rb pathway-deficient cancer cells. In some embodiments, the oncolytic virus is CG0070.

[0073] Thus, for example, in some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of adenovirus serotype 5 to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the endogenous E1a promoter of the native adenovirus is replaced by a human E2F-1 promoter, and the E3 19kD coding region of the native adenovirus is replaced by a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, e.g., GM-CSF). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents, including one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the adenovirus is administered directly into the tumor. In some embodiments, the adenovirus is administered to the tissue having the tumor. In some embodiments, the adenovirus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor.

[0074] In some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of CG0070 to the site of the tumor; and b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as at least two immune checkpoint inhibitors, at least two immune stimulants, or a combination of at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, CG0070 is administered directly into the tumor. In some embodiments, CG0070 is administered to the tissue having the tumor. In some embodiments, CG0070 is administered at a dose of about 1×10 8 ~ about 1×10 14 viral particles (vp) (such as about 1×10 8 ~ about 1×10 10 , about 1×10 10 ~ about 1×10 12 , or about 1×10 12 ~ about 1×10 14 vp). In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks, or 5 weeks). In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor.

[0075] In some embodiments, the above-described oncolytic virus and immunomodulatory agent (including combinations of immunomodulatory agents) are administered continuously, i.e., the administration of the oncolytic virus is administered before or after the administration of the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered prior to the administration of the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered within any of about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours before the administration of the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered over about several days or weeks (such as any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more) before the administration of the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered after the administration of the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered within any of about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours after the administration of the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered over about several days or weeks (such as any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more) after the administration of the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus and the immunomodulatory agent (including combinations of immunomodulatory agents) are administered such that one is administered immediately after the other (e.g., the time between the two administrations is within 5 minutes or less). For example, in some embodiments, the oncolytic virus is administered immediately before the administration of the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered immediately after the administration of the immunomodulatory agent (including combinations of immunomodulatory agents).

[0076] In some embodiments, the oncolytic virus and the immunomodulatory agent (including combinations of immunomodulatory agents) are administered simultaneously. In some embodiments, the oncolytic virus and the immunomodulatory agent (including combinations of immunomodulatory agents) are administered simultaneously by separate compositions.

[0077] The immunomodulatory agents discussed herein include both immunostimulatory agents and immune checkpoint inhibitors. The immunomodulatory agent can belong to any one of the molecular modalities well-known in the art, including but not limited to aptamers, mRNAs, siRNAs, microRNAs, shRNAs, peptides, antibodies, anticalins, spherical nucleic acids, TALENs, zinc finger nucleases, CRISPR / Cas9, and small molecules.

[0078] In some embodiments, the immunomodulatory agent is an immunostimulant. In some embodiments, the immunostimulant is a natural or artificial ligand of an immunostimulatory molecule, including, for example, ligands of OX40 (e.g., OX40L), CD-28 (e.g., CD80, CD86), ICOS (e.g., B7RP1), 4-1BB (e.g., 4-1BBL, Ultra4-1BBL), CD27 (e.g., CD70), CD40 (e.g., CD40L), and TCR (e.g., MHC class I or class II molecules, IMCgp100). In some embodiments, the immunostimulant is an antibody selected from the group consisting of anti-CD28 (e.g., TGN-1412), anti-OX40 (e.g., MEDI6469, MEDI-0562), anti-ICOS (e.g., MEDI-570), anti-GITR (e.g., TRX518, INBRX-110, NOV-120301), anti-41-BB (e.g., BMS-663513, PF-05082566), anti-CD27 (e.g., BION-1402, balstilimab, and hCD27.15), anti-CD40 (e.g., CP870,893, BI-655064, BMS-986090, APX005, APX005M), anti-CD3 (e.g., blinatumomab, muromonab), and anti-HVEM. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding partial sequences of a full-length antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single-domain antibody, a fusion protein containing an antibody portion, or any other functional variant or derivative thereof.

[0079] In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a natural or artificial ligand of an inhibitory immune checkpoint molecule, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), TIM3 (e.g., Galectin-9), A2a receptor (e.g., adenosine, regadenoson), LAG3 (e.g., MHC class I or MHC class II molecules), BTLA (e.g., HVEM, B7-H4), KIR (e.g., MHC class I or MHC class II molecules), PD-1 (e.g., PD-L1, PD-L2), IDO (e.g., NKTR-218, Indoximod, NLG919), CD47 (e.g., SIRP-α receptor), and CSF1R. In some embodiments, the immune checkpoint inhibitor is an antibody that targets an inhibitory immune checkpoint protein.In some embodiments, the immunomodulatory agent is an antibody selected from the group consisting of anti-CTLA-4 (e.g., ipilimumab, tremelimumab, KAHR-102), anti-TIM3 (e.g., F38-2E2, ENUM005), anti-LAG3 (e.g., BMS-986016, IMP701, IMP321, C9B7W), anti-KIR (e.g., lirilumab, IPH2101, IPH4102), anti-PD-1 (e.g., nivolumab, pidilizumab, pembrolizumab, BMS-936559, atezolizumab, ramucirumab, MK-3475, AMP-224, AMP-514, STI-A1110, TSR-042), anti-PD-L1 (e.g., KY-1003 (EP20120194977), MCLA-145, atezolizumab, BMS-936559, MEDI-4736, MSB0010718C, AUR-012, STI-A1010, PCT / US2001 / 020964, MPDL3280A, AMP-224, dapirolizumab pegol (CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42 (OSU-HDAC42, HDAC-42, AR42, AR 42,OSU-HDAC 42,OSU-HDAC-42,NSC D736012,HDAC-42,HDAC 42,HDAC42,NSCD736012,NSC-D736012), MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1, anti-B7-H4, anti-CD52 (such as alemtuzumab), anti-IL-10, anti-IL-35, anti-TGF-β (such as fresolumimab), anti-CSF1R (e.g., FPA008), anti-NKG2A (e.g., monalizumab), anti-MICA (e.g., IPH43), and anti-CD39. In some embodiments, the antibody is an antagonist antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a monoclonal antibody.In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of a full-length antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single-domain antibody, a fusion protein comprising an antibody moiety, or any other functional variant or derivative thereof.

[0080] In some embodiments, the method comprises systemic administration of a single immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the immunomodulatory agent is selected from the immunomodulatory agents listed in Table 1 and is administered by the same route of administration, and / or dose, and / or dosing frequency, and / or duration, and / or maintenance schedule as those listed in Table 1. In some embodiments, the immunomodulatory agent is selected from the immunomodulatory agents listed in Table 1 and is administered by a route of administration, and / or dose, and / or dosing frequency, and / or duration, and / or maintenance schedule different from those listed in Table 1. In some embodiments, the immunomodulatory agent is not a molecule selected from Table 1.

[0081] In some embodiments, the method comprises systemic administration of at least two (such as any of 2, 3, 4, 5, 6, or more) immunomodulatory agents. In some embodiments, all or a portion of the at least two immunomodulatory agents are administered simultaneously, such as within a single composition. In some embodiments, all or a portion of the at least two immunomodulatory agents are administered sequentially. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising an immune checkpoint inhibitor and an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising two or more (such as any of 2, 3, 4, 5, 6, or more) checkpoint inhibitors. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising two or more (such as any of 2, 3, 4, 5, 6, or more) immune stimulants. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising any number (such as 1, 2, 3, 4, 5, 6, or more) of immune checkpoint inhibitors and any number (such as any of 2, 3, 4, 5, 6, or more) of immune stimulants. In some embodiments, the at least two immunomodulatory agents comprise one or more immunomodulatory agents selected from Table 1. For example, in some embodiments, the method comprises: a) locally administering an effective amount of a oncolytic virus (such as a virus, e.g., an oncolytic virus) to the site of the tumor; b) systemically administering an effective amount of a first systemic immunomodulatory agent (such as an immune checkpoint inhibitor) to the individual; and c) systemically administering an effective amount of a second systemic immunomodulatory agent (such as an immune stimulant).

[0082] In some embodiments, the method further comprises local administration of any number (such as 1, 2, 3, 4, or more) of additional immunomodulatory agents (hereinafter referred to as "second immunomodulatory agents" or "local immunomodulatory agents") to the site of the tumor, and immunomodulatory agents in such context are referred to herein as "first immunomodulatory agents", "systemic immunomodulatory agents", or "immunomodulatory agents"). In some embodiments, the first immunomodulatory agent and the second immunomodulatory agent have the same target. In some embodiments, the first immunomodulatory agent and the second immunomodulatory agent are the same immunomodulatory agent molecule. In some embodiments, the first immunomodulatory agent and the second immunomodulatory agent have the same target but belong to different modalities. In some embodiments, the first immunomodulatory agent and the second immunomodulatory agent are different immunomodulatory agent molecules. In some embodiments, the first immunomodulatory agent and the second immunomodulatory agent do not have the same target. In some embodiments, the first immunomodulatory agent is an immune checkpoint inhibitor and the second immunomodulatory agent is an immune stimulant. In some embodiments, the first immunomodulatory agent is an immune checkpoint inhibitor and the second immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the first immunomodulatory agent is an immune stimulant and the second immunomodulatory agent is an immune stimulant. In some embodiments, the first immunomodulatory agent is an immune stimulant and the second immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the method comprises local administration of a combination of at least two local immunomodulatory agents. In cases where two or more systemic immunomodulatory agents and two or more local immunomodulatory agents are administered, any of the two or more systemic immunomodulatory agents may have the same target as the local immunomodulatory agent or may be the same immunomodulatory agent.

[0083] The administration of the immunomodulatory agents can be in any order, including co - administration of systemic administration of a first immunomodulatory agent and local administration of a second immunomodulatory agent, and sequential administration of immunomodulatory agents, wherein at least one immunomodulatory agent is administered systemically between the sequentially administered immunomodulatory agents. For example, after first locally administering a second immunomodulatory agent to the site of the tumor (such as intratumorally), systemic administration (such as intravenous administration) of a first immunomodulatory agent follows, or after first systemically administering a first immunomodulatory agent (such as intravenously), local administration (such as intratumoral administration) of a second immunomodulatory agent follows. Immunomodulatory agents co - administered simultaneously via the same route of administration may be administered as a single composition. For example, the immunomodulatory agents can be mixed prior to administration of the single composition (such as immediately before administration, for example, within about 10, 5, or less than 1 minute before administration).

[0084] The local administration of the oncolytic virus and the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents) can be done simultaneously or sequentially. In some embodiments, the oncolytic virus is administered before or after the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered prior to the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered within any of about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours prior to the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered over about several days or weeks (such as any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more) prior to the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered after the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered within any of about 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours after the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered over about several days or weeks (such as any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more) after the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus and the second immunomodulatory agent (including combinations of immunomodulatory agents) are administered such that one is administered immediately after the other (e.g., within 5 minutes or less between the two administrations). For example, in some embodiments, the oncolytic virus is administered immediately prior to the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the oncolytic virus is administered immediately after the local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents).

[0085] In some embodiments, the oncolytic virus and the second immunomodulatory agent (including combinations of immunomodulatory agents) are administered simultaneously. In some embodiments, the oncolytic virus and the second immunomodulatory agent (including combinations of immunomodulatory agents) are administered simultaneously by separate compositions. In some embodiments, the oncolytic virus and the second immunomodulatory agent (including combinations of immunomodulatory agents) are administered as a single composition. In some embodiments, the oncolytic virus and the second immunomodulatory agent (including combinations of immunomodulatory agents) are mixed prior to administration of the composition (e.g., within about 10, 5, or less than 1 minute before administration, such as immediately before administration). In some embodiments, the composition comprising the oncolytic virus and the second immunomodulatory agent (including combinations of immunomodulatory agents) is prepared in advance prior to administration and stored for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or longer.

[0086] Accordingly, in some embodiments, a method of treating a solid tumor or lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus, e.g., CG0070) to the site of the tumor; b) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents); and c) locally administering an effective amount of a second immunomodulatory agent (including a combination of immunomodulatory agents) to the site of the tumor, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for virus replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the heterologous gene is GM-CSF. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the second immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the second immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of the second immunomodulatory agent comprising one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant).In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously.

[0087] In some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus, e.g., CG0070) to the site of the tumor; b) systemically administering an effective amount of a first immunomodulatory agent; and c) locally administering an effective amount of a second immunomodulatory agent to the site of the tumor, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the heterologous gene is GM-CSF. In some embodiments, the first immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the first immunomodulatory agent is an immune stimulant. In some embodiments, the second immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the second immunomodulatory agent is an immune stimulant. In some embodiments, the oncolytic virus and / or the second immunomodulatory agent is directly administered into the tumor. In some embodiments, the oncolytic virus and / or the second immunomodulatory agent is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the first immunomodulatory agent is administered intravenously. In some embodiments, the second immunomodulatory agent and the oncolytic virus are administered simultaneously, such as in the same composition. In some embodiments, the second immunomodulatory agent and the oncolytic virus are administered sequentially. In some embodiments, the first immunomodulatory agent is administered after the administration of the second immunomodulatory agent. In some embodiments, the first immunomodulatory agent is administered before the administration of the second immunomodulatory agent.In some embodiments, the order of administration is as follows: local administration of the oncolytic virus (such as intratumoral administration), followed by local administration of the second immunomodulatory agent (such as intratumoral administration), followed by systemic administration of the first immunomodulatory agent (such as intravenous administration).

[0088] In some embodiments, provided is a method for treating a solid tumor or a lymphatic tumor in an individual, the method comprising: a) locally administering an effective amount of a oncolytic virus (such as an oncolytic adenovirus, e.g., CG0070) to the site of the tumor; b) systemically administering an effective amount of a first immunomodulatory agent; c) locally administering an effective amount of a second immunomodulatory agent to the site of the tumor; and d) administering an effective amount of a third immunomodulatory agent (such as systemically or locally to the site of the tumor), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the heterologous gene is GM-CSF. In some embodiments, the first and / or second and / or third immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the first and / or second and / or third immunomodulatory agent is an immune stimulant. In some embodiments, the oncolytic virus and / or the second immunomodulatory agent and / or the third immunomodulatory agent are directly administered into the tumor. In some embodiments, the oncolytic virus and / or the second immunomodulatory agent are administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the first immunomodulatory agent and / or the second immunomodulatory agent are administered intravenously. In some embodiments, the second immunomodulatory agent and / or the third immunomodulatory agent, and the oncolytic virus are administered simultaneously, such as in the same composition. In some embodiments, the second immunomodulatory agent and / or the third immunomodulatory agent, and the oncolytic virus are administered sequentially.In some embodiments, the first immunomodulatory agent is administered after the administration of the second immunomodulatory agent and / or the third immunomodulatory agent. In some embodiments, the first immunomodulatory agent is administered before the administration of the second immunomodulatory agent and / or the third immunomodulatory agent. In some embodiments, the order of administration is as follows: local administration of an oncolytic virus (such as intratumoral administration), followed by local administration of the second immunomodulatory agent (such as intratumoral administration), followed by local administration of the third immunomodulatory agent (such as intratumoral administration), followed by systemic administration of the first immunomodulatory agent (such as intravenous administration). In some embodiments, the order of administration is as follows: local administration of an oncolytic virus (such as intratumoral administration), followed by local administration of the second immunomodulatory agent and the third immunomodulatory agent (such as intratumoral administration, for example, in the same composition), followed by systemic administration of the first immunomodulatory agent (such as intravenous administration). In some embodiments, the order of administration is as follows: local administration of an oncolytic virus (such as intratumoral administration), followed by local administration of the second immunomodulatory agent (such as intratumoral administration), followed by systemic administration of the first immunomodulatory agent (such as intravenous administration), followed by systemic administration of the third immunomodulatory agent (such as intravenous administration). In some embodiments, the order of administration is as follows: local administration of an oncolytic virus (such as intratumoral administration), followed by local administration of the second immunomodulatory agent (such as intratumoral administration), followed by systemic administration of the first immunomodulatory agent and the third immunomodulatory agent (such as intravenous administration, for example, in the same composition).

[0089] The third immunomodulatory agent may be any of the immunomodulatory agents described herein. In some embodiments, the third immunomodulatory agent has the same target, such as being the same immunomodulatory agent molecule as the first immunomodulatory agent, and the third immunomodulatory agent is administered locally to the site of the tumor. In some embodiments, the third immunomodulatory agent has the same target, such as being the same immunomodulatory agent molecule as the second immunomodulatory agent, and the third immunomodulatory agent is administered systemically. In some embodiments, the first immunomodulatory agent, the second immunomodulatory agent, and the third immunomodulatory agent are different, for example, having different targets, being different types of immunomodulatory agents, and / or being different immunomodulatory agent molecules.

[0090] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody. In the present invention, any of the anti-CTLA-4 antibodies well-known in the art, including but not limited to ipilimumab, tremelimumab, and KAHR-102, may be used. In some embodiments, the anti-CTLA-4 antibody is YERVOY® (ipilimumab). In some embodiments, the anti-CTLA-4 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-CTLA-4 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of a full-length anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the anti-CTLA-4 antibody is a bispecific antibody, a multispecific antibody, a single-domain antibody, a fusion protein containing an antibody moiety, or any other functional variant or derivative thereof. In some embodiments, the inhibitor of CTLA-4 is an artificial lipocalin protein that specifically recognizes CTLA-4 (such as an anticarin molecule that specifically binds to CTLA-4). In some embodiments, the inhibitor of CTLA-4 is a natural or artificial ligand of CTLA-4, such as B7.1 or B7.2.

[0091] Thus, for example, in some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual (such as a human) is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an artificial lipocalin protein, e.g., an anticarin that specifically recognizes CTLA-4). The oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or a chemokine). In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in cancer cells, such as Rb pathway-deleted cancer cells. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, such as ipilimumab. In some embodiments, the inhibitor of CTLA-4 is an artificial lipocalin protein, such as an anticarin that specifically recognizes CTLA-4. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the inhibitor of CTLA-4 is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of CTLA-4 are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the inhibitor of CTLA-4. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of CTLA-4. In some embodiments, the oncolytic virus and the inhibitor of CTLA-4 are administered simultaneously. In some embodiments, the method further comprises locally administering a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant.In some embodiments, the method further comprises administering a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant, either systemically or locally to the site of the tumor.

[0092] In some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an artificial lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4). The oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or a chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0093] In some embodiments, a method for treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of adenovirus serotype 5 to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an artificial lipocalin protein, e.g., an anticarin that specifically recognizes CTLA-4), wherein the endogenous E1a promoter of the native adenovirus is replaced with a human E2F1 promoter, and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, e.g., GM-CSF). In some embodiments, the tumor-specific promoter is a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0094] In some embodiments, a method for treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of CG0070 to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an artificial lipocalin protein, e.g., an anticarin that specifically recognizes CTLA-4). In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, e.g., ipilimumab. In some embodiments, the inhibitor of CTLA-4 is an artificial lipocalin protein, e.g., an anticarin that specifically recognizes CTLA-4. In some embodiments, CG0070 is administered directly into the tumor. In some embodiments, CG0070 is administered to the tissue having the tumor. In some embodiments, CG007 is about 1×10 8 ~ about 1×10 14 virus particles (vp) (about 1×10 8 ~ about 1×10 10 , about 1×10 10 ~ about 1×10 12 , or about 1×10 12 ~ about 1×10 14It is administered at a dose such as any of those of vp). In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about one week to about six weeks (such as any of at least about three weeks, four weeks, or five weeks). In some embodiments, the CTLA-4 inhibitor is administered intravenously. In some embodiments, CG0070 and the CTLA-4 inhibitor are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the CTLA-4 inhibitor (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the CTLA-4 inhibitor (such as immediately after administration). In some embodiments, CG0070 and the CTLA-4 inhibitor are administered simultaneously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant. In some embodiments, the method further comprises administration (such as systemic administration or local administration to the site of the tumor) of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant.

[0095] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In the present invention, any of the anti-PD-1 antibodies well-known in the art, including but not limited to nivolumab, pembrolizumab, pidilizumab, BMS-936559, and atezolizumab, lambrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, and TSR-042, may be used. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-PD-1 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding partial sequences of a full-length anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the anti-PD-1 antibody is a bispecific antibody, a multispecific antibody, a single-domain antibody, a fusion protein containing an antibody portion, or any other variant or derivative thereof. In some embodiments, the inhibitor of PD-1 is a natural or artificial ligand of PD-1, such as PD-L1 or PD-L2. In some embodiments, the inhibitor of PD-1 is an inhibitor of the interaction between PD-1 and its ligand, for example, an inhibitor of the PD-1 / PD-L1 interaction or an inhibitor of the PD-1 / PD-L2 interaction. In some embodiments, the inhibitor of PD-1 is an inhibitor of a PD-1 ligand, such as an inhibitor of PD-L1 (e.g., an anti-PD-L1 antibody) or an inhibitor of PD-L2 (e.g., an anti-PD-L2 antibody). In the present invention, any of the inhibitors of the interaction between PD-1 and its ligand may be used, for example, see U.S. Patent Nos. 7,709,214, 7,432,059, 7,722,868, 8,217,149, 8,383,796, and 9,102,725. In some embodiments, the inhibitor of PD-1 is an Fc fusion protein containing a PD-1 ligand, such as an Fc fusion of PD-L2 (e.g., AMP-224).

[0096] Thus, for example, in some embodiments, a method of treating a solid tumor or lymphatic tumor in an individual (such as a human) is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of PD-1 (such as an anti-PD-1 antibody, e.g., nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, e.g., AMP-224, etc.). The oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus preferentially proliferates within cancer cells, such as Rb pathway-deleted cancer cells. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody, e.g., nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the inhibitor of PD-1 is an inhibitor of the interaction between PD-1 and its ligand, such as an inhibitor of the PD-1 / PD-L1 interaction or an inhibitor of the PD-1 / PD-L2 interaction. In some embodiments, the inhibitor of PD-1 is an Fc fusion protein comprising a PD-1 ligand, such as an Fc fusion of PD-L2 (e.g., AMP-224). In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the inhibitor of PD-1 is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of PD-1 are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the inhibitor of PD-1. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of PD-1. In some embodiments, the oncolytic virus and the inhibitor of PD-1 are administered simultaneously.In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor (such as a CTLA-4 inhibitor) or an immune stimulant (e.g., a CD40 activator or a 4-1BB activator). In some embodiments, the method further comprises administration (such as systemic administration or local administration to the site of the tumor) of a third immunomodulatory agent, such as an immune checkpoint inhibitor (such as a CTLA-4 inhibitor) or an immune stimulant (e.g., a CD40 activator or a 4-1BB activator).

[0097] In some embodiments, provided is a method of treating a solid tumor or a lymphatic tumor in an individual, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of PD-1 (such as an anti-PD-1 antibody, e.g., nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, e.g., AMP-224, etc.), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or a chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0098] In some embodiments, a method for treating solid tumors or lymphatic tumors in an individual is provided, the method comprising: a) locally administering an effective amount of adenovirus serotype 5 to the site of the tumor, wherein the endogenous E1a promoter of the native adenovirus is replaced by a human E2F1 promoter, and the E3 19kD coding region of the native adenovirus is replaced by a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, for example, GM-CSF); and b) systemically administering an effective amount of an inhibitor of PD-1 (an anti-PD-1 antibody, such as nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, such as AMP-224, etc.). In some embodiments, the tumor-specific promoter is a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0099] In some embodiments, a method for treating solid tumors or lymphatic tumors in an individual is provided, the method comprising: a) locally administering an effective amount of adenovirus serotype 5 to the site of the tumor, wherein the endogenous E1a promoter and the E3 19kD coding region of the native adenovirus are replaced by a human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (such as a cytokine or chemokine, for example, GM-CSF); and b) locally administering an effective amount of an inhibitor of PD-1 (an anti-PD-1 antibody, such as nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, such as AMP-224, etc.) to the site of the tumor. In some embodiments, the tumor-specific promoter is a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0100] In some embodiments, a method for treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of CG0070 to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of PD-1 (an anti-PD-1 antibody, such as nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, such as AMP-224, etc.). In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody, such as nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the inhibitor of PD-1 is an inhibitor of the interaction between PD-1 and its ligand, such as an inhibitor of the PD-1 / PD-L1 interaction or an inhibitor of the PD-1 / PD-L2 interaction. In some embodiments, the inhibitor of PD-1 is an Fc fusion protein comprising a PD-1 ligand, such as an Fc fusion of PD-L2 (such as AMP-224). In some embodiments, CG0070 is administered directly into the tumor. In some embodiments, CG0070 is administered to the tissue having the tumor. In some embodiments, CG007 is about 1×10 8 ~ about 1×10 14 virus particles (vp) (about 1×10 8 ~ about 1×10 10 , about 1×10 10 ~ about 1×10 12 , or about 1×10 12 ~ about 1×10 14It is administered at a dose such as any of those of vp). In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about one week to about six weeks (such as any of at least about three weeks, four weeks, or five weeks). In some embodiments, the PD-1 inhibitor is administered intravenously. In some embodiments, CG0070 and the PD-1 inhibitor are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the PD-1 inhibitor (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the PD-1 inhibitor (such as immediately after administration). In some embodiments, CG0070 and the PD-1 inhibitor are administered simultaneously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration (such as systemic administration or local administration to the site of the tumor) of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant.

[0101] In some embodiments, the immune checkpoint inhibitor is an inhibitor of the PD-1 ligand (e.g., PD-L1 and / or PD-L2). In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L1 antibody. In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L2 antibody. Exemplary anti-PD-L1 antibodies include, but are not limited to, KY-1003, MCLA-145, RG7446 (also known as atezolizumab), BMS935559 (also known as MDX-1105), MPDL3280A, MEDI4736, avelumab (also known as MSB0010718C), and STI-A1010. In some embodiments, the anti-PD-L1 or anti-PD-L2 is a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-PD-L1 or anti-PD-L2 is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of a full-length anti-PD-L1 or anti-PD-L2 antibody. In some embodiments, the anti-PD-L1 or anti-PD-L2 antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the anti-PD-L1 or anti-PD-L2 antibody is a bispecific antibody, a multispecific antibody, a single-domain antibody, a fusion protein containing an antibody portion, or any other variant or derivative thereof. In some embodiments, the inhibitor of the PD-1 ligand is an inhibitor of both PD-L1 and PD-L2 (e.g., a peptide, a protein, or a small molecule). Exemplary inhibitors of both PD-L1 and PD-L2 include, but are not limited to, AUR-012 and AMP-224. In some embodiments, the inhibitor of PD-L1 and the inhibitor of PD-L2 can be used interchangeably in any of the treatment methods described herein.

[0102] In some embodiments, provided is a method for treating a solid tumor or a lymphatic tumor in an individual (such as a human), the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of a PD-1 ligand (such as an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus preferentially proliferates in cancer cells, such as Rb pathway-deleted cancer cells. In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, atezolizumab, BMS935559, MPDL3280A, MEDI4736, avelumab, or STI-A1010. In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of the PD-1 ligand is an inhibitor of both PD-L1 and PD-L2 (such as a peptide, protein, or small molecule), such as AUR-012 and AMP-224. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to the tissue having the tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the inhibitor of the PD-1 ligand is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of the PD-1 ligand are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the inhibitor of the PD-1 ligand. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of the PD-1 ligand. In some embodiments, the oncolytic virus and the inhibitor of the PD-1 ligand are administered simultaneously.In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration (such as systemic administration or local administration to the site of the tumor) of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant.

[0103] In some embodiments, provided is a method of treating a solid tumor or a lymphatic tumor in an individual, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of a PD-1 ligand (such as an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or a chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the viral gene essential for virus replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0104] In some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of adenovirus serotype 5 to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of the PD-1 ligand (such as an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2), wherein the endogenous E1a promoter of the native adenovirus is replaced with a human E2F1 promoter, and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, e.g., GM-CSF). In some embodiments, the tumor-specific promoter is a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0105] In some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of CG0070 to the site of the tumor; and b) systemically administering an effective amount of an inhibitor of the PD-1 ligand (such as an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, atezolizumab, BMS935559, MPDL3280A, MEDI4736, avelumab, or STI-A1010. In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of the PD-1 ligand is an inhibitor of both PD-L1 and PD-L2 (such as a peptide, protein or small molecule), such as AUR-012 and AMP-224. In some embodiments, CG0070 is administered directly into the tumor. In some embodiments, CG0070 is administered to the tissue having the tumor. In some embodiments, CG007 is from about 1×10 8 ~ about 1×10 14 viral particles (vp) (from about 1×10 8 ~ about 1×10 10 , about 1×10 10 ~ about 1×10 12, or about 1×10 12 ~ about 1×10 14 vp, etc.) The dosage is administered. In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks, or 5 weeks). In some embodiments, the inhibitor of the PD-1 ligand is administered intravenously. In some embodiments, CG0070 and the inhibitor of the PD-1 ligand are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the inhibitor of the PD-1 ligand (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the inhibitor of the PD-1 ligand (such as immediately after administration). In some embodiments, CG0070 and the inhibitor of the PD-1 ligand are administered simultaneously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant. In some embodiments, the method further comprises administration of a third immunomodulatory agent (such as systemic administration or local administration to the site of the tumor), such as an immune checkpoint inhibitor or an immune stimulant.

[0106] In some embodiments, a method for treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) administering an effective amount of CG0070 into the tumor; b) administering an effective amount of an inhibitor of PD-L1 (such as an antagonistic anti-PD-L1 antibody, for example, atezolizumab) intravenously; and c) administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, for example, ipilimumab) into the tumor. In some embodiments, CG0070 is about 1×10 8 ~ about 1×10 14 viral particles (vp) (about 1×10 8 ~ about 1×10 10 , about 1×10 10 ~ about 1×10 12 , or about 1×10 12 ~ about 1×10 14It is administered at a dose such as any of those of vp. In some embodiments, CG0070 is administered once a week. In some embodiments, the PD-L1 inhibitor is administered at a dose of about 1 mg / kg to about 20 mg / kg, or about 750 mg to about 1200 mg. In some embodiments, the PD-L1 inhibitor is administered once every about 1 month to once every about 2 weeks (such as once every 2 weeks, once every 3 weeks, or once every 4 weeks, etc.). In some embodiments, the CTLA-4 inhibitor is administered at a dose of about 0.1 mg / kg to about 10 mg / kg (such as any of once a week, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg). In some embodiments, the CTLA-4 inhibitor is administered once a week. In some embodiments, the CTLA-4 inhibitor is administered immediately after the administration of CG0070 (for example, within 5 minutes after administration). In some embodiments, the PD-L1 inhibitor is an antagonist antibody of PD-L1, such as atezolizumab. In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 antibody, such as ipilimumab (for example, YERVOY®). In some embodiments, the CTLA-4 inhibitor is an artificial lipocalin protein, such as an anti-carlin that specifically recognizes CTLA-4. In some embodiments, to the individual, in combination with the administration of CG0070, an effective amount of DDM is further administered into the tumor as a transduction enhancer. In some embodiments, CG0070 and the CTLA-4 inhibitor are administered by injection into the tissue having the tumor. In some embodiments, CG0070 and the CTLA-4 inhibitor are directly injected into the tumor. In some embodiments, CG0070 and the PD-L1 inhibitor are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the PD-L1 inhibitor (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the PD-L1 inhibitor (such as immediately after administration). In some embodiments, CG0070 and the PD-L1 inhibitor are administered simultaneously. In some embodiments, CG0070 is administered over about 1 to about 6 weeks as one treatment unit.In some embodiments, the treatment unit is repeated every about 2 to about 3 months. In some embodiments, the solid tumor or lymphatic tumor is selected from the group consisting of head and neck cancer, breast cancer, colorectal cancer, liver cancer, pancreatic adenocarcinoma, gallbladder and bile duct cancer, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, bladder cancer, prostate cancer, bone cancer, mesothelioma, brain cancer, soft tissue sarcoma, uterine cancer, thyroid cancer, hypopharyngeal cancer, and melanoma. In some embodiments, the solid tumor or lymphatic tumor is resistant to prior treatment. In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune stimulant. In some embodiments, the second immunomodulatory agent is a CD40 activator, such as an agonist anti-CD40 antibody (e.g., APX005M). In some embodiments, the second immunomodulatory agent is a 4-1BB activator, such as an agonist anti-4-1BB antibody (e.g., PF-05082566).

[0107] In some embodiments, the immunostimulant is an activator of CD40. In some embodiments, the activator of CD40 is an agonist anti-CD40 antibody. In the present invention, any of the well-known anti-CD40 antibodies including, but not limited to, CP-870,893, dacetuzumab (also known as SGN-40), ChiLob 7 / 4, APX005, and APX005M, BI-655064, and BMS-986090 may be used. In some embodiments, the agonist anti-CD40 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the agonist anti-CD40 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of a full-length anti-CD40 antibody. In some embodiments, the agonist anti-CD40 antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the agonist anti-CD40 antibody is a bispecific antibody, a multispecific antibody, a single-domain antibody, a fusion protein comprising an antibody moiety, or any other variant or derivative thereof. In some embodiments, the activator of CD40 is a natural or artificial CD40 ligand such as CD40L. In some embodiments, the activator of CD40 is an inhibitor of the interaction between CD40 and CD40L. In some embodiments, the activator of CD40 increases the signal transduction of CD40.

[0108] Accordingly, for example, in some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual (such as a human) is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) an effective amount of an activator of CD40 (an agonist anti-CD40 antibody including administering systemically (e.g., CP-870,893, dacetuzumab, ChiLob 7 / 4, or APX005M), wherein the oncolytic virus comprises a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine), and is contained in a viral vector. In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus preferentially replicates within cancer cells, such as Rb pathway-deficient cancer cells. In some embodiments, the CD40 activator is an agonist anti-CD40 antibody, such as CP-870,893, dacetuzumab, ChiLob 7 / 4, or APX005M. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to a tissue having a tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the CD40 activator is administered intravenously. In some embodiments, the oncolytic virus and the CD40 activator are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the CD40 activator. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the CD40 activator. In some embodiments, the oncolytic virus and the CD40 activator are administered simultaneously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration of a third immunomodulatory agent (such as systemic administration or local administration to the site of the tumor), such as an immune checkpoint inhibitor or an immunostimulant.

[0109] In some embodiments, a method for treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an activator of CD40 (such as an agonist anti-CD40 antibody, for example, CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0110] In some embodiments, a method for treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of adenovirus serotype 5 to the site of the tumor; and b) systemically administering an effective amount of an activator of CD40 (such as an agonist anti-CD40 antibody, for example, CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M), wherein the endogenous E1a promoter of the native adenovirus is replaced with the human E2F1 promoter, and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, for example, GM-CSF). In some embodiments, the tumor-specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0111] In some embodiments, a method for treating a solid tumor or a lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of CG0070 to the site of the tumor; and b) systemically administering an effective amount of an activator of CD40 (such as an agonistic anti-CD40 antibody, for example, CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M). In some embodiments, the activator of CD40 is an agonistic anti-CD40 antibody, such as CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M. In some embodiments, CG0070 is administered directly into the tumor. In some embodiments, CG0070 is administered to the tissue having the tumor. In some embodiments, CG007 is administered at a dose of about 1×10 8 to about 1×10 14 viral particles (vp) (such as any of about 1×10 8 to about 1×10 10 , about 1×10 10 to about 1×10 12 , or about 1×10 12 to about 1×10 14 vp). In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks or 5 weeks). In some embodiments, the activator of CD40 is administered intravenously. In some embodiments, CG0070 and the activator of CD40 are administered continuously. In some embodiments, CG0070 is administered prior to (such as immediately before) the administration of the activator of CD40. In some embodiments, CG0070 is administered after (such as immediately after) the administration of the activator of CD40. In some embodiments, CG0070 and the activator of CD40 are administered simultaneously. In some embodiments, the method further comprises locally administering a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant. In some embodiments, the method further comprises administering (such as systemically or locally to the site of the tumor) a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant.

[0112] In some embodiments, the immune stimulant is an activator of OX40. In some embodiments, the activator of OX40 is an agonist anti-OX40 antibody. In the present invention, any of the well-known anti-OX40 antibodies including, but not limited to, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083, and InVivoMAb clone OX-86 may be used. In some embodiments, the agonist anti-OX40 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the agonist anti-OX40 antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding partial sequences of a full-length anti-OX40 antibody. In some embodiments, the agonist anti-OX40 antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the agonist anti-OX40 antibody is a bispecific antibody, a multispecific antibody, a single-domain antibody, a fusion protein containing an antibody moiety, or any other variant or derivative thereof. In some embodiments, the activator of OX40 is a natural or artificial OX40 ligand such as OX40L. In some embodiments, the activator of OX40 is an inhibitor of the interaction between OX40 and OX40L. In the present invention, any of the inhibitors of the interaction between OX40 and OX40L may be used. See, for example, U.S. Patent Nos. 8,283,450; 11,867,621; 7,547,438; 7,063,845; 7,537,763; and 5,801,227. In some embodiments, the activator of OX40 increases the signal transduction of OX40.

[0113] Thus, for example, in some embodiments, a method of treating a solid tumor or a lymphatic tumor in an individual (such as a human) is provided, the method comprising: a) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) to the site of the tumor; and b) an effective amount of an activator of OX40 (an agonist anti-OX40 antibody which comprises, for example, administering systemically a MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86), and wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in cancer cells, such as Rb pathway-deficient cancer cells. In some embodiments, the OX40 agonist is an agonist anti-OX40 antibody, such as MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86. In some embodiments, the oncolytic virus is administered directly into the tumor. In some embodiments, the oncolytic virus is administered to a tissue having a tumor. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the OX40 agonist is administered intravenously. In some embodiments, the oncolytic virus and the OX40 agonist are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the OX40 agonist. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the OX40 agonist. In some embodiments, the oncolytic virus and the OX40 agonist are administered simultaneously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration of a third immunomodulatory agent (such as systemic administration or local administration to the site of the tumor), such as an immune checkpoint inhibitor or an immunostimulant).

[0114] In some embodiments, a method of treating a solid tumor or lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of a tumor-lytic virus (such as a tumor-lytic adenovirus) to the site of the tumor; and b) systemically administering an effective amount of an activator of OX40 (such as an agonist anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86), wherein the tumor-lytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the viral gene essential for virus replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0115] In some embodiments, a method of treating a solid tumor or lymphatic tumor in an individual is provided, the method comprising: a) locally administering an effective amount of adenovirus serotype 5 to the site of the tumor; and b) systemically administering an effective amount of an activator of OX40 (such as an agonist anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86), wherein the endogenous E1a promoter of the native adenovirus is replaced with the human E2F1 promoter and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, for example, GM-CSF). In some embodiments, the tumor-specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO:1.

[0116] In some embodiments, provided is a method of treating a solid tumor or a lymphatic tumor in an individual, the method comprising: a) locally administering an effective amount of CG0070 to the site of the tumor; and b) systemically administering an activating agent of OX40 (such as an agonistic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86) in an effective amount. In some embodiments, the activating agent of OX40 is an agonistic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86. In some embodiments, CG0070 is administered directly into the tumor. In some embodiments, CG0070 is administered to the tissue having the tumor. In some embodiments, CG0070 is about 1×10 8 ~ about 1×10 14 virus particles (vp) (about 1×10 8 ~ about 1×10 10 、 about 1×10 10 ~ about 1×10 12 、 or about 1×10 12 ~ about 1×10 14It is administered at a dose such as any of those of vp. In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks, or 5 weeks). In some embodiments, the OX40 agonist is administered at a dose of about 0.001 mg / kg to about 10 mg / kg (such as any of about 0.003 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg). In some embodiments, the OX40 agonist is administered once a month to once a week (such as once a week, once every two weeks, or once every three weeks). In some embodiments, CG0070 and the OX40 agonist are administered continuously. In some embodiments, CG0070 is administered prior to (such as immediately before) the administration of the OX40 agonist. In some embodiments, CG0070 is administered after (such as immediately after) the administration of the OX40 agonist. In some embodiments, CG0070 and the OX40 agonist are administered simultaneously. In some embodiments, the method further comprises local administration of a second immunomodulatory agent such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration (such as systemic administration or local administration to the site of the tumor) of a third immunomodulatory agent such as an immune checkpoint inhibitor or an immunostimulant.

[0117] In some embodiments, provided is a method of treating a solid tumor or a lymphatic tumor in an individual, the method comprising: a) administering an effective amount of CG0070 intratumorally; b) administering an effective amount of an OX40 agonist (such as an agonistic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083, or InVivoMAb clone OX-86) intravenously; and c) administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, for example, ipilimumab) intratumorally. In some embodiments, CG0070 is about 1×10 8 ~ about 1×1014 Virus particles (vp) (about 1×10 8 ~ about 1×10 10 、about 1×10 10 ~ about 1×10 12 、or about 1×10 12 ~ about 1×10 14It is administered at a dose such as any of those of vp. In some embodiments, CG0070 is administered once a week. In some embodiments, the OX40 activator is administered at a dose of about 0.001 mg / kg to about 10 mg / kg (such as any of about 0.003 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg). In some embodiments, the activator of OX40 is administered once a month to once a week (such as once a week, once every two weeks, or once every three weeks). In some embodiments, the inhibitor of CTLA-4 is administered at a dose of about 0.1 mg / kg to about 10 mg / kg (such as any of once a week, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg). In some embodiments, the inhibitor of CTLA-4 is administered once a week. In some embodiments, the inhibitor of CTLA-4 is administered immediately after the administration of CG0070 (for example, within 5 minutes after administration). In some embodiments, the OX40 activator is an agonist antibody of OX40, such as GSK3174998. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, such as ipilimumab (for example, YERVOY (registered trademark)). In some embodiments, the inhibitor of CTLA-4 is an artificial lipocalin protein, such as an anticarin that specifically recognizes CTLA-4. In some embodiments, to the individual, in combination with the administration of CG0070, an effective amount of DDM is further administered intratumorally as a transduction enhancer. In some embodiments, CG0070 and the activator of OX40 are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the activator of OX40 (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the activator of OX40 (such as immediately after administration). In some embodiments, CG0070 and the activator of OX40 are administered simultaneously. In some embodiments, CG0070 and the inhibitor of CTLA-4 are administered by injection into the tissue having the tumor.In some embodiments, CG0070, and an inhibitor of CTLA-4 are administered directly into the tumor by injection. In some embodiments, CG0070 is administered over about 1 to about 6 weeks as one treatment unit. In some embodiments, the treatment unit is repeated every about 2 to about 3 months. In some embodiments, the solid tumor or lymphatic tumor is selected from the group consisting of head and neck cancer, breast cancer, colorectal cancer, liver cancer, pancreatic adenocarcinoma, gallbladder and bile duct cancer, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, bladder cancer, prostate cancer, bone cancer, mesothelioma, brain cancer, soft tissue sarcoma, uterine cancer, thyroid cancer, hypopharyngeal cancer, and melanoma. In some embodiments, the solid tumor or lymphatic tumor was resistant to prior treatment. In some embodiments, the method further comprises intratumoral administration of a second immunomodulatory agent, such as an immunostimulant. In some embodiments, the second immunomodulatory agent is a CD40 activator, such as an agonist anti-CD40 antibody (e.g., APX005M). In some embodiments, the second immunomodulatory agent is a 4-1BB activator, such as an agonist anti-4-1BB antibody (e.g., PF-05082566).

[0118] The method described herein may further comprise the step of locally administering a pretreatment composition to the site of the tumor prior to administration of the oncolytic virus. In some embodiments, the pretreatment composition comprises a transduction enhancer such as N-dodecyl-β-D-maltoside (DDM). DDM is a nonionic surfactant consisting of maltose derivatized with a single 12-carbon chain and acts as a neutral detergent and solubilizer. This has been used as a food additive and is known to enhance mucosal penetration in rodents, presumably by its action on membrane-bound GAGs and tight junctions.

[0119] The pretreatment composition can be administered directly into the tumor or to the tissue having the tumor. In some embodiments, the pretreatment composition comprises a solution of a transduction enhancer (such as DDM). Suitable concentrations of the pretreatment composition (such as DDM solution) include, but are not limited to, any one of about 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the transduction enhancer (such as DDM). In some embodiments, the pretreatment composition comprises a transduction enhancer (such as DDM) in any of about 0.01% to about 0.05%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 0.01% to about 1%, about 0.05% to about 2%, about 1% to about 5%, or about 0.1% to about 5%.

[0120] In some embodiments, the pretreatment (such as DDM) is administered immediately before the administration of the oncolytic virus (such as within 5 minutes before administration). In some embodiments, the pretreatment (such as DDM) is administered within any of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 90 minutes, 2 hours, 3 hours, or 4 hours before the administration of the oncolytic virus. In some embodiments, the pretreatment (such as DDM) is administered within about 2 hours before the administration of the oncolytic virus.

[0121] Suitable dosages of the pretreatment composition (such as DDM) include, but are not limited to, about 0.1 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 0.1 mg / kg to 0.5 mg / kg, 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 2 mg / kg, 2 mg / kg to 5 mg / kg, 5 mg / kg to 10 mg / kg, 10 mg / kg to 25 mg / kg, 25 mg / kg to 50 mg / kg, 50 mg / kg to 100 mg / kg, 100 mg / kg to 150 mg / kg, 150 mg / kg to 200 mg / kg, 200 mg / kg to 250 mg / kg, 250 mg / kg to 500 mg / kg, or about 0.5 mg / kg to about 5 mg / kg. In some embodiments, the suitable dosage of the pretreatment composition is any one of about 0.1 g, 0.2 g, 0.5 g, 0.75 g, 1 g, 1.5 g, 2 g, 2.5 g, 5 g, or 10 g of a transduction enhancer (such as DDM).

[0122] In some embodiments, the individual undergoes pretreatment (e.g., either systemically or only at the tumor site) prior to administration of the oncolytic virus and the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the pretreatment is a tumor site preparation using one or more (1, 2, 3, 4, 5, or more, etc.) treatment modalities including, but not limited to, radiation therapy, administration of one or more immune-related molecules, administration of other therapeutic agents, and combinations thereof. Adding other pretreatment preparations is thought to be able to increase the success rate of the above methods. Without being bound by any theory or hypothesis, for example, local radiation therapy (with or without lymphodepleting effects) or chemotherapy can increase the chance of infection progressing and can deplete more sensitive Treg cells at the tumor site, thereby resurrecting exhausted or tolerized T memory cells. Similarly, tumor site conditioning that precedes or is concurrent with administration of the combination of the present invention at the tumor site can include cytokines, chemokines, small molecules, and other well-known beneficial immunomodulatory agents such as IL2, IL12, OX40, CD40, and 4-1BB agonists. These tumor site conditioning modalities can be administered in combination or sequentially as needed.

[0123] In some embodiments, the pre-treatment is radiation therapy (with or without chemotherapy). In some embodiments, the radiation therapy is combined with chemotherapy. In some embodiments, the pre-treatment is whole-body radiation therapy. In some embodiments, the pre-treatment is radiation therapy only for the tumor site. In some embodiments, the pre-treatment is radiation therapy for the tissue having the tumor. In some embodiments, the pre-treatment is radiation therapy only for the site of the tumor selected for local administration of the oncolytic virus. In some embodiments, the pre-treatment is radiation therapy only for the tissue having the tumor selected for local administration of the oncolytic virus. In some embodiments, the dose of radiation therapy is insufficient to treat tumor cells. For example, a suitable dose of radiation therapy is any one of about 1 Gy, 5 Gy, 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, 70 Gy, 75 Gy, 80 Gy, 90 Gy, or 100 Gy. In some embodiments, the dose of radiation therapy is any one of about 1 Gy, 5 Gy, 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, 60 Gy, 65 Gy, 70 Gy, 75 Gy, 80 Gy, 90 Gy, or 100 Gy or less. In some embodiments, the dose of radiation therapy is any one of about 1 Gy to about 5 Gy, about 5 Gy to about 10 Gy, about 10 Gy to about 15 Gy, about 15 Gy to about 20 Gy, about 20 Gy to about 25 Gy, about 25 Gy to about 30 Gy, about 30 Gy to about 35 Gy, about 5 Gy to about 15 Gy, about 10 Gy to about 20 Gy, about 20 Gy to about 30 Gy, about 30 Gy to about 40 Gy, about 40 Gy to about 50 Gy, about 50 Gy to about 60 Gy, about 60 Gy to about 70 Gy, about 70 Gy to about 80 Gy, about 80 Gy to about 100 Gy, about 10 Gy to about 30 Gy, about 20 Gy to about 40 Gy, about 1 Gy to about 25 Gy, about 25 Gy to about 50 Gy, about 30 Gy to about 60 Gy, about 60 Gy to about 80 Gy, or about 10 Gy to about 60 Gy. A suitable dose of radiation therapy may also vary depending on the type, stage, and location of the tumor.

[0124] In some embodiments, radiation therapy is administered in two or more fractions, such as any one of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18, 20 or more fractions. In some embodiments, the fractions of radiation therapy are administered over any one of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or more. In some embodiments, the fractions of radiation therapy are administered over any one of about 1 day to about 5 days, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 5 weeks to about 6 weeks, about 6 weeks to about 7 weeks, about 2 weeks to about 4 weeks, about 4 weeks to about 6 weeks, or about 1 week to about 6 weeks. In some embodiments, radiation therapy is administered in about two fractions per day. In some embodiments, each fraction of radiation therapy is about 1.8 Gy to about 2 Gy per day for 5 days a week for adults, or about 1.5 Gy to about 1.8 Gy per day for 5 days a week for children. In some embodiments, each fraction of radiation therapy is any one of about 1 Gy, 1.5 Gy, 2 Gy, 2.5 Gy, 5 Gy, 10 Gy, 15 Gy, 20 Gy, 30 Gy, 40 Gy, 50 Gy or more. In some embodiments, each fraction of radiation therapy is any one of about 1 Gy to about 1.5 Gy, about 1.5 Gy to about 2 Gy, about 1 Gy to about 2.5 Gy, about 2.5 Gy to about 5 Gy, about 5 Gy to about 10 Gy, about 10 Gy to about 15 Gy, about 15 Gy to about 20 Gy, about 20 Gy to about 30 Gy, about 25 Gy to about 50 Gy, about 1 Gy to about 10 Gy, or about 2 Gy to about 20 Gy. In some embodiments, radiation therapy is administered in a single fraction.

[0125] In some embodiments, radiation therapy is aimed at achieving lymphopenia either as a single daily dose fraction or in multiple fractions over several days to several weeks. In some embodiments, lymphopenia radiation therapy is administered as total body irradiation. In some embodiments, lymphopenia is administered only to the local tumor site or to the tissue having the tumor. In some embodiments, lymphopenia radiation therapy is administered in two fractions per day. In some embodiments, each fraction of lymphopenia radiation therapy is about 1 Gy to about 2 Gy per day for 5 days a week for adults, or about 0.5 Gy to about 1.8 Gy per day for 5 days a week for children. In some embodiments, each fraction of radiation therapy is any one of about 1 Gy, 1.5 Gy, 2 Gy, 2.5 Gy, 5 Gy, 10 Gy, 15 Gy, 20 Gy, 30 Gy, 40 Gy, 50 Gy or more. In some embodiments, each fraction of radiation therapy is any one of about 1 Gy to about 1.5 Gy, about 1.5 Gy to about 2 Gy, about 1 Gy to about 2.5 Gy, about 2.5 Gy to about 5 Gy, about 5 Gy to about 10 Gy, about 10 Gy to about 15 Gy, about 15 Gy to about 20 Gy, about 20 Gy to about 30 Gy, about 25 Gy to about 50 Gy, about 1 Gy to about 10 Gy, or about 2 Gy to about 20 Gy. In some embodiments, lymphopenia radiation therapy is administered with or without the use of chemotherapeutic agents such as, but not limited to, cyclophosphamide and fludarabine.

[0126] In the present invention, any of the well-known methods of radiation therapy may be used, including but not limited to external beam radiation therapy (EBRT or XRT), teletherapy, brachytherapy, sealed source radiation therapy, systemic radioisotope therapy (RIT), unsealed source radiation therapy, intraoperative radiation therapy (IORT), targeted intraoperative radiation therapy (TARGIT), intensity modulated radiation therapy (IMRT), volumetric modulated arc therapy (VMAT), particle therapy, and Auger therapy.

[0127] In some embodiments, a method for treating an individual having a solid tumor or a lymphatic tumor comprises: (a) locally administering radiotherapy; (b) locally administering an effective amount of a oncolytic virus (such as an oncolytic adenovirus, e.g., CG0070) to the site of the tumor; and (c) systemically administering an effective amount of an immunomodulatory agent (including combinations of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for oncolytic virus replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF. In some embodiments, radiotherapy is administered prior to the administration of the oncolytic virus and / or the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, radiotherapy is administered about 1 day to about 1 week (e.g., about 2 days) prior to the administration of the oncolytic virus and the immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, radiotherapy and / or the oncolytic virus is directly administered to the solid tumor or lymphatic tumor. In some embodiments, radiotherapy and / or the oncolytic virus is administered to the tissue having the solid tumor or lymphatic tumor. In some embodiments, the immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an immune stimulant selected from the group consisting of activators of OX40, 4-1BB, and CD40.In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant. In some embodiments, the method further comprises administration of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant (e.g., systemic administration or local administration to the site of the tumor).

[0128] In some embodiments, the pre-treatment includes administration of a therapeutic agent. In some embodiments, the dosage of the therapeutic agent is sufficient to treat the tumor. In some embodiments, the dosage of the therapeutic agent is insufficient to treat the tumor. In some embodiments, the therapeutic agent is any one or any combination of chemotherapeutic agents well known in the art, for example, cyclophosphamide. In some embodiments, the therapeutic agent is any one or any combination of agents that target or block cell signaling pathways well known in the art, for example, a BRAF inhibitor. In some embodiments, the therapeutic agent is any one or any combination of cell therapies well known in the art, for example, TIL cells, CAR / T cells, and / or TCR / T cells. In some embodiments, the therapeutic agent is an agent that increases the concentration of cytokines involved in immunogenic pathways. Cytokines such as IL6, IL8, and IL18 (these cytokines can have either a pro-inflammatory and / or anti-inflammatory effect, or some can promote angiogenesis and tumor growth), chemokines (such as CCL21 which can promote tumor spread by proliferation of lymphoid tissue), growth factors (such as FLT3L), heat shock proteins, small molecule kinase inhibitors (such as JAK2 inhibitors), IAP inhibitors, STING activators (such as CDN), PRRago (such as CpG ODN (oligodeoxynucleotide), imiquimod, or poly I:C), TLR stimulants (such as GS-9620, AED-1419, CYT-003-QbG10, AVE-0675, or PF-7909), and RLR stimulants (such as RIG-I, Mda5, or LGP2 stimulants), among others, any of the immune-related molecules described herein may be used as a therapeutic agent. In some embodiments, the therapeutic agent is an agent that causes dysfunction or damage to the structural components of the tumor. Exemplary agents include, but are not limited to, anti-VEGF antibodies, hyaluronidase, and n-dodecyl-β-maltoside. In some embodiments, the therapeutic agent attracts immune cells such as dendritic cells, B cells, and T cells (such as follicular T helper cells).

[0129] For example, any of the therapeutic agents described herein, such as chemotherapeutic drugs, agents that target or block cell signaling pathways, cytokines, chemokines, cell therapies, etc., can be administered directly to the tumor site or indirectly (e.g., via intravenous administration), either alone or in combination.

[0130] In some embodiments, a method for treating an individual having a solid tumor or a lymphatic tumor comprises: (a) locally administering a therapeutic agent (such as a chemokine or PRRago); (b) locally administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus, e.g., CG0070) to the site of the tumor; and (c) systemically administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for the replication of the oncolytic virus is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF. In some embodiments, the therapeutic agent comprises a chemokine, such as CCL21. In some embodiments, the therapeutic agent is PRRago, such as CpG ODN (e.g., CpG 7909CCL21). In some embodiments, the therapeutic agent is within a nanocapsule. In some embodiments, the therapeutic agent is administered prior to the administration of the oncolytic virus. In some embodiments, the therapeutic agent is administered prior to the administration of the oncolytic virus and / or an immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the therapeutic agent is administered about 1 day to about 1 week (e.g., about 2 days) prior to the administration of the oncolytic virus and an immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the therapeutic agent and / or the oncolytic virus is directly administered to the solid tumor or lymphatic tumor. In some embodiments, the therapeutic agent and / or the oncolytic virus is administered to the tissue having the solid tumor or lymphatic tumor.In some embodiments, the immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an immunostimulant selected from the group consisting of activators of OX40, 4-1BB, and CD40. In some embodiments, the method further comprises local administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration of a third immunomodulatory agent (such as systemic administration or local administration to the site of the tumor), such as an immune checkpoint inhibitor or an immunostimulant.

[0131] The suitable dosage of the oncolytic virus depends on factors such as the nature of the oncolytic virus, the type of solid tumor or lymphoid tumor being treated, and the route of administration. As used herein, "particle" as related to an oncolytic virus means the collective number of physical single units of the oncolytic virus (such as a virus or bacterium). This number can be converted by an infectivity assay into another number that means an infectivity titer unit, such as plaque forming units (pfu) or international units, or is equivalent to such another number. In some embodiments, the oncolytic virus is about 1×10 5 particles, 1×10 6 particles, 1×10 7 particles, 1×10 8 particles, 1×10 9 particles, 1×10 10 particles, 2×10 10 particles, 5×10 10 particles, 1×10 11 particles, 2×10 11 particles, 5×10 11 particles, 1×10 12 particles, 2×10 12 particles, 5×10 12 particles, 1×10 13 particles, 2×10 13 particles, 5×10 13 particles, 1×10 14 particles, or 1×10 15It is administered at a dose of any one of the particles. In some embodiments, the oncolytic virus is about 1×10 5 particles to about 1×10 6 particles, about 1×10 6 particles to about 1×10 7 particles, about 1×10 7 particles to about 1×10 8 particles, about 1×10 8 particles to about 1×10 9 particles, about 1×10 9 particles to about 1×10 10 particles, about 1×10 10 particles to about 1×10 11 particles, about 1×10 11 particles to about 5×10 11 particles, about 5×10 11 particles to about 1×10 12 particles, about 1×10 12 particles to about 2×10 12 particles, about 2×10 12 particles to about 5×10 12 particles, about 5×10 12 particles to about 1×10 13 particles, about 1×10 13 particles to about 1×10 14 particles, or about 1×10 14 particles to about 1×10 15 particles and is administered at a dose of any one of them.

[0132] In some embodiments, the oncolytic virus is administered once a day. In some embodiments, the oncolytic virus is administered at any one of at least about once, twice, three times, four times, five times, six times, or seven times (i.e., daily) per week. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the oncolytic virus is administered once a week without interruption for two out of three weeks, once a week for three out of four weeks, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, once a month, or once every two to twelve months. In some embodiments, the interval between each administration is shorter than any one of about six months, three months, one month, twenty days, fifteen days, twelve days, ten days, nine days, eight days, seven days, six days, five days, four days, three days, two days, or one day. In some embodiments, the interval between each administration is longer than any one of about one month, two months, three months, four months, five months, six months, eight months, or twelve months. In some embodiments, there are no breaks in the dosing schedule. In some embodiments, the interval between each administration is about one week or less.

[0133] Administration of the oncolytic virus can occur over a long period of time, such as from about one month to about seven years. In some embodiments, the oncolytic virus is administered over a period of any one of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In some embodiments, the oncolytic virus is administered over a period of at least four weeks or six weeks. In some embodiments, the oncolytic virus is administered once a week for four weeks every three months. In some embodiments, the oncolytic virus is administered once a week for six weeks every three months.

[0134] The preferred dosage of an immunomodulatory agent (including the first, second, and third immunomodulatory agents, as well as combinations of immunomodulatory agents) depends on factors such as the nature of the immunomodulatory agent or combination of immunomodulatory agents, the type of solid tumor or lymphatic tumor being treated, and the route of administration. Exemplary dosages of an immunomodulatory agent (including the first, second, and third immunomodulatory agents, as well as combinations of immunomodulatory agents) include, but are not limited to, any one of about 1 mg / m 2 , 5 mg / m 2 , 10 mg / m 2 , 20 mg / m 2 , 50 mg / m 2 , 100 mg / m 2 , 200 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 750 mg / m 2 , 1000 mg / m 2 , or any one of more thereof. In some embodiments, the dosage of an immunomodulatory agent (including the first, second, and third immunomodulatory agents, as well as combinations of immunomodulatory agents) is included within any one of the following ranges: about 1 to about 5 mg / m 2 , about 5 to about 10 mg / m 2 , about 10 to about 20 mg / m 2 , about 20 to about 50 mg / m 2 , about 50 to about 100 mg / m 2 , about 100 mg / m 2 to about 200 mg / m 2 , about 200 to about 300 mg / m 2 , about 300 to about 400 mg / m 2 , about 400 to about 500 mg / m 2 , about 500 to about 750 mg / m 2 , or about 750 to about 1000 mg / m 2。In some embodiments, the dosage of the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is any one of about 1 μg / kg, 2 μg / kg, 5 μg / kg, 10 μg / kg, 20 μg / kg, 50 μg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg, or more. In some embodiments, the dosage of the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is any one of about 1 μg / kg to about 5 μg / kg, about 5 μg / kg to about 10 μg / kg, about 10 μg / kg to about 50 μg / kg, about 50 μg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.2 mg / kg to about 0.3 mg / kg, about 0.3 mg / kg to about 0.4 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 20 mg / kg to about 50 mg / kg, about 50 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 100 mg / kg. In some embodiments, the dosage of the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is any one of about 1 μg, 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 2 mg, 4 mg, 6 mg, 12 mg, 18 mg, 24 mg, 50 mg, 100 mg, 500 mg, or 1000 mg. In some embodiments, the dosage of the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is any one of about 1 μg to about 10 μg, about 10 μg to about 50 μg, about 50 μg to about 100 μg, about 100 μg to about 500 μg, about 500 μg to about 1 mg, about 1 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 25 mg, about 25 mg to about 50 mg, about 50 mg to about 100 mg, about 100 mg to about 500 mg, about 500 mg to about 1000 mg, about 1 μg to about 1 mg, about 1 mg to about 1000 mg, or about 1 μg to about 1000 mg.

[0135] When administered locally to the tumor site, in some embodiments, the dosage of the immunomodulatory agent (including the second and third immunomodulatory agents, and combinations of immunomodulatory agents) administered per tumor site is any one of about 10 μg, 50 μg, 100 μg, 500 μg, 1 mg, 2 mg, 4 mg, 6 mg, 12 mg, 18 mg, 24 mg, 50 mg, or 100 mg or less. In some embodiments, the dosage of the immunomodulatory agent (including the second and third immunomodulatory agents, and combinations of immunomodulatory agents) administered locally per tumor site is any one of about 10 μg to about 50 μg, about 50 μg to about 100 μg, about 100 μg to about 500 μg, about 100 μg to about 1 mg, about 1 mg to about 2 mg, about 2 mg to about 5 mg, about 5 mg to about 10 mg, about 10 mg to about 15 mg, about 10 mg to about 25 mg, about 25 mg to about 50 mg, about 50 mg to about 100 mg, about 1 mg to about 50 mg, or 100 μg to about 10 mg. In some embodiments, the dosage of the immunomodulatory agent (including the second and third immunomodulatory agents, and combinations of immunomodulatory agents) administered locally per tumor site is based on the size of the tumor.

[0136] In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is administered once a day. In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is administered at any one of at least about once, twice, three times, four times, five times, six times, or seven times (i.e., daily) per week. In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is administered once a week without interruption, once a week for two of three weeks, once a week for three of four weeks, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every eight weeks, once a month, or once every two to twelve months. In some embodiments, the interval between each administration is shorter than any one of about six months, three months, one month, twenty days, fifteen days, twelve days, ten days, nine days, eight days, seven days, six days, five days, four days, three days, two days, or one day. In some embodiments, the interval between each administration is longer than any one of about one month, two months, three months, four months, five months, six months, eight months, or twelve months. In some embodiments, there is no interruption in the dosing schedule. In some embodiments, the interval between each administration is about one week or less. In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is administered on the same dosing schedule as the oncolytic virus. In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is administered on a dosing schedule different from that of the oncolytic virus. In some embodiments, the oncolytic virus is administered once a week for four weeks.

[0137] Administration of an immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) can be over a long period, such as about 1 month to about 7 years. In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is administered over any one period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is administered over a period of at least 3 weeks or 6 weeks.

[0138] Exemplary routes of administration of the oncolytic virus, immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents), pre-treatment, and / or pretreatment composition include intratumoral, intravesical, intramuscular, intraperitoneal, intravenous, intraarterial, intracranial, intrapleural, subcutaneous, and epidermal routes, or routes of delivery into lymph nodes, body cavities, organs or tissues known to contain such living cancer cells (such as intrahepatic injection or intrapancreatic injection), but are not limited thereto. In some embodiments, local administration is performed by direct injection of the agent into the tumor. In some embodiments, local administration is performed by direct injection of the agent into a site proximal to the tumor cells. In some embodiments, systemic administration is by intravenous infusion. The specific route of administration depends on the nature of the solid tumor or lymphatic tumor and is further discussed below in relation to various types of solid tumors or lymphatic tumors.

[0139] In some embodiments, where an oncolytic virus and / or optionally additionally a second immunomodulatory agent (including combinations of immunomodulatory agents) is administered into a tumor (e.g., by intratumoral injection), the total volume administered is at most any one of about 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 5 mL, or 10 mL. In some embodiments, the volume of oncolytic virus and / or optionally additionally a second immunomodulatory agent (including combinations of immunomodulatory agents) for intratumoral administration (such as intratumoral injection) per tumor site is determined by the size of the tumor site. The tumor size can be measured as the tumor volume or the longest dimension of the tumor. For example, for a tumor having a longest dimension greater than about 5 cm, the intratumoral administration volume is about 2 mL or less; for a tumor having a longest dimension of about 2 cm to about 5 cm, the intratumoral administration volume is about 1 mL; for a tumor having a longest dimension of about 0.75 cm to about 2 cm, the intratumoral administration volume is about 0.5 mL; and for a tumor having a longest dimension less than about 0.75 cm, the intratumoral administration volume is about 0.1 mL. In some embodiments, the oncolytic virus and / or optionally additionally a second immunomodulatory agent (including combinations of immunomodulatory agents) is administered to all tumor sites. In some embodiments, the oncolytic virus and / or optionally additionally a second immunomodulatory agent (including combinations of immunomodulatory agents) is administered to any number of tumor sites of about 1, 2, 3, 4, 5, 6, or more. In some embodiments, the oncolytic virus and / or optionally additionally a second immunomodulatory agent (including combinations of immunomodulatory agents) is administered to the tumor site with the largest size.

[0140] The solid tumors or lymphoid tumors discussed in this specification include, but are not limited to, Hodgkin lymphoma, non-Hodgkin lymphoma, and sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sacronoma synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic lung cancer, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, epithelioma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0141] In some embodiments, the solid tumor or lymphoid tumor is selected from the group consisting of squamous cell carcinoma of the head and neck, breast cancer, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, prostate cancer, and melanoma. The method is applicable to solid tumors or lymphoid tumors at all stages, including stages I, II, III, and IV, according to the staging of the American Joint Committee on Cancer (AJCC). In some embodiments, the solid tumor or lymphoid tumor is an early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, cancer in remission, cancer in the adjuvant setting, or cancer in the neoadjuvant setting. In some embodiments, the solid tumor or lymphoid tumor is locally resectable, locally unresectable, or unresectable. In some embodiments, the solid tumor or lymphoid tumor has a locally resectable or resectable margin. In some embodiments, the cancer was resistant to prior treatment.

[0142] In some embodiments, the solid tumor or lymphatic tumor is a head and neck cancer. In some embodiments, the head and neck cancer is a squamous cell carcinoma within the head and neck. In some embodiments, the head and neck cancer is hypopharyngeal cancer, laryngeal cancer, lip and oral cavity cancer, metastatic squamous cell cervical cancer of unknown primary origin, nasopharyngeal cancer, oropharyngeal cancer, paranasal and nasal cavity cancer, or salivary gland cancer. In some embodiments, the squamous cell carcinoma of the head and neck is early stage head and neck cancer, non-metastatic head and neck cancer, advanced head and neck cancer, locally advanced head and neck cancer, metastatic head and neck cancer, remission stage head and neck cancer, adjuvant setting head and neck cancer, or neoadjuvant setting head and neck cancer. In some embodiments, the head and neck cancer is in the neoadjuvant setting. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the head and neck tissue having the head and neck tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the head and neck tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the head and neck tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the head and neck tissue adjacent to the head and neck tumor.

[0143] In some embodiments, the solid tumor or lymphatic tumor is breast cancer. In some embodiments, the breast cancer is early stage breast cancer, non-metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in the adjuvant setting, or breast cancer in the neoadjuvant setting. In some embodiments, the breast cancer is in the neoadjuvant setting. In some embodiments, the breast cancer is advanced. In some embodiments, the breast cancer (which can be HER2 positive or HER2 negative) includes, for example, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, and metastatic breast cancer. In some embodiments, the breast cancer is triple negative breast cancer. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intratumoral injection into breast tissue having a breast tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by intratumoral injection into the breast tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into a metastatic site of the breast tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intratumoral injection into breast tissue adjacent to the breast tumor.

[0144] In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is adenocarcinoma. In some embodiments, the renal cell carcinoma is clear cell renal cell carcinoma, papillary renal cell carcinoma (also referred to as chromophilic renal cell carcinoma), chromophobic renal cell carcinoma, collecting duct renal cell carcinoma, granular renal cell carcinoma, mixed granular renal cell carcinoma, renal angiomyolipoma, or spindle renal cell carcinoma. In some embodiments, the renal cell carcinoma is of stage I, II, III, or IV according to the American Joint Committee on Cancer (AJCC) staging. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intrarenal injection into renal tissue having a renal tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is directly performed by intrarenal injection into the renal tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is directly performed by injection into a metastatic site of the renal tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intrarenal injection into renal tissue adjacent to the renal tumor.

[0145] In some embodiments, the solid tumor or lymphatic tumor is prostate cancer. In some embodiments, the prostate cancer is adenocarcinoma. In some embodiments, the prostate cancer is a sarcoma, neuroendocrine tumor, small cell carcinoma, ductal carcinoma, or lymphoma. In some embodiments, the prostate cancer is any one of four stages A, B, C, or D according to the Jewett staging system. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intraprostatic injection into the prostatic tissue having a prostate tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by intraprostatic injection into the prostate tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the prostate tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intraprostatic injection into the prostatic tissue adjacent to the prostate tumor.

[0146] In some embodiments, the solid tumor or lymphatic tumor is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). Examples of NSCLC include, but are not limited to, large cell carcinoma, adenocarcinoma, neuroendocrine lung tumor, and squamous cell carcinoma. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intralung injection into lung tissue having a lung tumor. In some embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by intralung injection into the lung tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into a metastatic site of the lung tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intralung injection into lung tissue adjacent to the lung tumor.

[0147] In some embodiments, the solid tumor or lymphatic tumor is melanoma. In some embodiments, the melanoma is superficial spreading melanoma, lentigo maligna melanoma, nodular melanoma, mucosal melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue melanoma, or acral lentiginous melanoma. In some embodiments, the melanoma is American Joint It is of stage I, II, III, or IV according to the staging of the American Joint Committee on Cancer (AJCC). In some embodiments, the melanoma is recurrent. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the skin tissue having the melanoma tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the melanoma tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the melanoma tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the lung tissue adjacent to the melanoma tumor.

[0148] In some embodiments, the solid tumor or lymphatic tumor is ovarian cancer. In some embodiments, the ovarian cancer is ovarian epithelial cancer. In some embodiments, the ovarian cancer is in stage I (e.g., stage IA, IB, or IC), stage II (e.g., stage IIA, IIB, or IIC), stage III (e.g., stage IIIA, IIIB, or IIIC), or stage IV. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intraovarian injection into ovarian tissue having an ovarian tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by intraovarian injection into the ovarian tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into a metastatic site of the ovarian tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intraovarian injection into ovarian tissue adjacent to the ovarian tumor.

[0149] In some embodiments, according to any of the above methods, the solid tumor or lymphatic tumor is pancreatic cancer. In some embodiments, the pancreatic cancer is a serous cystic neoplasm, a mucinous cystic neoplasm, an intraductal papillary mucinous neoplasm, pancreatic adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma , it is signet ring cell carcinoma, undifferentiated carcinoma, undifferentiated carcinoma with giant cells, solid pseudopapillary tumor, papillary carcinoma, or pancreatic neuroendocrine tumor. In some embodiments, the pancreatic cancer is pancreatic adenocarcinoma. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intradermal injection into the pancreatic tissue having a pancreatic tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is directly performed by intradermal injection into the pancreatic tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is directly performed by injection into the metastatic site of the pancreatic tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intradermal injection into the pancreatic tissue adjacent to the pancreatic tumor.

[0150] In some embodiments, the solid tumor or lymphatic tumor is endometrial cancer. In some embodiments, the endometrial cancer is adenocarcinoma, carcinosarcoma, squamous cell carcinoma, undifferentiated carcinoma, small cell carcinoma, or transitional cell carcinoma. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intraendometrial injection into the endometrial tissue having an endometrial tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is directly performed by intraendometrial injection into the endometrial tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is directly performed by injection into the metastatic site of the endometrial tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intraendometrial injection into the endometrial tissue adjacent to the endometrial tumor.

[0151] In some embodiments, by any of the above methods, the solid tumor or lymphatic tumor is colorectal cancer. In some embodiments, the colorectal cancer is adenocarcinoma, gastrointestinal carcinoid, gastrointestinal stromal tumor, leiomysarcoma, melanoma, or squamous cell carcinoma. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the colorectal tissue having a colorectal tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the colorectal tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the colorectal tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the colorectal tissue adjacent to the colorectal tumor.

[0152] In some embodiments, by any of the above methods, the solid tumor or lymphatic tumor is hepatocellular carcinoma (HCC). In some embodiments, the HCC is early HCC, non-metastatic HCC, primary HCC, advanced HCC, locally advanced HCC, metastatic HCC, HCC in remission, or recurrent HCC. In some embodiments, the HCC is locally resected (i.e., a tumor limited in scope to a surgically resected portion of the liver), locally unresectable (i.e., the localized tumor may be unresectable because it infiltrates important vascular structures or causes damage to the liver), or unresectable (i.e., the tumor infiltrates all lobes of the liver and / or metastasizes to and infiltrates other organs (e.g., lungs, lymph nodes, bones)). In some embodiments, according to the TNM classification, the HCC is a stage I tumor (a single tumor without vascular invasion), a stage II tumor (a single tumor with vascular invasion or multiple tumors all less than 5 cm), a stage III tumor (multiple tumors all greater than 5 cm or a tumor infiltrating the main branches of the portal vein or hepatic vein), a stage IV tumor (direct invasion of adjacent organs other than the gallbladder or a tumor with perforation of the visceral peritoneum), an N1 tumor (regional lymph node metastasis), or an M1 tumor (distant metastasis). In some embodiments, according to the AJCC (American Joint Commission on Cancer) staging criteria, the HCC is stage T1, T2, T3, or T4 HCC. In some embodiments, the HCC is any one of hepatocellular carcinoma, the fibrolamellar variant of HCC, and mixed hepatocellular cholangiocarcinoma. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intrahepatic injection into the liver tissue having HCC. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by intrahepatic injection into the HCC.In some embodiments, administration of the oncolytic virus, and / or the second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic sites of HCC. In some embodiments, administration of the oncolytic virus, and / or the second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intrahepatic injection into the tissue adjacent to HCC.

[0153] In some embodiments, by any of the methods described above, the solid tumor or lymphatic tumor is lymphoma. In some embodiments, the lymphoma is a B cell tumor, a T cell tumor, and / or a putative NK cell tumor. Examples of B cell tumors include precursor B cell tumors (e.g., precursor B lymphoblastic leukemia / lymphoma) and peripheral B cell tumors (e.g., B cell chronic lymphocytic leukemia / prolymphocytic leukemia / small lymphocytic lymphoma (small lymphocytic (SL) NHL), lymphoplasmacytic lymphoma / immunocytoma, mantle cell lymphoma, follicular center lymphoma, follicular lymphoma (e.g., cytological malignancy: I (small cell), II (mixed small cell and large cell), III (large cell) and / or subtype: diffuse and small cell predominant), low grade / follicular non-Hodgkin lymphoma (NHL), intermediate grade / follicular NHL, marginal zone B cell lymphoma (e.g., extranodal (e.g., MALT type + / - monocytoid B cell) and / or nodal (e.g., + / - monocytoid B cell)), splenic marginal zone lymphoma (e.g., + / - villous lymphocytes), hairy cell leukemia, plasmacytoma / plasma cell myeloma (e.g., myeloma and multiple myeloma), diffuse large B cell lymphoma (e.g., primary mediastinal (thymic) B cell lymphoma), intermediate grade diffuse NHL, Burkitt lymphoma, high grade B cell lymphoma, Burkitt-like high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, large lesion NHL, AIDS-related lymphoma, and Waldenström macroglobulinemia), but are not limited thereto.Examples of T cell and / or putative NK cell tumors include precursor T cell tumors (precursor T lymphoblastic lymphoma / leukemia) and peripheral T cell and NK cell tumors (e.g., T cell chronic lymphocytic leukemia / prolymphocytic leukemia, and large granular lymphocytic leukemia (LGL) (e.g., T cell type and / or NK cell type), cutaneous T cell lymphoma (e.g., mycosis fungoides / Sézary syndrome), primary T cell lymphoma of unspecified type (e.g., cytological category (e.g., medium-sized cells, mixed medium and large cells, large cells, lymphoepithelioid cells, subtype hepatosplenic γδ T cell lymphoma, and subcutaneous panniculitis-like T cell lymphoma), angioimmunoblastic T cell lymphoma (AILD), angiocentric lymphoma, intestinal T cell lymphoma (e.g., + / - enteropathy-associated type), adult T cell lymphoma / leukemia (ATL), anaplastic large cell lymphoma (ALCL) (e.g., CD30+, T cell type and null cell type), anaplastic large cell type lymphoma, and Hodgkin-like), but are not limited thereto. In some embodiments, the lymphoma is Hodgkin's disease or non-Hodgkin lymphoma (NHL). For example, Hodgkin's disease can be lymphocyte-predominant type, nodular sclerosis type, mixed cellular type, lymphocyte-depleted type, and / or lymphocyte-rich type. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intralymphatic injection into a lymph node having a lymphoid tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by intralymphatic injection into the lymphoid tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into a metastatic site of the lymphoid tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intralymphatic injection into a tissue adjacent to the lymphoid tumor.

[0154] In some embodiments, by any of the methods described above, the solid tumor or lymphatic tumor is a mesothelioma. In some embodiments, the mesothelioma is a pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or a mesothelioma that has developed in the mesothelial tissue covering other organs. In some embodiments, the mesothelioma is a benign mesothelioma or a malignant mesothelioma. In some embodiments, the mesothelioma is an epithelial mesothelioma, sarcomatoid mesothelioma, biphasic mesothelioma, or papillary mesothelioma. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the mesothelial tissue having the mesothelioma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the mesothelioma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the mesothelioma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the mesothelial tissue adjacent to the mesothelioma.

[0155] In some embodiments, by any of the above methods, the solid tumor or lymphatic tumor is a brain tumor. In some embodiments, the brain tumor is a primary brain tumor or a secondary (or metastatic) brain tumor. In some embodiments, the brain tumor is a glioma (such as astrocytoma, oligodendroglioma, or ependymoma), meningioma, schwannoma, craniopharyngioma, germ cell tumor, or pineal tumor. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the brain tissue having the brain tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the brain tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the brain tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the brain tissue adjacent to the brain tumor.

[0156] In some embodiments, by any of the methods described above, the solid tumor or lymphatic tumor is a gallbladder and bile duct tumor. In some embodiments, the gallbladder and bile duct tumors are cancer, adenocarcinoma, cholangiocarcinoma, papillary tumor, small cell (neuroendocrine) carcinoma, adenocarcinoma with squamous metaplasia, or rhabdomyosarcoma. In some embodiments, the gallbladder and bile duct tumors are gallbladder cancer, cancer of the extrahepatic bile duct, or cancer of the intrahepatic bile duct. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the gallbladder or bile duct tissue having the gallbladder and bile duct tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the gallbladder and bile duct tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the gallbladder and bile duct tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the gallbladder or bile duct tissue adjacent to the gallbladder and bile duct tumor.

[0157] In some embodiments, by any of the methods described above, the solid tumor or lymphatic tumor is a soft tissue sarcoma. In some embodiments, the soft tissue sarcoma is adult fibrosarcoma, alveolar soft part sarcoma, angiosarcoma, clear cell sarcoma, desmoplastic small round cell tumor, epithelioid sarcoma, fibromyxoid sarcoma, liposarcoma, malignant mesenchymoma, malignant peripheral nerve sheath tumor (e.g., neurofibrosarcoma, malignant schwannoma, or neurogenic sarcoma), myxofibrosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, dermatofibrosarcoma protuberans, fibromatosis, hemangioendothelioma, pediatric fibrosarcoma, solitary fibrous tumor, elastofibroma, fibroma, fibrous histiocytoma, glomus tumor, granular cell tumor, hemangioma, brown tumor, lipoma, leiomyoma, lipoblastoma, lymphangioma, myxoma, neurofibroma, neuroma, perivascular epithelioid cell tumor, rhabdomyoma, schwannoma, giant cell tumor of tendon sheath, spindle cell tumor, or a tumor-like condition of soft tissue. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the tissue having the soft tissue sarcoma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the soft tissue sarcoma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the soft tissue sarcoma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the tissue adjacent to the soft tissue sarcoma.

[0158] In some embodiments, by any of the methods described above, the solid tumor or lymphatic tumor is a uterine tumor. In some embodiments, the uterine tumor is uterine cancer, uterine sarcoma (such as endometrial stromal sarcoma, undifferentiated sarcoma, or uterine leiomyosarcoma), or carcinosarcoma of the uterus (such as malignant mixed mesodermal tumor, or malignant Müllerian duct mixed tumor). In some embodiments, the uterine tumor is a fibroid, such as a leiomyoma, adenofibroma, or adenomyoma. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intrauterine injection into the uterine tissue having the uterine tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is directly performed by intrauterine injection into the uterine tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is directly performed by injection into the metastatic site of the uterine tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intrauterine injection into the uterine tissue adjacent to the uterine tumor.

[0159] In some embodiments, by any of the above methods, the solid tumor or lymphatic tumor is a neck tumor. In some embodiments, the neck tumor is squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intranodal injection into the nodal tissue having the neck tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by intranodal injection into the neck tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the neck tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by intranodal injection into the nodal tissue adjacent to the neck tumor.

[0160] In some embodiments, by any of the above methods, the solid tumor or lymphatic tumor is a thyroid tumor. In some embodiments, the thyroid tumor is a differentiated thyroid carcinoma (such as papillary carcinoma, follicular carcinoma, or Hurthle cell carcinoma), medullary thyroid carcinoma, undifferentiated carcinoma, thyroid lymphoma, thyroid sarcoma, or parathyroid tumor. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the thyroid tissue having the thyroid tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the thyroid tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the thyroid tumor. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the thyroid tissue adjacent to the thyroid tumor.

[0161] In some embodiments, by any of the methods described above, the solid tumor or lymphatic tumor is a nasopharyngeal carcinoma. In some embodiments, the nasopharyngeal carcinoma is a keratinizing squamous cell carcinoma, non-keratinizing differentiated carcinoma, or undifferentiated carcinoma (e.g., lymphoepithelioma), oral and oropharyngeal tumors, nasal and paranasal sinus tumors, or salivary gland tumors. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the nasopharyngeal tissue having nasopharyngeal carcinoma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the nasopharyngeal carcinoma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed directly by injection into the metastatic site of the nasopharyngeal carcinoma. In some embodiments, the administration of the oncolytic virus, and / or a second immunomodulatory agent (including combinations of immunomodulatory agents), and / or the pretreatment composition is performed by injection into the nasopharyngeal tissue adjacent to the nasopharyngeal carcinoma.

[0162] In some embodiments, the individual is a human individual. In some embodiments, the individual being treated for a solid tumor or a lymphatic tumor is identified as having one or more of the conditions described herein. Identification of the conditions as described herein by a skilled physician is routine in the art (e.g., by blood tests, X-rays, ultrasounds, CT scans, PET scans, PET / CT scans, MRI scans, PET / MRI scans, nuclear medicine radioisotope scans, endoscopies, biopsies, angiograms, CT angiograms, etc.) and may also be inferred by the individual or others, for example, by tumor growth, bleeding, ulceration, pain, enlarged lymph nodes, cough, jaundice, swelling, weight loss, cachexia, sweating, anemia, tumor-associated events, thrombosis, etc. In some embodiments, the individual is selected based on any one or more of a plurality of risk factors and / or diagnostic methods understood by those of skill in the art, including but not limited to gene profiling, family history, medical history (e.g., expression of relevant conditions and history of viral infections), lifestyle or habits, for any one of the treatment methods described herein.

[0163] In some embodiments, an individual is selected for any one of the treatment methods described herein based on the expression levels of one or more biomarkers including, but not limited to, immune checkpoint molecules, costimulatory molecules, cytokines, chemokines, other immune-related molecules, and HLA class II antigens. In some embodiments, an individual is selected for treatment based on the expression level (e.g., high expression level) of one or more inhibitory immune checkpoint molecules including, but not limited to, CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, 2B4, and their ligands. In some embodiments, an individual is selected for a treatment method based on the expression level (e.g., low expression level) of one or more stimulatory immune checkpoint molecules or costimulatory molecules including, but not limited to, OX40, 4-1BB, CD40, and their ligands. In some embodiments, an individual is selected for treatment based on the expression level (e.g., high expression level) of one or more biomarkers selected from the group consisting of PD-1, PD-L1, and PD-L2 in a tumor (such as tumor cells and / or immune cells within the tumor). In some embodiments, an individual is selected for treatment based on the expression level (e.g., high expression level) of one or more biomarkers selected from the group consisting of CD80, CD83, CD86, and HLA class II antigens in tumor-derived mature dendritic cells. In some embodiments, an individual is selected for treatment based on the expression level (e.g., high expression level) of one or more biomarkers selected from the group consisting of CXCL9, CXCL10, CXCL11, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1, HES4, MTIB, MTIE, MTIG, MTIH, GADD45A, LAMP3, and miR-155.

[0164] In some embodiments, the individual has high expression of one or more inhibitory immune checkpoint molecules. In some embodiments, the individual has low expression of one or more stimulatory immune checkpoint molecules and / or costimulatory molecules. In some embodiments, the individual has high expression of one or more biomarkers selected from the group consisting of PD-1, PD-L1, and PD-L2 in a tumor (such as tumor cells and / or immune cells within the tumor). In some embodiments, PD-L1 and PD-L2 can be used interchangeably as biomarkers for selecting patients or as ligands for inhibiting PD-1. In some embodiments, the individual has high expression of one or more biomarkers selected from the group consisting of CD80, CD83, CD86, and HLA class II antigens in tumor-derived mature dendritic cells. Exemplary HLA class II antigens include, but are not limited to, tumor-specific antigens and tumor-associated antigens expressed within solid tumors or lymphoid tumors, such as PSA for prostate tumors, alpha-fetoprotein for HCC, and CEA for adenocarcinoma. In some embodiments, the individual has high expression of one or more biomarkers selected from the group consisting of CXCL9, CXCL10, CXCL11, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1, HES4, MTIB, MTIE, MTIG, MTIH, GADD45A, LAMP3, and miR-155. In some embodiments, the method further comprises assessing the expression level of one or more biomarkers in the individual. In some embodiments, the method is adjusted based on the expression level of one or more biomarkers.

[0165] The expression level of a biomarker may be measured at the nucleic acid level (e.g., gene copy number, DNA methylation or chromatin remodeling level, mRNA level), or at the protein level, including the level of post-translational modification of the protein such as the phosphorylation level of the protein corresponding to the biomarker. The expression level can be determined using any of the methods well known in the art. For example, suitable methods for determining the mRNA expression level of a biomarker include, but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR, microarray, and RNA sequencing. For example, suitable methods for determining the protein expression level of a biomarker include, but are not limited to, immunohistochemistry, Western blotting, and mass spectrometry.

[0166] The expression level of a biomarker may be determined using fresh or preserved samples from an individual, including but not limited to solid tumor or lymphoid tumor tissue, normal tissue adjacent to solid tumor or lymphoid tumor tissue, normal tissue distal to solid tumor or lymphoid tumor tissue, or peripheral blood lymphocytes. In some embodiments, the sample is solid tumor or lymphoid tumor tissue. In some embodiments, the sample is a biopsy material containing tumor cells, such as aspirated tumor cells. In some embodiments, the biopsy-collected cells are centrifuged into pellets, fixed, and embedded in paraffin before analysis. In some embodiments, the biopsy-collected cells are snap-frozen before analysis. In some embodiments, the sample is a body fluid, such as a blood sample or plasma sample. In some embodiments, the sample contains circulating metastatic cancer cells. In some embodiments, the sample is obtained by sorting circulating tumor cells (CTCs) from blood.

[0167] In some embodiments, the expression level of one or more biomarkers within a particular cell population of an individual is determined using a sample derived from the individual. In some embodiments, the sample comprises immune cells isolated from or derived from a solid tumor or a lymphoid tumor. Exemplary immune cells suitable for biomarker expression determination include, but are not limited to, dendritic cells (immature or mature dendritic cells), B cells, T cells (Th1 cells, Th2 cells, Th17 cells, NK T cells, Treg cells, etc.), natural killer (NK) cells, monocytes, macrophages, neutrophils, and combinations thereof. In some embodiments, the sample comprises tumor infiltrating lymphocytes. In some embodiments, the sample comprises tumor-derived mature dendritic cells. The particular cell population can be isolated from a sample such as a tumor sample (e.g., tumor biopsy or resection) or a body fluid (e.g., blood sample) using methods well known in the art, such as flow cytometry based on the expression of specific cell surface molecules within the cell population.

[0168] The high or low expression level of a biomarker is determined by comparison with the standard expression level of the biomarker well-known in the art (e.g., clinically acceptable normal levels in a standard test), or with the expression level of the biomarker in a control sample. In some embodiments, the expression level of a biomarker in an individual is compared with the expression levels of the biomarker in a plurality of control samples. In some embodiments, the plurality of control samples are used to generate statistical values for classifying the levels of biomarkers in individuals with solid tumors or lymphatic tumors. Control samples can be obtained from the same source (e.g., individual and tissue) and method as non-control samples. In some embodiments, the control samples are obtained from different individuals (e.g., individuals without solid tumors or lymphatic tumors, individuals with solid tumors or lymphatic tumors in a benign or less advanced form, and / or individuals sharing the same race, age, and gender). In some embodiments, the control samples are cultured tissues or cells that have been determined to be appropriate controls. In some embodiments where the sample is solid tumor or lymphatic tumor tissue, the control sample can be a non-cancerous sample from the same individual. In some embodiments, a plurality of (e.g., from different individuals) control samples are used to determine the range of levels of a biomarker within a particular tissue, organ, or cell population. In some embodiments, the expression level of a biomarker in a sample from an individual is classified as high, medium, or low according to an evaluation system, such as an evaluation system based on immunohistochemical methods. In some embodiments, a biomarker with high expression is a biomarker in a sample from an individual at an expression level of at least about 1.5-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold or more compared to the control sample. In some embodiments, a biomarker with low expression is a biomarker in a sample from an individual at an expression level of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less compared to the control sample.In some embodiments, the expression levels of two or more biomarkers are combined, for example, using a statistical model to determine an expression score, to select or present an individual suitable for treatment. Method for treating bladder cancer by intravesical administration

[0169] For treating bladder cancer, any of the above methods may be used. In this regard, local administration of an oncolytic virus may include intravesical administration of the oncolytic virus. Systemic administration of an immunomodulatory agent (including combinations of immunomodulatory agents) may include intravenous administration of the immunomodulatory agent (including combinations of immunomodulatory agents). Further, local administration of a second immunomodulatory agent (including combinations of immunomodulatory agents) may include intravenous administration of the second immunomodulatory agent (including combinations of immunomodulatory agents).

[0170] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of an oncolytic virus into the bladder; and b) administering systemically an effective amount of an immunomodulatory agent (including combinations of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the oncolytic virus is selected from the group consisting of adenovirus, herpes simplex virus, vaccinia virus, mumps virus, Newcastle disease virus, poliovirus, measles virus, Seneca Valley virus, coxsackievirus, reovirus, vesicular stomatitis virus, Maraba and rhabdovirus, and parvovirus. In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation, or genetic modification). In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents, including one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the method further comprises administration (such as intravesical or systemic) of a third immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands.In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is an immunostimulant. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB or CD40.

[0171] In some embodiments, a method for treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) administering systemically an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the oncolytic virus comprises a viral vector including a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the oncolytic virus is replicative. In some embodiments, the oncolytic virus preferentially replicates in cancer cells, such as Rb pathway-deleted cancer cells. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents including one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the method further comprises administration (such as intravesical or systemic) of a third immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is an immune stimulant. In some embodiments, the immune stimulant is an activator of OX40, 4-1BB, or CD40.

[0172] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) administering an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents) systemically, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule. In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for the replication of the oncolytic virus is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents comprising one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the method further comprises administration (such as intravesical or systemic) of a third immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands.In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is an immunostimulant. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB or CD40.

[0173] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of adenovirus serotype 5 into the bladder; and b) administering systemically an effective amount of an immunomodulatory agent (including a combination of immunomodulatory agents), wherein the endogenous E1a promoter of the native adenovirus is replaced with a human E2F-1 promoter, and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, e.g., GM-CSF). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents, including one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the adenovirus is administered once a week. In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the method further comprises administration (such as intravesical or systemic) of a third immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is an immune stimulant. In some embodiments, the immune stimulant is an activator of OX40, 4-1BB or CD40.

[0174] In some embodiments, methods of treating bladder cancer in an individual are provided, the methods comprising: a) administering an effective amount of CG0070 intravesically; and b) administering systemically an effective amount of an immunomodulatory agent (including combinations of immunomodulatory agents). In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant. In some embodiments, the method comprises systemic administration of a combination of immunomodulatory agents, including one or more immune checkpoint inhibitors and / or one or more immune stimulants (such as at least two immune checkpoint inhibitors, at least two immune stimulants, or a combination of at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, CG0070 is from about 1×10 8 to about 1×10 14 virus particles (vp) (from about 1×10 8 to about 1×10 10 ; from about 1×10 10 to about 1×10 12 ; or from about 1×10 12 to about 1×10 14It is administered at a dose such as any of those of vp. In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks, or 5 weeks). In some embodiments, the immunomodulatory agent (including a combination of immunomodulatory agents) is administered intravenously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the method further comprises administration (such as intravesical or systemic) of a third immunomodulatory agent (including a combination of immunomodulatory agents). In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent, and / or the second immunomodulatory agent, and / or the third immunomodulatory agent is an immune stimulant. In some embodiments, the immune stimulant is an activator of OX40, 4-1BB, or CD40.

[0175] The methods described herein can be used to treat various bladder cancer states. In some embodiments, the bladder cancer is low-grade bladder cancer. In some embodiments, the bladder cancer is high-grade bladder cancer. In some embodiments, the bladder cancer is muscle-invasive (e.g., T2, T3, or T4). In some embodiments, the bladder cancer is non-invasive (e.g., Ta, T1 Cis, Ta and / or Cis having T1).

[0176] In some embodiments, the bladder cancer is a transitional cell carcinoma or urothelial carcinoma (such as metastatic urothelial carcinoma), including but not limited to papillary tumors and flat carcinomas. In some embodiments, the bladder cancer is metastatic urothelial carcinoma. In some embodiments, the bladder cancer is urothelial carcinoma of the bladder. In some embodiments, the bladder cancer is urothelial carcinoma of the ureter. In some embodiments, the bladder cancer is urothelial carcinoma of the urethra. In some embodiments, the bladder cancer is urothelial carcinoma of the renal pelvis.

[0177] In some embodiments, the bladder cancer is squamous cell carcinoma. In some embodiments, the bladder cancer is non-squamous cell carcinoma. In some embodiments, the bladder cancer is adenocarcinoma. In some embodiments, the bladder cancer is small cell carcinoma.

[0178] In some embodiments, the bladder cancer is early-stage bladder cancer, non-metastatic bladder cancer, non-invasive bladder cancer, muscle-invasive non-invasive bladder cancer, primary bladder cancer, advanced bladder cancer, locally advanced bladder cancer (such as locally unresectable advanced bladder cancer), metastatic bladder cancer, or bladder cancer in remission. In some embodiments, the bladder cancer is locally resectable, locally unresectable, or unresectable. In some embodiments, the bladder cancer is a high-grade muscle-invasive non-invasive cancer that is resistant to standard intravesical instillation (intravesical) therapy.

[0179] The methods provided herein can be used to treat an individual (e.g., a human) diagnosed with or suspected of having bladder cancer. In some embodiments, the individual has undergone tumor resection. In some embodiments, the individual has refused surgery. In some embodiments, the individual is medically inoperable. In some embodiments, the individual is in a clinical stage of Ta, Tis, T1, T2, T3a, T3b, or T4 bladder cancer. In some embodiments, the individual is in a clinical stage of Tis, CIS, Ta, or T1.

[0180] In some embodiments, the individual has been previously treated for bladder cancer (also referred to as "prior treatment"). In some embodiments, the individual has been previously treated for bladder cancer with standard treatment. In some embodiments, the prior standard treatment is treatment with BCG. In some embodiments, the prior standard treatment is treatment with mitomycin C. In some embodiments, the prior standard treatment is treatment with interferon (such as interferon alpha). In some embodiments, the individual has non-invasive bladder cancer, advanced bladder cancer, or recurrent bladder cancer. In some embodiments, the individual is resistant to treatment of bladder cancer with other agents (such as platinum-based agents, BCG, mitomycin C, and / or interferon). In some embodiments, the individual was initially responsive to treatment of bladder cancer with other agents (such as platinum-based agents or BCG), but the cancer has progressed after treatment.

[0181] In some embodiments, the individual has recurrent bladder cancer (such as bladder cancer at clinical stages Ta, Tis, T1, T2, T3a, T3b, or T4) after prior treatment (such as prior standard treatment, for example treatment with BCG). For example, the individual may initially be responsive to prior treatment, but develop bladder cancer about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 60 months after discontinuing the prior treatment.

[0182] Any of the immunomodulatory agents described herein, including immunostimulants and immune checkpoint inhibitors, may be used for systemic or intravesical administration in combination therapy. The immunomodulatory agents (including first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) may belong to any one of the molecular modalities well known in the art, including but not limited to aptamers, mRNAs, siRNAs, microRNAs, shRNAs, peptides, antibodies, anticalins, spherical nucleic acids, TALENs, zinc finger nucleases, CRISPR / Cas9, and small molecules.

[0183] In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is an immunostimulant. In some embodiments, the immunostimulant is a natural or artificial ligand of an immunostimulatory molecule, which includes, for example, ligands of OX40 (e.g., OX40L), CD-28 (e.g., CD80, CD86), ICOS (e.g., B7RP1), 4-1BB (e.g., 4-1BBL, Ultra4-1BBL), CD27 (e.g., CD70), CD40 (e.g., CD40L), and TCR (e.g., MHC class I or class II molecules, IMCgp100). In some embodiments, the immunostimulant is an antibody selected from the group consisting of anti-CD28 (e.g., TGN-1412), anti-OX40 (e.g., MEDI6469, MEDI-0562), anti-ICOS (e.g., MEDI-570), anti-GITR (e.g., TRX518, INBRX-110, NOV-120301), anti-41-BB (e.g., BMS-663513, PF-05082566), anti-CD27 (e.g., BION-1402, varlilumab, and hCD27.15), anti-CD40 (e.g., CP870,893, BI-655064, BMS-986090, APX005, APX005M), anti-CD3 (e.g., blinatumomab, muromonab), and anti-HVEM. In some embodiments, the antibody is an agonist antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding partial sequences of a full-length antibody. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single-domain antibody, a fusion protein containing an antibody portion, or any other functional variant or derivative thereof.

[0184] In some embodiments, the immunomodulatory agent (including the first, second, and third immunomodulatory agents, and combinations of immunomodulatory agents) is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a natural or artificial ligand of an inhibitory immune checkpoint molecule, which includes, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM3 (e.g., Galectin-9), ligands of A2a receptor (e.g., adenosine, regadenoson), ligands of LAG3 (e.g., MHC class I or MHC class II molecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g., MHC class I or MHC class II molecules), ligands of PD-1 (e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, Indoximod, NLG919), ligands of CD47 (e.g., SIRP-α receptor), and ligands of CSF1R. In some embodiments, the immune checkpoint inhibitor is an antibody that targets an inhibitory immune checkpoint protein.In some embodiments, the immunomodulatory agent is an antibody selected from the group consisting of anti-CTLA-4 (e.g., ipilimumab, tremelimumab, KAHR-102), anti-TIM3 (e.g., F38-2E2, ENUM005), anti-LAG3 (e.g., BMS-986016, IMP701, IMP321, C9B7W), anti-KIR (e.g., lirilumab, IPH2101, IPH4102), anti-PD-1 (e.g., nivolumab, pidilizumab, pembrolizumab, BMS-936559, atezolizumab, ramucirumab, MK-3475, AMP-224, AMP-514, STI-A1110, TSR-042), anti-PD-L1 (e.g., KY-1003 (EP20120194977), MCLA-145, atezolizumab, BMS-936559, MEDI-4736, MSB0010718C, AUR-012, STI-A1010, PCT / US2001 / 020964, MPDL3280A, AMP-224, dupilumab pegol (CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42 (OSU-HDAC42, HDAC-42, AR42, AR 42,OSU-HDAC 42,OSU-HDAC-42,NSC D736012,HDAC-42,HDAC 42,HDAC42,NSCD736012,NSC-D736012), MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1, anti-B7-H4, anti-CD52 (such as alemtuzumab), anti-IL-10, anti-IL-35, anti-TGF-β (such as fresolimumab), anti-CSF1R (e.g., FPA008), anti-NKG2A (e.g., monalizumab), anti-MICA (e.g., IPH43), and anti-CD39. In some embodiments, the antibody is an antagonist antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab’, F(ab’)2, Fv, scFv, and other antigen-binding subsequences of a full-length antibody.In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody moiety, or any other functional variant or derivative thereof.

[0185] In some embodiments, the method comprises systemic administration (such as intravenous administration) of a single immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant.

[0186] In some embodiments, the method comprises systemic administration (such as intravenous administration) of at least two (such as any of 2, 3, 4, 5, 6, or more) immunomodulatory agents. In some embodiments, all or a portion of the at least two immunomodulatory agents are administered simultaneously, such as within a single composition. In some embodiments, all or a portion of the at least two immunomodulatory agents are administered sequentially. In some embodiments, the method comprises systemic administration (such as intravenous administration) of a combination of immunomodulatory agents comprising an immune checkpoint inhibitor and an immune stimulant. In some embodiments, the method comprises systemic administration (such as intravenous administration) of a combination of immunomodulatory agents comprising two or more (such as any of 2, 3, 4, 5, 6, or more) checkpoint inhibitors. In some embodiments, the method comprises systemic administration (such as intravenous administration) of a combination of immunomodulatory agents comprising two or more (such as any of 2, 3, 4, 5, 6, or more) immune stimulants. In some embodiments, the method comprises systemic administration (such as intravenous administration) of a combination of immunomodulatory agents comprising any number (such as 1, 2, 3, 4, 5, 6, or more) of immune checkpoint inhibitors and any number (such as any of 2, 3, 4, 5, 6, or more) of immune stimulants. In some embodiments, the method comprises systemic administration (such as intravenous administration) of an OX40 inhibitor (such as an agonistic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86).

[0187] In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent (including combinations of immunomodulatory agents).

[0188] In some embodiments, the method further comprises intravesical administration of a single immunomodulatory agent. In some embodiments, the immunomodulatory agent is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immune stimulant.

[0189] In some embodiments, the method further comprises intravesical administration of at least two (such as any of 2, 3, 4, 5, 6, or more) immunomodulatory agents. In some embodiments, all or a portion of the at least two immunomodulatory agents are administered simultaneously, such as within a single composition. In some embodiments, all or a portion of the at least two immunomodulatory agents are administered sequentially. In some embodiments, the method comprises intravesical administration of a combination of immunomodulatory agents comprising an immune checkpoint inhibitor and an immune stimulant. In some embodiments, the method comprises intravesical administration of a combination of immunomodulatory agents comprising two or more (such as any of 2, 3, 4, 5, 6, or more) checkpoint inhibitors. In some embodiments, the method comprises intravesical administration of a combination of immunomodulatory agents comprising two or more (such as any of 2, 3, 4, 5, 6, or more) immune stimulants. In some embodiments, the method comprises intravesical administration of a combination of immunomodulatory agents comprising any number (such as 1, 2, 3, 4, 5, 6, or more) of immune checkpoint inhibitors and any number (such as any of 2, 3, 4, 5, 6, or more) of immune stimulants. In some embodiments, the method comprises intravesical administration of a CTLA-4 inhibitor (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an engineered lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4) and a CD40 agonist (such as an agonist anti-CD40 antibody, e.g., APX005M). In some embodiments, the method comprises intravesical administration of a CTLA-4 inhibitor (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an engineered lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4) and a 4-1BB agonist (such as an agonist anti-4-1BB antibody, e.g., PF-05082566).

[0190] Accordingly, for example, in some embodiments, a method of treating bladder cancer in an individual (such as a human) is provided, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) systemically administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an artificial lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4). The oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the oncolytic virus is attenuated (e.g., by multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in cancer cells, such as Rb pathway-deficient cancer cells. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, e.g., ipilimumab. In some embodiments, the inhibitor of CTLA-4 is an artificial lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the inhibitor of CTLA-4 is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of CTLA-4 are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the inhibitor of CTLA-4. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of CTLA-4. In some embodiments, the oncolytic virus and the inhibitor of CTLA-4 are administered simultaneously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant. In some embodiments, the method further comprises administration (such as systemic or intravesical administration) of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant.

[0191] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering into the bladder an effective amount of an oncolytic virus (such as an oncolytic adenovirus); and b) systemically administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an engineered lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0192] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering into the bladder an effective amount of adenovirus serotype 5; and b) systemically administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an engineered lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4), wherein the endogenous E1a promoter of the native adenovirus is replaced with the human E2F1 promoter and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, e.g., GM-CSF). In some embodiments, the tumor-specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0193] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of CG0070 into the bladder; and b) administering systemically an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an artificial lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4). In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, such as ipilimumab. In some embodiments, the inhibitor of CTLA-4 is an artificial lipocalin protein, such as an anti-carlin that specifically recognizes CTLA-4. In some embodiments, CG007 is administered at a dose of about 1×10 8 to about 1×10 14 viral particles (vp) (such as about 1×10 8 to about 1×10 10 , about 1×10 10 to about 1×10 12 , or about 1×10 12 to about 1×10 14 vp, etc.). In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks, or 5 weeks). In some embodiments, the inhibitor of CTLA-4 is administered intravenously. In some embodiments, CG0070 and the inhibitor of CTLA-4 are administered continuously. In some embodiments, CG0070 is administered prior to (such as immediately before) the administration of the inhibitor of CTLA-4. In some embodiments, CG0070 is administered after (such as immediately after) the administration of the inhibitor of CTLA-4. In some embodiments, CG0070 and the inhibitor of CTLA-4 are administered simultaneously. In some embodiments, the method further comprises administering into the bladder a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administering (such as systemically or into the bladder) a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant.

[0194] In some embodiments, a method for treating bladder cancer in an individual (such as a human) is provided, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) administering an effective amount of an inhibitor of PD-1 (such as an anti-PD-1 antibody, for example, nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, for example, AMP-224, etc.) systemically, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the oncolytic virus is attenuated (such as by multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus preferentially proliferates within cancer cells, such as Rb pathway-deficient cancer cells. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody, for example, nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the inhibitor of PD-1 is an inhibitor of the interaction between PD-1 and its ligand, such as an inhibitor of the PD-1 / PD-L1 interaction or an inhibitor of the PD-1 / PD-L2 interaction. In some embodiments, the inhibitor of PD-1 is an Fc fusion protein comprising a PD-1 ligand, such as an Fc fusion of PD-L2 (for example, AMP-224). In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the inhibitor of PD-1 is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of PD-1 are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the inhibitor of PD-1. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of PD-1. In some embodiments, the oncolytic virus and the inhibitor of PD-1 are administered simultaneously. In some embodiments, the method further comprises administering into the bladder a second immunomodulatory agent, such as an immune checkpoint inhibitor (such as a CTLA-4 inhibitor) or an immune stimulant (for example, a CD40 activator or a 4-1BB activator).In some embodiments, the method further comprises administration (e.g., systemic or intravesical administration) of a third immunomodulatory agent, such as an immune checkpoint inhibitor (e.g., a CTLA-4 inhibitor) or an immune stimulant (e.g., a CD40 activator or a 4-1BB activator).

[0195] In some embodiments, provided is a method of treating bladder cancer in an individual, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) intravesically; and b) administering systemically an effective amount of an inhibitor of PD-1 (such as an anti-PD-1 antibody, e.g., nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, e.g., AMP-224), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0196] In some embodiments, a method for treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of adenovirus serotype 5 into the bladder, wherein the endogenous E1a promoter of the native adenovirus is replaced with a human E2F1 promoter, and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, e.g., GM-CSF); and b) administering an effective amount of an inhibitor of PD-1 (an anti-PD-1 antibody, such as nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, such as AMP-224, etc.) systemically. In some embodiments, the tumor-specific promoter is a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0197] In some embodiments, a method for treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of adenovirus serotype 5 into the bladder, wherein the endogenous E1a promoter and the E3 19kD coding region of the native adenovirus are replaced with a human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (such as a cytokine or chemokine, e.g., GM-CSF); and b) administering an effective amount of an inhibitor of PD-1 (an anti-PD-1 antibody, such as nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand, such as AMP-224, etc.) into the bladder. In some embodiments, the tumor-specific promoter is a human E2F-1 promoter or an E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0198] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of CG0070 intravesically; and b) administering systemically an effective amount of an inhibitor of PD-1 (an anti-PD-1 antibody such as nivolumab, pembrolizumab, or pidilizumab, or an Fc fusion protein of a PD-1 ligand such as AMP-224, etc.). In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody such as nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the inhibitor of PD-1 is an inhibitor of the interaction between PD-1 and its ligand, such as an inhibitor of the PD-1 / PD-L1 interaction or an inhibitor of the PD-1 / PD-L2 interaction. In some embodiments, the inhibitor of PD-1 is an Fc fusion protein comprising a PD-1 ligand, such as an Fc fusion of PD-L2 (e.g., AMP-224). In some embodiments, CG007 is from about 1×10 8 to about 1×10 14 viral particles (vp) (from about 1×10 8 to about 1×10 10 , from about 1×10 10 to about 1×10 12 , or from about 1×10 12 to about 1×10 14It is administered at a dose such as any of those of vp. In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about one week to about six weeks (such as any of at least about three weeks, four weeks, or five weeks). In some embodiments, the PD-1 inhibitor is administered intravenously. In some embodiments, CG0070 and the PD-1 inhibitor are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the PD-1 inhibitor (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the PD-1 inhibitor (such as immediately after administration). In some embodiments, CG0070 and the PD-1 inhibitor are administered simultaneously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration (such as systemic administration or intravesical administration) of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant.

[0199] In some embodiments, a method for treating bladder cancer in an individual (such as a human) is provided, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) systemically administering an effective amount of an inhibitor of a PD-1 ligand (such as an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the oncolytic virus is attenuated (such as by multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in cancer cells, such as Rb pathway-deficient cancer cells. In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, atezolizumab, BMS935559, MPDL3280A, MEDI4736, avelumab, or STI-A1010. In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of the PD-1 ligand is an inhibitor of both PD-L1 and PD-L2 (such as a peptide, protein, or small molecule), such as AUR-012 and AMP-224. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the inhibitor of the PD-1 ligand is administered intravenously. In some embodiments, the oncolytic virus and the inhibitor of the PD-1 ligand are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the inhibitor of the PD-1 ligand. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the inhibitor of the PD-1 ligand. In some embodiments, the oncolytic virus and the inhibitor of the PD-1 ligand are administered simultaneously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immune stimulant.In some embodiments, the method further comprises administering a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant, either systemically or intravesically.

[0200] In some embodiments, provided is a method of treating bladder cancer in an individual, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) intravesically; and b) administering an effective amount of an inhibitor of a PD-1 ligand (such as an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2) systemically, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0201] In some embodiments, provided is a method of treating bladder cancer in an individual, the method comprising: a) administering an effective amount of adenovirus serotype 5 intravesically; and b) administering an effective amount of an inhibitor of a PD-1 ligand (such as an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2) systemically, wherein the endogenous E1a promoter of the native adenovirus is replaced with the human E2F1 promoter, and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, e.g., GM-CSF). In some embodiments, the tumor-specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0202] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of CG0070 into the bladder; and b) administering systemically an effective amount of an inhibitor of the PD-1 ligand (such as an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, atezolizumab, BMS935559, MPDL3280A, MEDI4736, avelumab, or STI-A1010. In some embodiments, the inhibitor of the PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of the PD-1 ligand is an inhibitor of both PD-L1 and PD-L2 (such as a peptide, protein or small molecule), such as AUR-012 and AMP-224. In some embodiments, CG007 is about 1×10 8 ~ about 1×10 14 viral particles (vp) (about 1×10 8 ~ about 1×10 10 、 about 1×10 10 ~ about 1×10 12 、 or about 1×10 12 ~ about 1×10 14administered at a dose such as any of those of vp. In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks, or 5 weeks). In some embodiments, the inhibitor of the PD-1 ligand is administered intravenously. In some embodiments, CG0070 and the inhibitor of the PD-1 ligand are administered continuously. In some embodiments, CG0070 is administered prior to (such as immediately before) the administration of the inhibitor of the PD-1 ligand. In some embodiments, CG0070 is administered after (such as immediately after) the administration of the inhibitor of the PD-1 ligand. In some embodiments, CG0070 and the inhibitor of the PD-1 ligand are administered simultaneously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent such as an immune checkpoint inhibitor or an immune stimulant. In some embodiments, the method further comprises administration (such as systemic administration or intravesical administration) of a third immunomodulatory agent such as an immune checkpoint inhibitor or an immune stimulant.

[0203] In some embodiments, provided is a method of treating bladder cancer in an individual, the method comprising: a) administering an effective amount of CG0070 intravesically; b) administering an effective amount of an inhibitor of PD-L1 (such as an antagonistic anti-PD-L1 antibody, for example, atezolizumab) intravenously; and c) administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, for example, ipilimumab) intravesically. In some embodiments, CG0070 is about 1×10 8 to about 1×10 14 viral particles (vp) (about 1×10 8 to about 1×10 10 , about 1×10 10 to about 1×10 12 , or about 1×10 12 to about 1×10 14It is administered at a dose such as any of those of vp. In some embodiments, CG0070 is administered once a week. In some embodiments, the PD-L1 inhibitor is administered at a dose of about 1 mg / kg to about 20 mg / kg, or about 750 mg to about 1200 mg. In some embodiments, the PD-L1 inhibitor is administered once every about one month to once every about two weeks (such as once every two weeks, once every three weeks, or once every four weeks, etc.). In some embodiments, the CTLA-4 inhibitor is administered at a dose of about 0.1 mg / kg to about 10 mg / kg (such as any of once a week, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg). In some embodiments, the CTLA-4 inhibitor is administered once a week. In some embodiments, the CTLA-4 inhibitor is administered immediately after the administration of CG0070 (for example, within 5 minutes after administration). In some embodiments, the PD-L1 inhibitor is an antagonist antibody of PD-L1, such as atezolizumab. In some embodiments, the CTLA-4 inhibitor is an anti-CTLA-4 antibody, such as ipilimumab (for example, YERVOY (registered trademark)). In some embodiments, the CTLA-4 inhibitor is an artificial lipocalin protein, such as an anti-calirin that specifically recognizes CTLA-4. In some embodiments, CG0070 and the PD-L1 inhibitor are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the PD-L1 inhibitor (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the PD-L1 inhibitor (such as immediately after administration). In some embodiments, CG0070 and the PD-L1 inhibitor are administered simultaneously. In some embodiments, to the individual, in combination with the administration of CG0070, an effective amount of DDM is further administered intravesically as a transduction enhancer. In some embodiments, CG0070 is administered over about 1 to about 6 weeks as one treatment unit. In some embodiments, the treatment unit is repeated every about 2 to about 3 months. In some embodiments, the method further includes the intravesical administration of a second immunomodulatory agent, such as an immunostimulant.In some embodiments, the second immunomodulatory agent is a CD40 activator, such as an agonist anti-CD40 antibody (e.g., APX005M). In some embodiments, the second immunomodulatory agent is a 4-1BB activator, such as an agonist anti-4-1BB antibody (e.g., PF-05082566).

[0204] In some embodiments, a method of treating bladder cancer in an individual (such as a human) is provided, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) administering systemically an effective amount of an activator of CD40 (such as an agonist anti-CD40 antibody, for example, CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for virus replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the oncolytic virus is attenuated (such as by multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in cancer cells, such as Rb pathway-deficient cancer cells. In some embodiments, the activator of CD40 is an agonist anti-CD40 antibody, for example, CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the activator of CD40 is administered intravenously. In some embodiments, the oncolytic virus and the activator of CD40 are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the activator of CD40. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the activator of CD40. In some embodiments, the oncolytic virus and the activator of CD40 are administered simultaneously. In some embodiments, the method further comprises administering into the bladder a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administering a third immunomodulatory agent (such as systemically or into the bladder), such as an immune checkpoint inhibitor or an immunostimulant.

[0205] In some embodiments, a method for treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) administering systemically an effective amount of an activator of CD40 (such as an agonist anti-CD40 antibody, for example, CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0206] In some embodiments, a method for treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of adenovirus serotype 5 into the bladder; and b) administering systemically an effective amount of an activator of CD40 (such as an agonist anti-CD40 antibody, for example, CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M), wherein the endogenous E1a promoter of the native adenovirus is replaced with the human E2F1 promoter and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, for example, GM-CSF). In some embodiments, the tumor-specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

[0207] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of CG0070 intravesically; and b) administering systemically an effective amount of an activator of CD40 (such as an agonistic anti-CD40 antibody, for example, CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M). In some embodiments, the activator of CD40 is an agonistic anti-CD40 antibody, such as CP-870,893, dacetuzumab, ChiLob 7 / 4 or APX005M. In some embodiments, CG007 is administered at a dose of about 1×10 8 to about 1×10 14 viral particles (vp) (such as any of about 1×10 8 to about 1×10 10 , about 1×10 10 to about 1×10 12 , or about 1×10 12 to about 1×10 14 vp). In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks or 5 weeks). In some embodiments, the activator of CD40 is administered intravenously. In some embodiments, CG0070 and the activator of CD40 are administered continuously. In some embodiments, CG0070 is administered prior to (such as immediately before) the administration of the activator of CD40. In some embodiments, CG0070 is administered after (such as immediately after) the administration of the activator of CD40. In some embodiments, CG0070 and the activator of CD40 are administered simultaneously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration (such as systemic or intravesical administration) of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant.

[0208] In some embodiments, provided is a method of treating bladder cancer in an individual (such as a human), the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) administering systemically an effective amount of an activator of OX40 (such as an agonist anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the oncolytic virus is attenuated (such as by multiple passages, inactivation or genetic modification). In some embodiments, the oncolytic virus preferentially replicates in cancer cells, such as Rb pathway-deleted cancer cells. In some embodiments, the activator of OX40 is an agonist anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86. In some embodiments, the oncolytic virus is administered once a week. In some embodiments, the activator of OX40 is administered intravenously. In some embodiments, the oncolytic virus and the activator of OX40 are administered continuously. In some embodiments, the oncolytic virus is administered prior to (such as immediately before) the administration of the activator of OX40. In some embodiments, the oncolytic virus is administered after (such as immediately after) the administration of the activator of OX40. In some embodiments, the oncolytic virus and the activator of OX40 are administered simultaneously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration (such as systemic or intravesical administration) of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant.

[0209] In some embodiments, a method for treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of an oncolytic virus (such as an oncolytic adenovirus) into the bladder; and b) administering systemically an effective amount of an activator of OX40 (such as an agonist anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86), wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine). In some embodiments, the tumor-specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments, the viral gene essential for viral replication is selected from the group consisting of E1A, E1B, and E4. In some embodiments, the heterologous gene is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

[0210] In some embodiments, a method for treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of adenovirus serotype 5 into the bladder; and b) administering systemically an effective amount of an activator of OX40 (such as an agonist anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86), wherein the endogenous E1a promoter of the native adenovirus is replaced with the human E2F1 promoter and the E3 19kD coding region of the native adenovirus is replaced with a heterologous gene encoding an immune-related molecule (such as a cytokine or chemokine, for example, GM-CSF). In some embodiments, the tumor-specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO:1.

[0211] In some embodiments, a method of treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of CG0070 intravesically; and b) administering systemically an effective amount of an activator of OX40 (such as an agonistic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86). In some embodiments, the activator of OX40 is an agonistic anti-OX40 antibody, for example, MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083 or InVivoMAb clone OX-86. In some embodiments, CG0070 is from about 1×10 8 to about 1×10 14 virus particles (vp) (from about 1×10 8 to about 1×10 10 , from about 1×10 10 to about 1×10 12 , or from about 1×10 12 to about 1×10 14It is administered at a dose such as any of those of vp). In some embodiments, CG0070 is administered once a week. In some embodiments, CG0070 is administered over a period of about 1 week to about 6 weeks (such as any of at least about 3 weeks, 4 weeks, or 5 weeks). In some embodiments, the OX40 activator is administered at a dose of about 0.001 mg / kg to about 10 mg / kg (such as any of about 0.003 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg). In some embodiments, the OX40 activator is administered once a month to once a week (such as once a week, once every two weeks, or once every three weeks). In some embodiments, CG0070 and the OX40 activator are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the OX40 activator (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the OX40 activator (such as immediately after administration). In some embodiments, CG0070 and the OX40 activator are administered simultaneously. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant. In some embodiments, the method further comprises administration (such as systemic administration or intravesical administration) of a third immunomodulatory agent, such as an immune checkpoint inhibitor or an immunostimulant.

[0212] In some embodiments, a method for treating bladder cancer in an individual is provided, the method comprising: a) administering an effective amount of CG0070 intravesically; b) administering an effective amount of an OX40 agonist (such as an agonistic anti-OX40 antibody, e.g., MEDI6469, MEDI0562, MEDI6383, GSK3174998, KHK4083, or InVivoMAb clone OX-86) intravenously; and c) administering an effective amount of an inhibitor of CTLA-4 (such as an anti-CTLA-4 antibody, e.g., ipilimumab, or an artificial lipocalin protein, e.g., an anti-carlin that specifically recognizes CTLA-4) intravesically. In some embodiments, CG0070 is from about 1×10 8 to about 1×10 14 virus particles (vp) (from about 1×10 8 to about 1×10 10 , from about 1×10 10 to about 1×10 12 , or from about 1×10 12 to about 1×10 14It is administered at a dose such as any of those of vp. In some embodiments, CG0070 is administered once a week. In some embodiments, the OX40 activator is administered at a dose of about 0.001 mg / kg to about 10 mg / kg (such as any of about 0.003 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg). In some embodiments, the activator of OX40 is administered once a month to once a week (such as once a week, once every two weeks, or once every three weeks). In some embodiments, the inhibitor of CTLA-4 is administered at a dose of about 0.1 mg / kg to about 10 mg / kg (such as any of once a week, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, or about 5 mg / kg to about 10 mg / kg). In some embodiments, the inhibitor of CTLA-4 is administered once a week. In some embodiments, the inhibitor of CTLA-4 is administered immediately after the administration of CG0070 (for example, within 5 minutes after administration). In some embodiments, the OX40 activator is an agonist antibody of OX40, such as GSK3174998. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, such as ipilimumab (for example, YERVOY (registered trademark)). In some embodiments, the inhibitor of CTLA-4 is an artificial lipocalin protein, such as an anti-carlin that specifically recognizes CTLA-4. In some embodiments, CG0070 and the OX40 activator are administered continuously. In some embodiments, CG0070 is administered prior to the administration of the OX40 activator (such as immediately before administration). In some embodiments, CG0070 is administered after the administration of the OX40 activator (such as immediately after administration). In some embodiments, CG0070 and the OX40 activator are administered simultaneously. In some embodiments, to the individual, in combination with the administration of CG0070, an effective amount of DDM is further administered into the bladder as a transduction enhancer. In some embodiments, CG0070 is administered over about 1 to about 6 weeks as one treatment unit.In some embodiments, the treatment unit is repeated every about 2 to about 3 months. In some embodiments, the method further comprises intravesical administration of a second immunomodulatory agent, such as an immunostimulant. In some embodiments, the second immunomodulatory agent is a CD40 activator, such as an agonist anti-CD40 antibody (e.g., APX005M). In some embodiments, the second immunomodulatory agent is a 4-1BB activator, such as an agonist anti-4-1BB antibody (e.g., PF-05082566).

[0213] Intravesical administration of the oncolytic virus and / or optionally an additional second immunomodulatory agent (including combinations of immunomodulatory agents) provides a unique opportunity to relatively simply and effectively expose intravesical tumors to the oncolytic virus and / or any additional second immunomodulatory agent (including combinations of immunomodulatory agents), while potentially reducing toxicity to other tissues. Suitable dosages and dosing frequencies of the oncolytic virus and immunomodulatory agents (including first, second, third immunomodulatory agents, and combinations of immunomodulatory agents) are within the same ranges respectively described above for local administration of the oncolytic virus and immunomodulatory agents (including first, second, third immunomodulatory agents, and combinations of immunomodulatory agents).

[0214] In some embodiments, the oncolytic virus and / or optionally additionally a second immunomodulatory agent (including combinations of immunomodulatory agents) are administered via a catheter as a solution by infusion. In some embodiments, the total volume of the solution used for intravesical installation is any one of about 1 mL, 10 mL, 50 mL, 75 mL, 100 mL, 125 mL, 150 mL, 200 mL, 250 mL, 300 mL, 400 mL or 500 mL. In some embodiments, the total volume of the solution used for intravesical installation is any one of about 1 mL to about 10 mL, about 10 mL to about 50 mL, about 50 mL to about 75 mL, about 75 mL to about 100 mL, about 100 mL to about 125 mL, about 75 mL to about 125 mL, about 100 mL to about 150 mL, about 150 mL to about 200 mL, about 200 mL to about 300 mL, about 300 mL to about 400 mL, about 400 mL to about 500 mL, about 50 mL to about 500 mL, about 50 mL to about 250 mL, or about 100 mL to about 250 mL.

[0215] In some embodiments, the oncolytic virus is about 1×10 8 to about 1×10 15 particles (such as about 1×10 11 to about 1×10 14 particles, for example about 1×10 12 particles) and is administered at a dose. In some embodiments, the oncolytic virus is administered by infusion in a volume of about 50 to about 500 mL (such as about 100 mL).

[0216] In some embodiments, the second immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravesically at a dose of about 0.1 mg / kg to about 100 mg / kg (such as about 0.1 mg / kg to about 0.3 mg / kg, about 0.1 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1 mg / kg, about 1 mg / kg to about 10 mg / kg, about 10 mg / kg to about 50 mg / kg, about 50 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 100 mg / kg). In some embodiments, the second immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravesically at a dose of any one of about 500 mg, 400 mg, 300 mg, 200 mg, 100 mg, 80 mg, 60 mg, 40 mg, 20 mg, or 10 mg or less per administration. In some embodiments, the second immunomodulatory agent (including combinations of immunomodulatory agents) is administered intravesically by infusion in a volume of about 1 mL to about 500 mL (such as about 100 mL).

[0217] A solution of an oncolytic virus and / or optionally additionally a second immunomodulatory agent (including combinations of immunomodulatory agents) may be retained in the bladder for a period of time prior to urination to achieve a uniform distribution or sufficient exposure of the oncolytic virus, optionally additionally the second immunomodulatory agent (including combinations of immunomodulatory agents) among bladder tumor cells. In some embodiments, the solution is retained in the individual's bladder for any of at least about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, or more. In some embodiments, the solution is retained in the individual's bladder for any of about 5 minutes to about 10 minutes, about 10 minutes to about 15 minutes, about 10 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 30 minutes to about 45 minutes, about 45 minutes to about 50 minutes, about 50 minutes to about 1 hour, about 5 minutes to about 15 minutes, about 10 minutes to about 30 minutes, about 30 minutes to about 1 hour, or about 1 hour to about 2 hours. In some embodiments, the oncolytic virus (such as an oncolytic virus, e.g., CG0070) is retained in the individual's bladder for about 45 minutes to about 50 minutes. In some embodiments, the second immunomodulatory agent (including combinations of immunomodulatory agents) is retained in the bladder for about 45 minutes to 1 hour. In some embodiments, the efficiency of intradetrusor administration of the oncolytic virus is further enhanced by pretreatment including intradetrusor administration of an effective amount of a transduction enhancer (such as DDM).

[0218] In some embodiments, the pretreatment step is performed by contacting the luminal side of the bladder within the individual with a pretreatment composition prior to administration of the oncolytic virus. For example, the pretreatment composition may comprise a transduction enhancer (such as DDM) of about 0.01% to about 0.5% (such as 0.05 to about 0.2%, for example about 0.1%). In some embodiments, the total volume of the pretreatment composition (such as DDM) is about 10 mL to about 1000 mL (such as about 10 mL to about 100 mL, about 100 mL to about 500 mL, or about 500 mL to about 1000 mL). In some embodiments, a suitable dosage of the pretreatment composition is any one of about 0.1 g, 0.2 g, 0.5 g, 0.75 g, 1 g, 1.5 g, 2 g, 2.5 g, 5 g, or 10 g of a transduction enhancer (such as DDM). In some embodiments, an effective amount of the pretreatment composition is about 1 g of DDM (for example, a 0.1% DDM solution of 100 mL).

[0219] In some embodiments, the pretreatment composition (such as DDM) is administered immediately before administration of the oncolytic virus (such as within 5 minutes before administration). In some embodiments, the pretreatment composition (such as DDM) is administered within any one of about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 90 minutes, 2 hours, 3 hours, or 4 hours before administration of the oncolytic virus. In some embodiments, the pretreatment composition (such as DDM) is administered within about 2 hours before administration of the oncolytic virus. In some embodiments, the pretreatment composition (such as a DDM solution) is retained in the bladder for at least any one of about 5 minutes, 10 minutes, 15 minutes, or 20 minutes. In some embodiments, the pretreatment composition (such as a DDM solution) is retained in the bladder for any one of about 5 minutes to about 10 minutes, about 10 minutes to about 15 minutes, about 12 minutes to about 15 minutes, about 15 minutes to about 20 minutes, or about 10 minutes to about 20 minutes. In some embodiments, the pretreatment composition (such as a DDM solution) is retained in the bladder for about 12 minutes to about 15 minutes.

[0220] In some embodiments, the pretreatment step is performed by contacting the luminal side of the bladder within the individual with a pretreatment composition prior to administration of the oncolytic virus.

[0221] In some embodiments, the method further comprises flushing the luminal side of the bladder that contacts the pretreatment composition. In some embodiments, the method further comprises flushing the luminal side of the bladder prior to administration of the oncolytic virus after contacting the bladder with the pretreatment composition....

Claims

1. 1. A combination for use in a method of treating bladder cancer in an individual, comprising an oncolytic virus and an immunomodulatory agent, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for replication of the virus and a heterologous gene encoding an immune-related molecule, the immunomodulatory agent comprising an anti-PD-L1 antibody or an anti-CTLA4 antibody, the method comprising: a) administering an effective amount of the oncolytic virus intravesically; and b) administering an effective amount of said immunomodulatory agent intravenously. A combination comprising:

2. The combination for use according to claim 1, wherein the oncolytic virus is an oncolytic adenovirus.

3. The combination for use according to claim 1, wherein the oncolytic virus is one in which the endogenous E1a promoter of native adenovirus serotype 5 has been replaced with a human E2F-1 promoter, and the endogenous E3 19 kD coding region of native adenovirus serotype 5 has been replaced with a nucleic acid encoding human GM-CSF.

4. The combination for use according to claim 3, wherein the oncolytic virus is CG0070.

5. The oncolytic virus is about 1×10 ８ ~Approx. 1×10 １４ The combination for use according to claim 1, administered in a dose of viral particles.

6. The combination for use according to claim 1, wherein the method comprises administering the oncolytic virus for about 1 week to about 6 weeks.

7. The combination for use according to claim 1 , wherein the oncolytic virus and the immunomodulatory agent are administered sequentially.

8. The combination for use according to claim 1 , wherein the oncolytic virus and the immunomodulatory agent are administered simultaneously.

9. 2. The combination for use of claim 1, wherein the method further comprises locally administering a pretreatment composition to the site of the tumor prior to the administration of the oncolytic virus.

10. The combination for use according to claim 9, wherein the pretreatment composition comprises a transduction enhancing agent.

11. The combination for use according to claim 10, wherein the transduction enhancer is N-dodecyl-β-D-maltoside (DDM).

12. The combination for use according to claim 1 , wherein the individual has undergone pretreatment prior to the administration of the oncolytic virus and the immunomodulatory agent.

13. 13. The combination for use according to claim 12, wherein the prior treatment is provided at a dose insufficient to treat the tumor.

14. The combination for use according to claim 12, wherein the individual has bladder cancer that is resistant to the prior treatment or recurrent after the prior treatment.

15. The combination for use according to claim 12, wherein the pre-treatment is treatment with Bacillus Calmette-Guerin (BCG).

16. a. the individual is resistant to treatment of bladder cancer with BCG; b. The individual initially responds to treatment of bladder cancer with BCG but progresses after treatment; or c. the individual has recurrent bladder cancer after treatment with BCG. A combination for use according to claim 15.

17. The combination for use according to claim 1, wherein the bladder cancer is non-muscle invasive bladder cancer.

18. 18. The combination for use according to claim 17, wherein the non-muscle invasive bladder cancer comprises: a) carcinoma in situ (Cis); b) Ta, T1 or a combination thereof; or c) both (a) and (b).

19. The combination for use according to any one of claims 1 to 18, wherein the immunomodulatory agent comprises an anti-PD-L1 antibody.

20. The combination for use according to claim 19, wherein the anti-PD-L1 antibody is atezolizumab, avelumab, MEDI-4736, KY-1003, MCLA-145, BMS-936559, AUR-012, STI-A1010, AMP-224, or MEDI-4920.

21. The combination for use according to any one of claims 1 to 18, wherein the immunomodulatory agent comprises an anti-CTLA4 antibody.

22. The combination for use according to claim 21, wherein the anti-CTLA4 antibody is ipilimumab, tremelimumab or KAHR-102.

23. 1. A kit for treating bladder cancer in an individual, comprising: a) an oncolytic virus, said oncolytic virus comprising a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for replication of said virus and a heterologous gene encoding an immune-related molecule; b) an immunomodulatory agent, wherein the immunomodulatory agent comprises an anti-PD-L1 antibody or an anti-CTLA4 antibody, and wherein the immunomodulatory agent is formulated for intravenous administration; and c) a device for intravesical administration of the oncolytic virus to the site of a tumor. Includes a kit.

24. 1. A composition for use in a method of treating bladder cancer in an individual, comprising an oncolytic virus, said oncolytic virus comprising a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for replication of said virus, and a heterologous gene encoding an immune-related molecule, said method comprising: a) intravesically administering an effective amount of the oncolytic virus to the site of a tumor; and b) intravenously administering an effective amount of an immunomodulatory agent, wherein the immunomodulatory agent comprises an anti-PD-L1 antibody or an anti-CTLA4 antibody. A composition comprising:

25. 1. A composition for use in a method of treating bladder cancer in an individual, comprising an immunomodulatory agent, wherein the immunomodulatory agent comprises an anti-PD-L1 antibody or an anti-CTLA4 antibody, the method comprising: a) intravesically administering to the site of a tumor an effective amount of an oncolytic virus, wherein the oncolytic virus comprises a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for replication of the virus and a heterologous gene encoding an immune-related molecule; and b) administering an effective amount of said immunomodulatory agent intravenously. A composition comprising:

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