WO2020228828A1

WO2020228828A1 - Composition for inducing immune cell activity and method for treating disease using same

Info

Publication number: WO2020228828A1
Application number: PCT/CN2020/090631
Authority: WO
Inventors: 韩志强; 李文; 邢风云; 万娜
Original assignee: 郑州威瑞生物技术有限公司
Priority date: 2019-05-15
Filing date: 2020-05-15
Publication date: 2020-11-19
Also published as: CN113891747A

Abstract

A composition for inducing immune cell activity in a subject in need thereof, comprising a tumor cell lysate which is autologous to the subject, an OX40 agonist, and a TLR agonist. Also provided is a method for treating a condition in a subject by using the composition.

Description

Composition for inducing immune cell activity and method for treating disease using the composition

Background technique

Immunotherapy (Immunotherapy) is a method to treat diseases by regulating the immune system, which includes activating immunotherapy and suppressing immunotherapy. In recent years, immunotherapy has received more and more attention, especially various immunotherapies developed for tumor treatment. Compared with conventional methods such as chemotherapy or radiotherapy, immunotherapy usually has fewer side effects.

In some immunotherapy against tumors, tumors are targeted by inducing or regulating the activity of immune cells such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), cytotoxic T lymphocytes (CTL), etc. Cells to achieve the effect of treating tumors. However, there are often big differences in the immune response between patients, and the use of general immunomodulators often fails to achieve the desired effect in many individuals.

Summary of the invention

At present, there is still a need in the art for the development of effective anti-tumor immunotherapy, especially individualized tumor immunotherapy for patients. The composition and method provided by the present invention solve the above-mentioned needs. The present invention provides a composition, method and kit for inducing immune cell activity in a subject.

In one aspect, the present application provides a composition for inducing immune cell activity in a subject in need, comprising: a lysate of tumor cells autologous to the subject, an OX40 agonist, and a TLR agonist Agent.

In some embodiments, the immune cell activity is selected from the group consisting of: proliferation of immune cells; differentiation, dedifferentiation or transdifferentiation of immune cells; release of immune cell cytokines; cytotoxicity of immune cells; immune cell movement and/or Transportation; exhaustion of immune cells and their combinations.

In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are T cells. In some embodiments, the lymphocytes are NK cells. In some embodiments, the immune cells are macrophages.

In some embodiments, the subject's autologous tumor cell lysate is derived from the subject's autologous tumor cell or its progeny cells. In some embodiments, the subject's autologous tumor cell lysate is derived from an autologous tumor cell of a solid tumor of the subject or its progeny cells. In some embodiments, the subject's autologous tumor cell lysate is derived from autologous tumor cells of one or more lesions of the subject or their progeny cells. In some embodiments, the subject's autologous tumor cell lysate is derived from autologous tumor cells of one or more organs of the subject or their progeny cells. In some embodiments, the subject's autologous tumor cell lysate is derived from an autologous tumor cell or its progeny cells in one or more tissues of the subject. In some embodiments, the subject's autologous tumor cell lysate is derived from tumor cells that have lost the ability to proliferate. In some embodiments, the subject's autologous tumor cell lysate is derived from fresh tumor tissue, frozen or fixed tumor tissue. In some embodiments, the fixing solution comprises formalin, formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, and any combination thereof.

In some embodiments, the tumor cell lysate in the composition is obtained by a method selected from the group consisting of homogenization, ultrasound, or a combination of the two. In some embodiments, the lysate of the tumor cells in the composition is obtained by a process of first homogenizing and then sonicating.

In some embodiments, the tumor cell lysate is obtained from 1×10 ⁴ -1× ^{10 8} number of tumor cells. In some embodiments, the tumor cell lysate is obtained from 1×10 ⁵ -1× ^{10 7} tumor cells.

In some embodiments, the OX40 agonist includes OX40 antibody or OX40 ligand fusion protein (OX40L).

In some embodiments, the TLR agonist includes CpG oligonucleotides (oligodeoxynucleotides, CpG ODN), Resiquimod (R848), Imiquimod (R837), dsRNA, and combinations thereof.

In some embodiments, the composition further comprises other immune activators. In some embodiments, the other immune activator is an agonist of one or more molecules selected from the group consisting of CD27, CD28, CD40, CD122, CD137 and GITR. In some embodiments, the immune activator is an antagonist of one or more molecules selected from the group consisting of A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1 , TIM-3, VISTA, SIGLEC7 and SIGLEC9. In some embodiments, the other immune activator is an antibody that specifically binds to an epitope of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA , CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the other immune activator is one or more cytokines selected from the group consisting of IL-2, IL-12, IL15, IL6, IL18, IFN-ɑ, TNF-β, IFN- γ, GM-CSF, M-CSF and any combination thereof.

In some embodiments, the composition is administered to the subject in situ or ex situ. In some embodiments, the in situ administration includes intratumoral injection, peritumoral injection, and combinations thereof. In some embodiments, the ex situ administration includes subcutaneous injection, intramuscular injection, intraperitoneal injection, thoracic injection, intravenous injection, arterial injection, and combinations thereof. In some embodiments, the composition is administered to the subject by single-point or multiple-point subcutaneous injection.

In another aspect, the present disclosure provides a method for treating tumors by inducing immune cell activity in a subject in need, which comprises administering to the subject a composition comprising: autologously identical to the subject Source tumor cell lysates, OX40 agonists and TLR agonists.

In some embodiments, the treatment includes tumor regression, inhibition of tumor progression and/or metastasis, and prevention of its recurrence and/or metastasis. In some embodiments, the method is for treating a condition in which the subject is selected from the group consisting of lung cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer , Esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, multiple myeloma, liver cancer, gallbladder cancer, lymphoma, melanoma, mesothelioma, Medulloblastoma, oral cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, mediastinal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.

In some embodiments, the immune cell activity is selected from: proliferation of immune cells; differentiation, dedifferentiation, or transdifferentiation of immune cells; release of immune cell cytokines; cytotoxicity of immune cells; immune cell movement and/or Transportation; exhaustion of immune cells and their combinations.

In another aspect, the present disclosure provides a kit for inducing immune cell activity in a subject in need, which comprises an OX40 agonist, a TLR agonist, and instructions, wherein the instructions indicate to obtain from the subject And/or a method of preparing autologous tumor cells, preparing a lysate of the tumor cell, and co-administering the lysate of the tumor cell with an OX40 agonist and a TLR agonist to the subject.

In another aspect, the present disclosure provides a composition for inducing immune cell activity in a subject in need, comprising: an oncolytic virus, an OX40 agonist and a TLR agonist, or an oncolytic virus expressing an OX40 agonist and TLR agonist.

In some embodiments, the oncolytic virus is a modified virus. In some embodiments, the oncolytic virus is a modified attenuated virus. In some embodiments, the oncolytic virus is selected from the group consisting of Herpesvirus, Adenovirus, Reovirus, Vaccinia Virus, Coxsackievirus , Measles Virus, Poliovirus, Retrovirus, Parvovirus H1 (Parvovirus H1), Vesicular Stomatitis Virus, Newcastle Disease Virus (Newcastle Disease Virus) ) And M1 oncolytic virus (Oncolytic virus M1). In some embodiments, the herpes virus is herpes simplex virus (Herpes simplex virus). In some embodiments, the herpes simplex virus is selected from HSV-1 or HSV-2. In some embodiments, the oncolytic virus is herpes simplex virus HSV-1 modified to lack γ34.5 function. In some embodiments, the oncolytic virus is modified to encode the OX40 agonist.

In some embodiments, the amount of oncolytic virus in the composition is 1×10 ⁵ -1× ^{10 9} pfu. In some embodiments, the amount of oncolytic virus in the composition is 1×10 ⁷ -1× ^{10 8} pfu.

In some embodiments, the OX40 agonist and/or the TLR agonist are expressed by the oncolytic virus as a vector.

In another aspect, the present disclosure provides a method for treating tumors by inducing immune cell activity in a subject in need thereof, which comprises administering to the subject a composition comprising: an oncolytic virus, an OX40 agonist, and TLR agonists, or oncolytic viruses expressing OX40 agonists and TLR agonists.

In another aspect, the present disclosure provides a kit for inducing immune cell activity in a subject in need, comprising an oncolytic virus, an OX40 agonist and a TLR agonist, or an oncolytic virus expressing an OX40 agonist and a TLR Agonist, and instructions.

On the other hand, the present disclosure provides a composition for inducing immune cell activity in a subject in need, which comprises a tumor cell autologous to the subject, an oncolytic virus, and one or more Immune activator.

In some embodiments, the oncolytic virus is a modified virus. In some embodiments, the oncolytic virus is a modified attenuated virus. In some embodiments, the oncolytic virus is selected from the group consisting of Herpesvirus, Adenovirus, Reovirus, Vaccinia Virus, Coxsackievirus , Measles Virus, Poliovirus, Retrovirus, Parvovirus H1 (Parvovirus H1), Vesicular Stomatitis Virus, Newcastle Disease Virus (Newcastle Disease Virus) ) And M1 oncolytic virus (Oncolytic virus M1). In some embodiments, the herpes virus is herpes simplex virus (Herpes simplex virus). In some embodiments, the herpes simplex virus is selected from HSV-1 or HSV-2. In some embodiments, the oncolytic virus is herpes simplex virus HSV-1 modified to lack the function of γ34.5. In some embodiments, the oncolytic virus is modified to encode genes for the one or more immune activators.

In some embodiments, the immune activator is an agonist of one or more molecules selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, and GITR. In some embodiments, the immune activator is an OX40 agonist. In some embodiments, the immune activator is an antagonist of one or more molecules selected from the group consisting of A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1 , TIM-3, VISTA, SIGLEC7 and SIGLEC9.

In some embodiments, the immune activator comprises an antibody that specifically binds to an epitope of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the antibody is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody (scFv), single-chain antibody (scFv-FC), minibody, diabody, single domain antibody , Full-length antibody.

In some embodiments, the immune activator comprises an anti-OX40 antibody. In some embodiments, the immune activator comprises a recombinant OX40 ligand fusion protein.

In some embodiments, the immune activator further comprises a ligand of Toll-Like Receptor (TLR). In some embodiments, the ligand of the TLR is a TLR agonist. In some embodiments, the TLR agonist includes CpG oligonucleotides (oligodeoxynucleotides, CpG ODN), Resiquimod (R848), Imiquimod (R837), dsRNA, and combinations thereof.

In some embodiments, the immune activator comprises an anti-OX40 antibody and CpG ODN.

In some embodiments, the immune activator further comprises one or more cytokines. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-12, IL15, IL6, IL18, IFN-ɑ, TNF-β, IFN-γ, GM-CSF, M-CSF, and random combination.

In some embodiments, the tumor cell is an autologous tumor cell isolated from the subject or its progeny cells. In some embodiments, the tumor cell is an autologous tumor cell isolated from a solid tumor of the subject or its progeny cells. In some embodiments, the tumor cell is an autologous tumor cell or its progeny cells isolated from one or more lesions of the subject. In some embodiments, the tumor cell is an autologous tumor cell isolated from one or more organs of the subject or its progeny cells. In some embodiments, the tumor cell is an autologous tumor cell or its progeny cells isolated from one or more tissues of the subject. In some embodiments, the tumor cell is a tumor cell that has lost the ability to proliferate.

In some embodiments, the number of tumor cells in the composition is 1×10 ⁴ -1× ^{10 8} . In some embodiments, the number of tumor cells in the composition is 1×10 ⁵ -1× ^{10 7} .

In another aspect, the present disclosure provides a method for treating tumors by inducing immune cell activity in a subject in need, which comprises administering to the subject a composition comprising: autologously identical to the subject Source tumor cells, oncolytic viruses and one or more immune activators.

In some embodiments, the treatment includes tumor regression, inhibition of tumor progression and/or metastasis, and prevention of its recurrence and/or metastasis. In some embodiments, the method is for treating a condition in which the subject is selected from the group consisting of lung cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer , Esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, multiple myeloma, liver cancer, gallbladder cancer, lymphoma, melanoma, mesothelioma, Medulloblastoma, oral cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, mediastinal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer. In some embodiments, the immune cell activity is selected from the group consisting of: proliferation of immune cells; differentiation, dedifferentiation or transdifferentiation of immune cells; release of immune cell cytokines; cytotoxicity of immune cells; immune cell movement and/or Transportation; exhaustion of immune cells and their combinations.

In some embodiments, the oncolytic virus is a modified virus. In some embodiments, the oncolytic virus is a modified attenuated virus. In some embodiments, the oncolytic virus is selected from the group consisting of Herpesvirus, Adenovirus, Reovirus, Vaccinia Virus, Coxsackievirus , Measles Virus, Poliovirus, Retrovirus, Parvovirus H1 (Parvovirus H1), Vesicular Stomatitis Virus, Newcastle Disease Virus (Newcastle Disease Virus) ) And M1 oncolytic virus. In some embodiments, the herpes virus is herpes simplex virus (Herpes simplex virus).

In some embodiments, the herpes simplex virus is selected from HSV-1 or HSV-2. In some embodiments, the oncolytic virus is herpes simplex virus HSV-1 modified to lack the function of γ34.5. In some embodiments, the oncolytic virus is modified to encode genes for the one or more immune activators.

In some embodiments, the immune activator is an agonist of one or more molecules selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, and GITR. In some embodiments, the immune activator is an OX40 agonist. In some embodiments, the immune activator is an antagonist of one or more molecules selected from the group consisting of A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1 , TIM-3, VISTA, SIGLEC7 and SIGLEC9. In some embodiments, the immune activator comprises an antibody that specifically binds to an epitope of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the antibody is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody scFv, single-chain antibody scFv-FC, minibody, diabody, single domain antibody, full-length antibody .

In some embodiments, the immune activator comprises OX40 antibody. In some embodiments, the immune activator comprises a recombinant OX40 ligand fusion protein OX40L.

In some embodiments, the method includes administering the composition to the subject in situ or ex situ. In some embodiments, the in situ administration includes intratumoral injection, peritumoral injection, and combinations thereof. In some embodiments, the ex situ administration includes subcutaneous injection, intramuscular injection, intraperitoneal injection, thoracic injection, intravenous injection, arterial injection, and combinations thereof. In some embodiments, the method includes administering the composition to the subject by single-point or multiple-point subcutaneous injection.

In some embodiments, the method further comprises pre-treating the tumor cells with the oncolytic virus in vitro before administering the composition to the subject. In some embodiments, the pretreatment time is less than 12 hours. In some embodiments, the pretreatment time is less than 2 hours.

In another aspect, the present disclosure provides a kit for inducing immune cell activity in a subject in need, which comprises an oncolytic virus, one or more immune activators, and instructions, wherein the instructions indicate A method for obtaining and/or preparing autologous tumor cells in a subject, and co-administering the tumor cells, the oncolytic virus and one or more immune activators to the subject.

Description of the drawings

The embodiments of the present invention can be described with reference to the drawings.

Figure 1 exemplarily shows the construction of the recombinant vector used in the present invention. Figure 1A exemplarily shows the oncolytic virus vector H1 with the double-copy γ34.5 gene deleted. Figure 1B exemplarily shows the oncolytic virus vector H1 recombined into the DsRed reporter gene expression cassette H1-DsRed. Figure 1C exemplarily shows the oncolytic virus vector H1 recombined into the immune activator IAA gene expression cassette H1-IAA. Figure ID exemplarily shows the gene expression cassette of the OX40 single chain antibody scFv-FC. Figure 1E exemplarily shows the gene expression cassette of the OX40 full-length antibody. Figure 1F exemplarily shows the gene expression cassette of the FC fusion protein of OX40 ligand OX40L.

Figure 2 exemplarily shows the infection and killing effect of the oncolytic virus used in the present invention on tumor cells. Figure 2A exemplarily shows the fluorescence observation of H1-DsRed infected tumor cells of different mice. Figure 2B exemplarily shows the fluorescence observation of H1-DsRed infected different human tumor cells. Figure 2C exemplarily shows the killing effect of oncolytic virus H1-DsRed on mouse tumor cells. 2D exemplarily shows the killing effect of oncolytic virus H1-DsRed on human tumor cells.

Figure 3 exemplarily shows the inhibitory effect of intratumoral administration of H1 and TLR agonists (HG (H1+GM-CSF), HR (H1+R848), HC (H1+CpG ODN)) on tumors in mice. Figure 3A exemplarily shows the growth curve of tumors in mice after administration of a composition of H1 and different TLR agonists. Figure 3B exemplarily shows pictures of mice and tumors and their tumors after administration of a composition of H1 and different TLR agonists. Figure 3C exemplarily shows the average weight of tumors in mice after administration of the composition of H1 and different TLR agonists.

Figure 4 exemplarily shows the inhibitory effect of subcutaneous administration of the composition HOC (H1+OX40mab+autologous tumor cells) of the present invention on tumors in mice. Figure 4A exemplarily shows a schematic diagram of the construction of a tumor model in mice and the method of subcutaneous administration. 4B exemplarily shows the growth curve of tumors in mice after subcutaneous injection of H1+CT-26 tumor cells+OX40mab, and Fig. 4C shows the average weight of tumors in mice after subcutaneous injection of H1+CT-26 tumor cells+OX40mab.

Figure 5 exemplarily shows the inhibitory effect of intratumoral administration of the composition HOR (H1+OX40mab+R848) of the present invention on tumors in mice. Figure 5A exemplarily shows the tumor growth curve in mice after intratumoral injection of HOR (H1+OX40mab+R848). Figure 5B exemplarily shows the overall picture of mouse tumors after intratumoral injection of HOR (H1+OX40mab+R848). Figure 5C exemplarily shows a picture of mouse tumor size after intratumor injection of HOR (H1+OX40mab+R848). Figure 5D exemplarily shows the average weight of tumors in mice after intratumoral injection of HOR (H1+OX40mab+R848).

Figure 6 exemplarily shows the inhibitory effect of subcutaneous administration of the composition of the present invention OR(OX40mab+R848)+CT-26 autologous tumor tissue lysate (CT-26TL) on tumors in mice. Figure 6A exemplarily shows the tumor growth curve in mice after subcutaneous injection of OR+CT-26TL, Figure 6B exemplarily shows the overall picture of the tumor in mice after subcutaneous injection of OR+CT-26TL, and Figure 6C exemplarily shows the subcutaneous Picture of tumor size in mice after OR+CT-26TL injection. Figure 6D shows the average weight of tumors in mice after subcutaneous injection of OR+CT-26TL.

Fig. 7 exemplarily shows the therapeutic effect of subcutaneous administration of OR(OX40mab+R848)+A20 autologous tumor tissue lysate (A20TL) on lymphoma in mice. Fig. 7A exemplarily shows the tumor growth curve in mice after subcutaneous injection of OR+A20TL, Fig. 7B exemplarily shows the overall tumor picture of mice after subcutaneous injection of OR+A20TL, and Fig. 7C exemplarily shows the subcutaneous injection of OR+A20TL After the mouse tumor size picture, Figure 7D exemplarily shows the average weight of the tumor in the mouse after subcutaneous injection of OR+A20TL.

Figure 8 exemplarily shows the therapeutic effect of subcutaneous administration of the composition of the present invention OR(OX40mab+R848)+LLC autologous tumor tissue lysate (LLC TL) mice with lung cancer. Figure 8A exemplarily shows the tumor growth curve in mice after subcutaneous injection of OR+LLC TL. Figure 8B exemplarily shows the overall picture of the tumor of the mouse after subcutaneous injection of OR+LLC TL. Figure 8C exemplarily shows a picture of mouse tumor size after subcutaneous injection of OR+LLC TL. Figure 8D exemplarily shows the average tumor weight in mice after subcutaneous injection of OR+LLC TL.

Figure 9 exemplarily shows the long-acting anti-tumor immune memory in mice after subcutaneous co-injection of the composition OR(OX40mab+R848)+CT-26TL of the present invention.

Figure 10 exemplarily shows the composition of OR and fresh autologous tumor tissue homogenate (CT-26TL) and formalin-fixed autologous tumor tissue homogenate (CT-26FTL) after subcutaneous injection Comparison of anti-tumor effects.

Detailed description of the invention

Although various embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that these embodiments are only provided in an exemplary manner. Without departing from the present invention, those skilled in the art can think of many changes, changes and substitutions. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

Unless otherwise stated, the practice of some embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FMAusubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PC 2: A Practical Approach (MJMacPherson, BD Hames and GRTaylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technology Applications, 6th Edition (RIFreshney, ed. (2010)).

definition

As used in the specification and claims, the singular forms "a", "an" and "said" include plural references unless the context clearly dictates otherwise. For example, the term "immune activator" includes one or more immune activators.

The term "about" or "approximately" refers to within the acceptable error range of a particular value determined by a person of ordinary skill in the art, which will depend in part on how the value is measured or determined, that is, the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Or, especially for biological systems or processes, the term may represent an order of magnitude of the value, preferably within 5 times, more preferably within 2 times. Where specific values are described in the application and claims, unless otherwise stated, it should be assumed that the term "about" means within the acceptable error range of the specific value.

The term "immune cells" as used herein refers to cells of the immune system that are present in a subject's body. Immune cells further include lymphocytes, neutrophils, and monocytes/macrophages. The term "lymphocyte" as used herein refers to a subtype of white blood cells present in the immune system of a subject. Lymphocytes include T cells, natural killer cells, and B cells.

The term "T cell" as used herein is a lymphocyte developed in the thymus, which plays an important role in the immune response. T cell receptors exist on the surface of T cells, and T cell receptors can be used to distinguish T cells from other lymphocytes.

As used herein, the term "T helper cell" (T helper cell, Th cell), also known as CD4+ T cell, releases cytokines to help other immune cells activate. In addition, helper T cells are also necessary in B cell antibody class switching, activation and growth of cytotoxic T cells, and maximizing the activity of phagocytes such as macrophages.

As used herein, the term "Cytotoxic T cell" (Cytotoxic T cell, CTL) is also called killer T cell or CD8+ T cell, which destroys virus-infected cells and tumor cells, and also participates in transplant rejection. Cytotoxic T cells also produce cytokines such as IL-2 and IFNγ to affect the effector functions of other cells, especially macrophages and NK cells.

The term "Nature Killer T cell" (Nature Killer T cell) as used herein is a heterogeneous T cell that shares the characteristics of T cells and natural killer cells. After activation, natural killer T cells can produce large amounts of interferon gamma, IL-4 and granulocyte-macrophage colony stimulating factor, as well as a variety of other cytokines and chemokines such as IL-2, IL-13, and IL- 17. IL-21 and TNF-α, etc.

The term "immune cell activity" as used herein refers to the process by which immune cells change from a resting state to an active state. Immune cell activity may include, but is not limited to: proliferation of immune cells; differentiation, dedifferentiation or transdifferentiation of immune cells; release of immune cell cytokines; cytotoxicity of immune cells; movement and/or transportation of immune cells; Failure and its combination

The term "autologous" as used herein refers to cells removed from a donor and administered to a recipient, where the donor and recipient are the same individual.

The term "tumor" as used herein refers to all neoplastic cells and all precancerous and cancerous cells and tissues, whether malignant or benign. The terms "cancer" and "cancerous" refer to conditions in which cell growth is not regulated. The term "tumor cell" or "cancer cell" as used herein refers to a cell that undergoes malignant transformation such that it is pathological to the host organism. The definition of cancer cells as used herein includes not only primary cancer cells, but also any cells derived from cancer cells such as metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells.

The term "oncolytic virus" as used herein is a virus that preferentially infects and kills cancer cells. Infected cancer cells are destroyed by oncolytic virus and will release new infectious virus particles or virions to destroy the remaining tumors. Oncolytic viruses not only cause the direct destruction of tumor cells, but also stimulate the host's anti-tumor immune system to respond.

The term "immune activation agent" (Immune activation agent, IAA) as used herein refers to any substance that can improve or strengthen the body's immune response, for example, ligands of immune cell surface molecules such as agonists or antagonists. The immune activator can be either an innate immune activator or a molecule prepared by genetic engineering that has a considerable degree of homology with the innate immune activator.

The term "agonist" as used herein refers to a ligand that binds directly or indirectly to the receptor and activates the receptor or activates its biological response. The term "antagonist" as used herein is a ligand that binds directly or indirectly to the receptor and blocks the receptor or inhibits its biological response.

The term "antibody" is used in the broadest sense herein and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments, as long as they can exhibit antigen-binding activity. Antibodies that can be used in the present disclosure include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, and antibody fragments. The antibody fragments include but are not limited to: Fab, Fab', F(ab')2, Fv, single-chain antibody scFv, single-chain antibody scFv-FC, minibody, diabody, single domain antibody, full-length antibody.

The term "cytokine" as used herein refers to a class of biological molecules that affect immune cells. Exemplary cytokines used in the practice of the present invention include but are not limited to interferon-α (IFN-α), interferon-β (IFN-β) and interferon-γ (IFN-γ), interleukin (such as IL -2), tumor necrosis factor (for example, TNF-α and TNF-β), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulation Factor (GM-CSF) and so on.

"Treatment" as used herein refers to an attempt to change the natural course of a disease in the treated individual, and may be for prevention or clinical intervention implemented during the course of clinical pathology. The desired effects of treatment include but are not limited to preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or reducing the disease state and/or improving the prognosis.

An "effective amount" as used herein refers to at least the minimum amount required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary with the patient's disease state, age, sex, weight and other factors. An effective amount is also an amount where the therapeutic benefit exceeds any toxic or adverse effects of the treatment. In the treatment of cancer or tumors, the effective amount of the drug can have the following effects: reducing the number of cancer cells, reducing tumor size, inhibiting the infiltration of cancer cells into peripheral organs, inhibiting tumor metastasis, inhibiting tumor growth to a certain extent and/or To a certain extent alleviate one or more symptoms related to the disease. The effective amount can be administered in one or more applications.

"Subject", "individual" and "patient" as used herein are used interchangeably herein and refer to vertebrates, preferably mammals, such as humans. Mammals include, but are not limited to, mice, apes, humans, farm animals, sports animals, and pets.

The term "kit" as used herein refers to a combination packaged for common use or commercially available. For example, the kit of the present disclosure may include the composition of the present disclosure, and instructions for using the composition or the kit. The term "instructions" refers to the explanatory inserts usually contained in commercial packages of therapeutic products, which contain information about indications, use, dosage, administration, combination therapy, contraindications, and/or warnings about the use of such therapeutic products.

combination

In one aspect, the present application provides a composition for inducing immune cell activity in a subject in need, comprising: a lysate of tumor cells autologous to the subject, an OX40 agonist and a TLR agonist .

In another aspect, the present disclosure provides a composition for inducing immune cell activity in a subject in need, which comprises a tumor cell autologous to the subject, an oncolytic virus, and one or more immune cells. Activator.

In some embodiments, the activity of the immune cells includes the proliferation of immune cells. In some embodiments, the proliferation refers to an increase in the number of immune cells. In some embodiments, the number of immune cells is quantified by methods such as flow cytometry and/or blood count. In some embodiments, the composition of the present disclosure makes the proliferation of immune cells in the subject higher than the proliferation of immune cells in the subject before the administration of the composition. In some embodiments, the composition of the present disclosure enables the proliferation of immune cells in a subject to be higher than the proliferation of immune cells in another subject to which the composition has not been administered.

In some embodiments, the activity of the immune cell includes the release of cytokines by the immune cell. In some embodiments, the cytokine includes but is not limited to TNFα, TGFβ, IFNγ, CSF, IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-21, IL-22, GM-CSF, etc. In some embodiments, the cytokine can be quantified using ELISA, flow cytometry, and/or western blotting. In some embodiments, the composition of the present disclosure makes the amount of cytokine released by immune cells in the subject higher than the amount of cytokine released by immune cells in the subject before the administration of the composition. In some embodiments, the composition of the present disclosure enables the amount of cytokines released by immune cells in a subject to be higher than the amount of cytokines released by immune cells in another subject to which the composition has not been administered.

In some embodiments, the activity of the immune cell includes the cytotoxicity of the immune cell. In some embodiments, the cytotoxicity is used to kill tumor cells. In some embodiments, the cytotoxicity can induce tumor cells to undergo apoptosis. In some embodiments, the cytotoxicity further includes the release of cytotoxic cytokines such as IFNγ. In some embodiments, the cytotoxicity can be quantified by a cytotoxicity assay such as ELISPOT and the like. In some embodiments, the composition of the present disclosure makes the cytotoxic amount of immune cells in the subject higher than the cytotoxic amount of immune cells in the subject before the administration of the composition. In some embodiments, the composition of the present disclosure makes the cytotoxic amount of immunity in a subject higher than the amount of cytotoxicity released by immune cells in another subject to which the composition has not been administered.

In some embodiments, the activity of the immune cell includes differentiation, dedifferentiation, and transdifferentiation of the immune cell. The differentiation, dedifferentiation or transdifferentiation can be determined by the expression of relevant markers on the surface of the immune cells through flow cytometry evaluation. In some embodiments, the composition of the present disclosure makes the differentiation ability of immune cells in a subject higher than the differentiation ability of immune cells in the subject before administration of the composition. In some embodiments, the composition of the present disclosure makes the differentiation ability of immune cells in a subject higher than the differentiation ability of immune cells in another subject to which the composition has not been administered. In some embodiments, the composition of the present disclosure enables the dedifferentiation ability of immune cells in a subject to be higher than the dedifferentiation ability of immune cells in the subject before the administration of the composition. In some embodiments, the composition of the present disclosure enables the dedifferentiation ability of immune cells in a subject to be higher than the dedifferentiation ability of immune cells in another subject to which the composition has not been administered. In some embodiments, the composition of the present disclosure enables the transdifferentiation ability of immune cells in a subject to be higher than the transdifferentiation ability of immune cells in the subject before administration of the composition. In some embodiments, the composition of the present disclosure enables the transdifferentiation ability of immune cells in a subject to be higher than the transdifferentiation ability of immune cells in another subject to which the composition has not been administered.

In some embodiments, the activity of the immune cell includes the motility and/or transportation capacity of the immune cell. In some embodiments, the motility and/or transportation capacity can be determined by measuring the transfer of the immune cells to the target site. In some embodiments, the determination can be performed by determining the number of immune cells such as tumor infiltrating lymphocytes (TIL) in the lesion. In some embodiments, the composition of the present disclosure enables the motility and/or transportation capacity of immune cells in the subject to be higher than the motility and/or transportation capacity of immune cells in the subject before the administration of the composition. In some embodiments, the composition of the present disclosure enables the motility and/or transportation capacity of immune cells in a subject to be higher than the motility and/or transportation capacity of immune cells in another subject to which the composition has not been administered .

In some embodiments, the activity of the immune cells includes the failure of immune cells. In some embodiments, the failure is analyzed by flow cytometry. In some embodiments, the failure is determined by the expression level of immune cell markers. In some embodiments, the markers include but are not limited to PD1, 2B4, LAG3, CD160, IFNγ and the like. In some embodiments, the composition of the present disclosure makes the depletion capacity of immune cells in a subject higher than the depletion capacity of immune cells in the subject before the administration of the composition. In some embodiments, the composition of the present disclosure makes the depletion capacity of immune cells in a subject higher than the depletion capacity of immune cells in another subject to which the composition has not been administered.

In some embodiments, the immune cells described in the present disclosure are lymphocytes. In some embodiments, the lymphocytes include, but are not limited to, T cells, natural killer cells, and B cells. In some embodiments, the T cells include, but are not limited to, helper T cells, cytotoxic T cells, and natural killer T cells. In some embodiments, the immune cells described in the present disclosure are macrophages.

In some embodiments, the oncolytic virus used in the composition of the present disclosure can be any oncolytic virus suitable for use in the composition of the present application. In some embodiments, the oncolytic virus is selected from the group consisting of myoviridae, herpesviridae, retroviridae, adenoviridae, barnaviridae, and barnaviridae. ) Parvoviridae, baculoviridae, microviridae, siphoviridae, podpviridae, corticoviridae, budding virus (plasmavifidae), lipoviridae (lipothrixviridae), poxviridae, iridoviridae, phycodnaviridae, papovaviridae, polydnaviridae , Inoviridae, Geminiviridae, Circoviridae, Hepadnaviridae, Reoviridae, Bimaviridae, Paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, light Leviviridae, Picornaviridae, Caliciviridae, Astroviridae, Nodaviridae, Tetraviridae, Coronavirus coronaviridae) or a member of the togaviridae family.

In some embodiments, oncolytic viruses that can be used in the composition of the present disclosure include, but are not limited to, Herpesvirus, Adenovirus, Reovirus, Vaccinia Virus, Cosa Coxsackievirus, Measles Virus, Polio Virus, Retrovirus, Parvovirus H1, Vesicular Stomatitis Virus, Newtown Newcastle Disease Virus and M1 oncolytic virus.

In some embodiments, the oncolytic viruses described in the present disclosure may be naturally occurring or modified oncolytic viruses. In some embodiments, the "naturally occurring" oncolytic virus refers to an oncolytic virus that is isolated from a natural source and has not been intentionally modified in the laboratory. For example, the oncolytic virus may be derived from a "natural source", that is, from a subject infected with an oncolytic virus.

In some embodiments, the oncolytic virus may be a modified oncolytic virus. In some embodiments, the modified oncolytic virus retains its ability to infect tumor cells. In some embodiments, the oncolytic virus contains mutations, insertions and/or deletions in one or more genome segments, or engineered genes that are designed to encode, such as one or more of the genes described in this disclosure. An immune activator gene. In some embodiments, the modified oncolytic virus is an attenuated oncolytic virus. In some embodiments, the oncolytic virus is a recombinant oncolytic virus. In some embodiments, the recombinant oncolytic virus is produced by recombination/rearrangement of genome segments of two or more different oncolytic viruses.

In some embodiments, the oncolytic virus is herpes simplex virus. In some embodiments, the herpes simplex virus is selected from HSV-1 or HSV-2. In some embodiments, the oncolytic virus is herpes simplex virus HSV-1 modified to lack the function of γ34.5. In some embodiments, the herpes simplex virus modified to delete the function of γ34.5 includes, but is not limited to, the herpes simplex virus containing mutations, insertions and/or deletions in the γ34.5 gene.

In some embodiments, an OX40 agonist and a TLR agonist are included in the composition of the invention. In some embodiments, the OX40 agonist included in the composition of the present invention includes OX40 antibody, OX40 ligand fusion protein (OX40L). In some embodiments, the OX40 agonist includes an OX40 antibody. The OX40 antibody includes but is not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies. In some embodiments, the OX40 antibody is a full-length antibody. In some embodiments, the OX40 antibody is an antibody fragment. In some embodiments, the OX40 antibody is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody scFv, single-chain antibody scFv-FC, minibody, diabody, single domain antibody, full-length antibody antibody. In some embodiments, the OX40 agonist includes an OX40 ligand fusion protein. In some embodiments, the OX40 ligand fusion protein is an Fc fusion protein.

In some embodiments, the TLR agonists included in the composition of the present invention include, but are not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and / Or TLR13 ligand. In some embodiments, the ligand of the TLR is a TLR agonist. In some embodiments, the TLR ligand includes, but is not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13 agonists. In some embodiments, the TLR agonist includes CpG oligonucleotides (oligodeoxynucleotides, CpG ODN), Resiquimod (R848), Imiquimod (R837), dsRNA, and combinations thereof. In some embodiments, the CpG ODN is unmethylated CpG ODN.

In some embodiments, the composition of the present disclosure includes one or more immune activating agents. In some embodiments, the immune activator comprises a molecule capable of binding to immune cell surface receptors and activating downstream signaling pathways of the immune cells. In some embodiments, the immune activator comprises a ligand that is capable of binding to a surface receptor of an immune cell and activating the downstream signaling pathway of the immune cell.

In some embodiments, the immune activator comprises one or more agonists of immune cell surface receptors. In some embodiments, the immune activator comprises an agonist of one or more molecules selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, and GITR. In some embodiments, the immune activator comprises an agonist of OX40.

In some embodiments, the immune activator comprises one or more antagonists of immune cell surface receptors. In some embodiments, the immune activator comprises an antagonist of one or more molecules selected from the group consisting of A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1 , TIM-3, VISTA, SIGLEC7 and SIGLEC9.

In some embodiments, the immune activator comprises a ligand of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the immune activator comprises a fusion protein of OX40 ligand (OX40L).

In some embodiments, the immune activator comprises a fusion protein of a ligand of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4 , IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the immune activator comprises a fusion protein of OX40 ligand (OX40L).

In some embodiments, the immune activator comprises an antibody that specifically binds to an epitope of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the immune activator comprises an antibody to OX40.

In some embodiments, the antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody scFv, single-chain antibody scFv-FC, minibody, diabody, single domain antibody, full-length antibody . .

In some embodiments, the immune activator described in the present disclosure further comprises a ligand of Toll-Like Receptor (TLR). In some embodiments, the TLR ligand includes, but is not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13 ligand. In some embodiments, the ligand of the TLR is a TLR agonist. In some embodiments, the TLR ligand includes, but is not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13 agonists.

In some embodiments, the TLR agonist includes CpG oligonucleotides (oligodeoxynucleotides, CpG ODN), Resiquimod (R848), Imiquimod (R837), dsRNA, and combinations thereof. In some embodiments, the CpG ODN is unmethylated CpG ODN.

In some embodiments, the immune activator further comprises a single-stranded or double-stranded nucleic acid molecule. In some embodiments, the single-stranded or double-stranded nucleic acid molecule may be any nucleic acid molecule that can directly and/or indirectly modulate the immune response of immune cells. In some embodiments, the length of the nucleic acid is 2-100 bases. In some embodiments, the length of the nucleic acid is 6-100 nucleic acids. In some embodiments, the nucleic acid is 8-100 nucleic acids in length. In some embodiments, the nucleic acid is 2-50 bases in length. In some embodiments, the length of the nucleic acid is 6-50 nucleic acids. In some embodiments, the length of the nucleic acid is 8-50 nucleic acids.

In some embodiments, the immune activator further comprises one or more cytokines. In some embodiments, the cytokine includes but is not limited to: IL-1, IL-2, IL-5, IL-6, IL-12, IL-13, IL15, IL16, IL-17, IL18, IL -21, IL-22, IFN-ɑ, TNF-β, IFN-γ, GM-CSF, M-CSF and any combination thereof. In some embodiments, the cytokine is selected from the group consisting of IL-2, IL-12, IL15, IL6, IL18, IFN-ɑ, TNF-β, IFN-γ, GM-CSF, M-CSF And any combination.

In some embodiments, the genes of one or more immune activators in the composition of the present disclosure are encoded and expressed by the oncolytic virus. In some embodiments, the gene of the OX40 agonist in the composition of the present disclosure is encoded and expressed by an oncolytic virus. In some embodiments, the gene of the TLR agonist in the composition of the present disclosure is encoded and expressed by an oncolytic virus. In some embodiments, the genes of the OX40 agonist and TLR agonist in the composition of the present disclosure are encoded and expressed by an oncolytic virus. In some embodiments, one or more immune activators in the composition of the present disclosure are not encoded or expressed by the oncolytic virus. In some embodiments, part of the immune activator in the composition of the present disclosure is encoded and expressed by the oncolytic virus.

In some embodiments, the amount of oncolytic virus in the composition is 1×10 ⁵ -1× ^{10 9} pfu. In some embodiments, the amount of oncolytic virus in the composition is 1×10 ⁷ -1× ^{10 8} pfu. In some embodiments, the amount of the oncolytic virus may be about ^{^{^{1x10 5, 5x10 5, 1x10 6}}} , 5x10 6, 1x10 7, 5x10 7, 1x10 8 or 5x10 ⁸ pfu.

In some embodiments, the composition described in the present disclosure comprises tumor cells that are autologous to the subject. In some embodiments, the tumor cell is an autologous tumor cell isolated from the subject or its progeny cells. In some embodiments, the tumor cell is an autologous tumor cell isolated from a solid tumor of the subject or its progeny cells. In some embodiments, the tumor cell is an autologous tumor cell or its progeny cells isolated from one or more lesions of the subject. In some embodiments, the tumor cell is an autologous tumor cell isolated from one or more organs of the subject or its progeny cells. In some embodiments, the tumor cell is an autologous tumor cell or its progeny cells isolated from one or more tissues of the subject. In some embodiments, the lesions include but are not limited to: lung cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, glioma, Head and neck cancer, kidney cancer, multiple myeloma, lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, liver cancer, gallbladder cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, oral cancer, nasopharyngeal carcinoma , Laryngeal cancer, thyroid cancer, mediastinal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, tracheal cancer and vulvar cancer. In some embodiments, the tumor cell is an autologous tumor cell or its progeny cells isolated from the body fluid of the subject. In some embodiments, the body fluids include, but are not limited to: whole blood, plasma, effusion, ascites, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions including Sputum and breast fluid.

In some embodiments, the tumor cells used in the composition are derived from fresh tumor tissue. In some embodiments, the tumor cells used in the composition are derived from frozen tumor tissue. In some embodiments, the tumor cells are derived from tumor tissues fixed with fixative. In some embodiments, the fixative solution comprises formalin, formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, or any combination thereof.

In some embodiments, the tumor cell is a tumor cell that has lost the ability to proliferate. In some embodiments, the tumor cell is a tumor cell that has been genetically modified to lose the ability to proliferate. In some embodiments, the tumor cells are tumor cells that have mutations, deletions, and/or insertions at the gene level to lose the ability to proliferate. In some embodiments, the tumor cells are tumor cells that have been treated such as radiation to lose the ability to proliferate.

In some embodiments, the number of tumor cells used in the composition is 1×10 ⁴ -1× ^{10 8} . In some embodiments, the number of tumor cells in the composition is 1×10 ⁵ -1× ^{10 7} . In some embodiments, the number of tumor cells in the composition is ^{^{^{1x10 4, 5x10 4, 1x10 5}}} , 5x10 5, 1x10 6, 5x10 6, 1x10 7, 5x10 7, 1x10 8.

In some embodiments, the composition described in the present disclosure comprises a tumor cell lysate that is autologous to the subject. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from tumor cells that are autologous to the subject. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from an autologous tumor cell isolated from the subject or its progeny cells.

In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from an autologous tumor cell of a solid tumor of the subject or its progeny cells. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from autologous tumor cells or progeny cells of one or more lesions of the subject. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from autologous tumor cells of one or more organs of the subject or their progeny cells. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from an autologous tumor cell or its progeny cells in one or more tissues of the subject. In some embodiments, the lesions include but are not limited to: lung cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, glioma, Head and neck cancer, kidney cancer, multiple myeloma, lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, liver cancer, gallbladder cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, oral cancer, nasopharyngeal carcinoma , Laryngeal cancer, thyroid cancer, mediastinal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, tracheal cancer and vulvar cancer. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from autologous tumor cells of the subject's body fluid or progeny cells thereof. In some embodiments, the body fluids include, but are not limited to: whole blood, plasma, effusion, ascites, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions including Sputum and breast fluid.

In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from fresh tumor tissue. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from frozen tumor tissue. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from tumor tissue fixed with a fixative. In some embodiments, the fixative solution comprises formalin, formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, or any combination thereof.

In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from tumor cells that have lost the ability to proliferate. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from tumor cells that have been genetically modified to lose the ability to proliferate. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from tumor cells that have mutations, deletions, and/or insertions at the gene level to lose the ability to proliferate. In some embodiments, the tumor cell lysate used in the composition of the present disclosure is derived from tumor cells that have been treated such as radiation to lose their ability to proliferate.

In some embodiments, the tumor cell lysate used in the composition of the present application is derived from 1×10 ⁴ -1× ^{10 8} number of tumor cells. In some embodiments, the tumor cell lysate used in the composition of the present application is derived from 1×10 ⁵ -1× ^{10 7} tumor cells. In some embodiments, the compositions used in the present application, a tumor cell lysate from ^{^{^{1x10 4, 5x10 4, 1x10 5}}} , 5x10 5, 1x10 6, 5x10 6, 1x10 7, 5x10 7, 1x10 8 number of tumor cells .

In some embodiments, the tumor cell lysate used in the composition of the present application is obtained by a method selected from the group consisting of homogenization, ultrasound, or a combination of the two. In some embodiments, the tumor cell lysate used in the composition of the present application is obtained by homogenization. In some embodiments, the tumor cell lysate used in the composition of the present application is obtained by ultrasound. In some embodiments, the lysate of the tumor cells in the composition is obtained by a process of homogenization and then ultrasound. In some embodiments, the homogenization time does not exceed 30 seconds. In some embodiments, the homogenization time does not exceed 1 minute. In some embodiments, the homogenization time does not exceed 2 minutes. In some embodiments, the homogenization time does not exceed 3 minutes. In some embodiments, the homogenization time does not exceed 4 minutes. In some embodiments, the homogenization time does not exceed 5 minutes. In some embodiments, the homogenization time does not exceed 6 minutes. In some embodiments, the homogenization time does not exceed 7 minutes. In some embodiments, the homogenization time does not exceed 8 minutes. In some embodiments, the homogenization time does not exceed 9 minutes.

In some embodiments, the homogenization includes multiple homogenizations, wherein the time of each homogenization does not exceed 30 seconds, does not exceed 1 minute, does not exceed 2 minutes, does not exceed 3 minutes, does not exceed 4 minutes, and does not exceed 5 minutes, no more than 6 minutes, no more than 7 minutes, no more than 8 minutes, no more than 9 minutes, no more than 10 minutes. In some embodiments, the homogenization includes multiple homogenizations, wherein the interval between each homogenization is no more than 30 seconds, no more than 1 minute, no more than 2 minutes, no more than 3 minutes, no more than 4 minutes, no More than 5 minutes, no more than 6 minutes, no more than 7 minutes, no more than 8 minutes, no more than 9 minutes, no more than 10 minutes.

In some embodiments, the working time of the ultrasound does not exceed 0.5 seconds. In some embodiments, the working time of the ultrasound does not exceed 1 second. In some embodiments, the working time of the ultrasound does not exceed 1.5 seconds. In some embodiments, the working time of the ultrasound does not exceed 2 seconds. In some embodiments, the working time of the ultrasound does not exceed 2.5 seconds. In some embodiments, the working time of the ultrasound does not exceed 3 seconds. In some embodiments, the working time of the ultrasound does not exceed 3.5 seconds. In some embodiments, the working time of the ultrasound does not exceed 4 seconds. In some embodiments, the working time of the ultrasound does not exceed 4.5 seconds. In some embodiments, the working time of the ultrasound does not exceed 5 seconds.

In some embodiments, the intermittent time of each operation of the ultrasound is not less than 0.5 seconds. In some embodiments, the intermittent time of each ultrasound operation is not less than 1 second. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 1.5 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 2 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 2.5 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 3 seconds. In some embodiments, the intermittent time of each ultrasound operation is not less than 3.5 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 4 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 4.5 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 5 seconds.

In some embodiments, the working times of the ultrasound are at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, at least 16 times, at least 17 times, at least 18 times, at least 19 times, at least 20 times.

method

In another aspect, the present disclosure provides a method for treating tumors by inducing immune cell activity in a subject in need, which comprises administering an effective amount of the composition of the application to the subject. In some embodiments, the composition comprises a lysate of tumor cells autologous to the subject, an OX40 agonist, and a TLR agonist. In some embodiments, the composition comprises an oncolytic virus, an OX40 agonist and a TLR agonist, or an oncolytic virus expressing an OX40 agonist and a TLR agonist. In some embodiments, the composition comprises tumor cells autologous to the subject, an oncolytic virus, and one or more immune activators. In some embodiments, the treatment includes tumor regression, inhibition of tumor progression and/or metastasis, and prevention of its recurrence and/or metastasis.

In some embodiments, the method is used to treat solid tumors in the subject. In some embodiments, the method is used to treat hematological tumors in the subject. In some embodiments, the method is for treating a condition in which the subject is selected from the group consisting of lung cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer , Esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, multiple myeloma, liver cancer, gallbladder cancer, lymphoma, melanoma, mesothelioma, Medulloblastoma, oral cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, mediastinal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.

In some embodiments of the method, the immune cell is a lymphocyte. In some embodiments, the lymphocytes include, but are not limited to, T cells, natural killer cells, and B cells. In some embodiments, the T cells include, but are not limited to, helper T cells, cytotoxic T cells, and natural killer T cells. In some embodiments, the immune cells described in the present disclosure are macrophages.

In some embodiments of the method, the oncolytic virus used in the method can be any suitable oncolytic virus. In some embodiments, the oncolytic virus used in the method is selected from the group consisting of myoviridae, herpesviridae, retroviridae, adenoviridae, rod-shaped RNA Viridae (barnaviridae) Parvoviridae, Baculoviridae, Microviridae, Siphoviridae, Podpviridae, Corticoviridae , Plasmavifidae, lipoviridae, poxviridae, iridoviridae, phycodnaviridae, papovaviridae, multiple DNA viruses Family (polydnaviridae), filoviridae (inoviridae), geminiviridae (geminiviridae), circoviridae (circoviridae), hepadnaviridae (hepadnaviridae), reoviridae (reoviridae), double RNA virus family (bimaviridae), paramyxoviridae, rhabdoviridae, filoviridae, orthomyxoviridae, bunyaviridae, arenaviridae ( arenaviridae), leviviridae, picornaviridae, caliciviridae, astroviridae, nodaviridae, tetraviridae, Coronaviridae (coronaviridae), togaviridae (togaviridae) or members of. In some embodiments, the oncolytic virus used in the method is selected from the group consisting of Herpesvirus, Adenovirus, Reovirus, Vaccinia Virus, Coxsackievirus ), Measles Virus, Poliovirus, Retrovirus, Parvovirus H1 (Parvovirus H1), Vesicular Stomatitis Virus, Newcastle Disease Virus (Newcastle Disease) Virus) and M1 oncolytic virus.

In some embodiments of the methods, the oncolytic viruses used in the methods of the present disclosure may be naturally occurring or modified oncolytic viruses. In some embodiments of the method, the modified oncolytic virus retains its ability to infect tumor cells. In some embodiments, the modified oncolytic virus is an attenuated oncolytic virus. In some embodiments, the oncolytic virus used in the methods of the present disclosure is a recombinant oncolytic virus. In some embodiments, the recombinant oncolytic virus is produced by recombination/rearrangement of genome segments of two or more different oncolytic viruses. In some embodiments, the oncolytic virus contains mutations, insertions and/or deletions in one or more genome segments, or engineered genes that are designed to encode, such as one or more of the genes described in this disclosure. An immune activator gene.

In some embodiments of the methods, the oncolytic virus used in the methods of the present disclosure is herpes simplex virus. In some embodiments, the herpes simplex virus includes but is not limited to HSV-1 and HSV-2. In some embodiments, the oncolytic virus used in the methods of the present disclosure is a herpes simplex virus modified to lack the function of γ34.5. In some embodiments, the herpes simplex virus modified to delete the function of γ34.5 includes, but is not limited to, the herpes simplex virus containing mutations, insertions and/or deletions in the γ34.5 gene.

In some embodiments, the composition used in the method includes an OX40 agonist and a TLR agonist. In some embodiments, the OX40 agonist includes OX40 antibody or OX40 ligand fusion protein OX40L. In some embodiments, the OX40 agonist includes an OX40 antibody. The OX40 antibody includes but is not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies. In some embodiments, the OX40 antibody is a full-length antibody. In some embodiments, the OX40 antibody is an antibody fragment. In some embodiments, the OX40 antibody is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody scFv, single-chain antibody scFv-FC, minibody, diabody, single domain antibody, full-length antibody antibody. In some embodiments, the OX40 agonist includes the OX40 ligand fusion protein OX40L. In some embodiments, the OX40 ligand fusion protein is the Fc fusion protein OX40L.

In some embodiments, the TLR agonist includes the TLR agonist including but not limited to TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13. body. In some embodiments, the ligand of the TLR is a TLR agonist. In some embodiments, the TLR ligand includes, but is not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13 agonists. In some embodiments, the TLR agonist includes CpG oligonucleotides (oligodeoxynucleotides, CpG ODN), Resiquimod (R848), Imiquimod (Imiquimod, R837) _, dsRNA, and combinations thereof. In some embodiments, the CpG ODN is an unmethylated CpG ODN.

In some embodiments of the method, the composition used in the method comprises one or more immune activators. In some embodiments, the immune activator comprises a molecule capable of binding to immune cell surface receptors and activating downstream signaling pathways of the immune cells. In some embodiments, the immune activator comprises a ligand that is capable of binding to a surface receptor of an immune cell and activating the downstream signaling pathway of the immune cell. In some embodiments, the immune activator comprises one or more agonists of immune cell surface receptors. In some embodiments, the immune activator comprises an agonist of one or more molecules selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, and GITR. In some embodiments, the immune activator comprises an agonist of OX40. In some embodiments, the immune activator comprises one or more antagonists of immune cell surface receptors. In some embodiments, the immune activator comprises an antagonist of one or more molecules selected from the group consisting of A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1 , TIM-3, VISTA, SIGLEC7 and SIGLEC9.

In some embodiments, the immune activator agent used in the method of the present disclosure comprises a ligand of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA , CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the immune activator comprises a ligand of OX40. In some embodiments, the immune activator comprises a fusion protein of a ligand of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4 , IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the immune activator comprises a fusion protein of OX40 ligand.

In some embodiments, the immune activator used in the methods of the present disclosure comprises an antibody that specifically binds to an epitope of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the immune activator comprises an antibody to OX40.

In some embodiments, the immune activator used in the method of the present disclosure further comprises a ligand of Toll-Like Receptor (TLR). In some embodiments, the TLR ligand includes, but is not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13 ligand. In some embodiments, the ligand of the TLR is a TLR agonist. In some embodiments, the TLR ligand includes but is not limited to TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13 agonists.

In some embodiments, one or more cytokines used in the methods of the present disclosure are encoded and expressed by the oncolytic virus. In some embodiments, the gene of the OX40 agonist used in the methods of the present disclosure is encoded and expressed by an oncolytic virus. In some embodiments, the gene of the TLR agonist used in the method of the present disclosure is encoded and expressed by an oncolytic virus. In some embodiments, the genes of the OX40 agonist and TLR agonist used in the methods of the present disclosure are encoded and expressed by an oncolytic virus. In some embodiments, one or more cytokines used in the methods of the present disclosure are not encoded and expressed by the oncolytic virus. In some embodiments, one or more cytokines used in the methods of the present disclosure are partially encoded and expressed by the oncolytic virus.

In some embodiments, the amount of oncolytic virus used in the method is 1×10 ⁵ -1× ^{10 9} pfu. In some embodiments, the amount of oncolytic virus in the composition is 1×10 ⁷ -1× ^{10 8} pfu. In some embodiments, the amount of the oncolytic virus may be about ^{^{^{1x10 5, 5x10 5, 1x10 6}}} , 5x10 6, 1x10 7 .5x10 7 1x10 8 or 5x10 ⁸ pfu.

In some embodiments of the method, the tumor cells used in the method are autologous tumor cells of the subject. In some embodiments, the tumor cell is an autologous tumor cell isolated from the subject or its progeny cells.

In some embodiments, the tumor cell used in the method is an autologous tumor cell isolated from a solid tumor of the subject or its progeny cells. In some embodiments, the tumor cell is an autologous tumor cell or its progeny cells isolated from one or more lesions of the subject. In some embodiments, the tumor cell is an autologous tumor cell isolated from one or more organs of the subject or its progeny cells. In some embodiments, the tumor cell is an autologous tumor cell or its progeny cells isolated from one or more tissues of the subject. In some embodiments, the lesions include but are not limited to: lung cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, glioma, Head and neck cancer, kidney cancer, multiple myeloma, lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, liver cancer, gallbladder cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, oral cancer, nasopharyngeal carcinoma , Laryngeal cancer, thyroid cancer, mediastinal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, tracheal cancer and vulvar cancer. In some embodiments, the tumor cell is an autologous tumor cell or its progeny cells isolated from the body fluid of the subject. In some embodiments, the body fluids include, but are not limited to: whole blood, plasma, effusion, ascites, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions including Sputum and breast fluid.

In some embodiments, the tumor cells used in the method are derived from fresh tumor tissue. In some embodiments, the tumor cells used in the method are derived from frozen tumor tissue. In some embodiments, the tumor cells are derived from tumor tissues fixed with fixative. In some embodiments, the fixing solution is selected from formalin, formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, or any combination thereof.

In some embodiments of the method, the tumor cells used in the method are tumor cells that have lost the ability to proliferate. In some embodiments, the tumor cell is a tumor cell that has been genetically modified to lose the ability to proliferate. In some embodiments, the tumor cells are tumor cells that have mutations, deletions, and/or insertions at the gene level to lose the ability to proliferate. In some embodiments, the tumor cells are tumor cells that have been treated such as radiation to lose the ability to proliferate.

In some embodiments, the number of tumor cells used in the method is 1×10 ⁴ -1× ^{10 8} . In some embodiments, the number of tumor cells in the composition is 1×10 ⁵ -1× ^{10 7} . In some embodiments, the number of tumor cells in the composition is ^{^{^{1x10 4, 5x10 4, 1x10 5}}} , 5x10 5, 1x10 6, 5x10 6, 1x10 7, 5x10 7, 1x10 8.

In some embodiments, the composition used in the method comprises a tumor cell lysate that is autologous to the subject. In some embodiments, the tumor cell lysate used in the method is derived from tumor cells that are autologous to the subject. In some embodiments, the tumor cell lysate used in the method is derived from an autologous tumor cell or its progeny cells isolated from the subject.

In some embodiments, the tumor cell lysate used in the method is derived from an autologous tumor cell of a solid tumor of the subject or its progeny cells. In some embodiments, the tumor cell lysate used in the method is derived from an autologous tumor cell or its progeny cells from one or more lesions of the subject. In some embodiments, the tumor cell lysate used in the method is derived from autologous tumor cells or progeny cells of one or more organs of the subject. In some embodiments, the tumor cell lysate used in the method is derived from autologous tumor cells or progeny cells of one or more tissues of the subject. In some embodiments, the lesions include but are not limited to: lung cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, rectal cancer, colorectal cancer, esophageal cancer, gastric cancer, glioma, Head and neck cancer, kidney cancer, multiple myeloma, lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, liver cancer, gallbladder cancer, lymphoma, melanoma, mesothelioma, medulloblastoma, oral cancer, nasopharyngeal carcinoma , Laryngeal cancer, thyroid cancer, mediastinal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, tracheal cancer and vulvar cancer. In some embodiments, the tumor cell lysate used in the method is derived from autologous tumor cells or progeny cells of the subject's body fluid. In some embodiments, the body fluids include, but are not limited to: whole blood, plasma, effusion, ascites, cerebrospinal fluid, cervical secretions, vaginal secretions, endometrial secretions, gastrointestinal secretions, bronchial secretions including Sputum and breast fluid.

In some embodiments, the tumor cell lysate used in the method is derived from fresh tumor tissue. In some embodiments, the tumor cell lysate used in the method is derived from frozen tumor tissue. In some embodiments, the tumor cell lysate used in the method is derived from tumor tissue fixed with a fixative. In some embodiments, the fixative solution comprises formalin, formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, or any combination thereof.

In some embodiments, the tumor cell lysate used in the method is derived from tumor cells that have lost the ability to proliferate. In some embodiments, the tumor cell lysate used in the method is derived from tumor cells that have been genetically modified to lose the ability to proliferate. In some embodiments, the tumor cell lysate used in the method is derived from tumor cells that have mutations, deletions, and/or insertions at the gene level to lose the ability to proliferate. In some embodiments, the tumor cell lysate used in the method is derived from tumor cells that have been treated such as radiation to lose the ability to proliferate.

In some embodiments, the tumor cell lysate used in the method is derived from 1×10 ⁴ -1× ^{10 8} number of tumor cells. In some embodiments, the tumor cell lysate used in the method is derived from 1×10 ⁵ -1× ^{10 7} tumor cells. In some embodiments, the method used in tumor cell lysates from ^{^{^{1x10 4, 5x10 4, 1x10 5}}} , 5x10 5, 1x10 6, 5x10 6, 1x10 7, 5x10 7, 1x10 8 number of tumor cells.

In some embodiments, the tumor cell lysate used in the method is obtained by a method selected from the group consisting of homogenization, ultrasound, or a combination of the two. In some embodiments, the tumor cell lysate used in the composition of the present application is obtained by homogenization. In some embodiments, the tumor cell lysate used in the method is obtained by ultrasound. In some embodiments, the lysate of the tumor cells used in the method is obtained by a process of homogenization and then ultrasound.

In some embodiments, the homogenization time does not exceed 30 seconds. In some embodiments, the homogenization time does not exceed 1 minute. In some embodiments, the homogenization time does not exceed 2 minutes. In some embodiments, the homogenization time does not exceed 3 minutes. In some embodiments, the homogenization time does not exceed 4 minutes. In some embodiments, the homogenization time does not exceed 5 minutes. In some embodiments, the homogenization time does not exceed 6 minutes. In some embodiments, the homogenization time does not exceed 7 minutes. In some embodiments, the homogenization time does not exceed 8 minutes. In some embodiments, the homogenization time does not exceed 9 minutes.

In some embodiments, the intermittent time of each operation of the ultrasound is not less than 0.5 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 1 second. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 1.5 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 2 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 2.5 seconds. In some implementation schemes, the intermittent time of each ultrasound operation is not less than 3 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 3.5 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 4 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 4.5 seconds. In some embodiments, the intermittent time of each operation of the ultrasound is not less than 5 seconds.

In some embodiments, the method of the application comprises administering the composition to the subject in situ or ex situ. In some embodiments, the in situ administration includes intratumoral injection, peritumoral injection, and combinations thereof. In some embodiments, the ex situ administration includes subcutaneous injection, intramuscular injection, intraperitoneal injection, thoracic injection, intravenous injection, arterial injection, and combinations thereof.

In some embodiments, the method includes a single administration of the composition of the present disclosure to the subject to treat the subject. In some embodiments, the method includes multiple administrations of the composition of the present disclosure to the subject to treat the subject. In some embodiments, the time interval for each of the multiple administrations is 1 day to 3 months, for example, the time interval can be 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months or 3 months.

In some embodiments, the method includes administering the composition to the subject by single-point or multiple-point injection. In some embodiments, the method includes administering the composition to the subject by single-point or multiple-point subcutaneous injection. In some embodiments, the multiple injections can be performed at the same time or each of the multiple injections can have a certain time interval. In some embodiments, the time interval is 1 day to 3 months, for example, the time interval can be 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months or 3 months. In some embodiments, the method further comprises pretreating the tumor cells with the oncolytic virus in vitro before administering the composition to the subject. In some embodiments, the pretreatment time is 15 minutes to 12 hours. In some embodiments, the pretreatment time is less than or equal to 12 hours. In some embodiments, the pretreatment time is less than or equal to 11 hours. The method according to claim 79, wherein the pretreatment time is less than 10 hours. In some embodiments, the pretreatment time is less than or equal to 9 hours. In some embodiments, the pretreatment time is less than or equal to 8 hours. In some embodiments, the pretreatment time is less than or equal to 7 hours. In some embodiments, the pretreatment time is less than or equal to 6 hours. In some embodiments, the pretreatment time is less than or equal to 5 hours. In some embodiments, the pretreatment time is less than or equal to 4 hours. In some embodiments, the pretreatment time is less than or equal to 3 hours. In some embodiments, the pretreatment time is less than or equal to 2 hours. In some embodiments, the pretreatment time is less than or equal to 1 hour. In some embodiments, the pretreatment time is less than or equal to 0.5 hours.

In some embodiments, the method further includes administering the composition directly to the subject in admixture with autologous tumor cells. In some embodiments, the method further includes mixing and administering the composition directly to the subject with a lysate of autologous tumor cells.

Reagent test kit

On the other hand, the present disclosure provides a kit for inducing immune cell activity in a subject in need, which includes the composition of the present application and instructions. In some embodiments, the composition includes an OX40 agonist and a TLR agonist. In some embodiments, the composition comprises an oncolytic virus, an OX40 agonist and a TLR agonist, or an oncolytic virus expressing an OX40 agonist and a TLR agonist. In some embodiments, the composition comprises an oncolytic virus and one or more immune activators.

In some embodiments, the instructions instruct to obtain and/or prepare autologous tumor cells from the subject, prepare a lysate of the tumor cell, and combine the tumor cell lysate, an OX40 agonist A method of co-administering the subject with a TLR agonist. In some embodiments, the instructions indicate a method of co-administering an oncolytic virus, an OX40 agonist, and a TLR agonist to the subject. In some embodiments, the instructions instruct to obtain and/or prepare autologous tumor cells from the subject, and to combine the tumor cells with the oncolytic virus and one or more immune activators Methods of co-administration to the subject.

In some embodiments, the kit contains HSV-1 or HSV-2 oncolytic virus.

In some embodiments, the kit includes one or more immune activators. In some embodiments, the immune activator in the kit comprises a fusion protein of a ligand of a molecule selected from the following group: CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9. In some embodiments, the immune activator comprises a fusion protein of OX40 ligand.

In some embodiments, the immune activator in the kit comprises an antibody that specifically binds to an epitope of a molecule selected from the group consisting of CD27, CD28, CD40, CD122, CD137, OX40, GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2, PD-1, TIM-3, VISTA, SIGLEC7, CD47 and SIGLEC9.

In some embodiments, the immune activator in the kit further comprises a TLR agonist such as unmethylated CpG ODN.

In some embodiments, the kit includes an OX40 agonist and a TLR agonist. In some embodiments, the OX40 agonist includes OX40 antibody, OX40 ligand fusion protein. In some embodiments, the OX40 agonist includes an OX40 antibody. The OX40 antibody includes but is not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies. In some embodiments, the OX40 antibody is a full-length antibody. In some embodiments, the OX40 antibody is an antibody fragment. In some embodiments, the OX40 antibody is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody scFv, single-chain antibody scFv-FC, minibody, diabody, single domain antibody, full-length antibody antibody. . In some embodiments, the OX40 agonist includes an OX40 ligand fusion protein. In some embodiments, the OX40 ligand fusion protein is an Fc fusion protein.

In some embodiments, the TLR agonist includes the TLR agonist including but not limited to TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13. body. In some embodiments, the ligand of the TLR is a TLR agonist. In some embodiments, the TLR ligand includes, but is not limited to, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 and/or TLR13 agonists. In some embodiments, the TLR agonist includes CpG oligonucleotides (oligodeoxynucleotides, CpG ODN), Resiquimod (R848), Imiquimod (R 837), dsRNA or a combination thereof. In some embodiments, the CpG ODN is unmethylated CpG ODN.

In some embodiments, the kit further comprises reagents and/or elements for obtaining tumor cells and/or tumor cell lysates from the subject. In some embodiments, the reagent includes a buffer. In some embodiments, the buffer includes cell lysates. In some embodiments, the element can be used to obtain tumor cells from different organs or tissues of the subject's body. In some embodiments, the kit further comprises a container for collecting the obtained tumor cells. In some embodiments, the kit further includes a device for administering each component of the kit to a subject. In some embodiments, the use device includes a syringe, a needle, and the like.

Example

The following examples further illustrate the invention. These examples are only intended to illustrate the present invention and should not be construed as limiting the present invention.

Example 1 Construction and preparation of oncolytic virus used in the present invention

The materials used in Example 1 are shown in Table 1-1:

Table 1-1

项目project	来源source
HSV-1病毒株HSV-1 virus strain	临床分离株Clinical isolate
2×Phanta Max Master Mix2×Phanta Max Master Mix	南京诺唯赞生物科技有限公司Nanjing Nuoweizan Biotechnology Co., Ltd.
pRedTKI、PMDISI、PMDIAIpRedTKI, PMDISI, PMDIAI	AddgeneAddgene

DMEM培养基DMEM medium	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.
胎牛血清Fetal Bovine Serum	浙江天杭生物科技股份有限公司Zhejiang Tianhang Biological Technology Co., Ltd.
胰蛋白胨Tryptone	OxOid公司OxOid Corporation
酵母粉yeast	OxOid公司OxOid Corporation
低熔点琼脂糖Low melting point agarose	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.
琼脂糖Agarose	西班牙BIOWEST公司BIOWEST Spain
IPIGIPIG	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.
L-阿拉伯糖L-arabinose	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.
75cm ²塑料培养瓶 75cm ² plastic culture flask	广州洁特生物过滤股份有限公司Guangzhou Jiete Biological Filtration Co., Ltd.
182cm ²塑料培养瓶 182cm ² plastic culture flask	广州洁特生物过滤股份有限公司Guangzhou Jiete Biological Filtration Co., Ltd.
1900cm ²塑料培养转瓶 1900cm ² plastic culture roller bottle	广州洁特生物过滤股份有限公司Guangzhou Jiete Biological Filtration Co., Ltd.
96孔细胞培养板96-well cell culture plate	广州洁特生物过滤股份有限公司Guangzhou Jiete Biological Filtration Co., Ltd.
DMEM培养基DMEM medium	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.
磷酸盐缓冲液(PBS)干粉Phosphate buffered saline (PBS) dry powder	北京索来宝生物科技有限公司Beijing Suolabao Biotechnology Co., Ltd.
甘油glycerin	天津是凯通化学试剂有限公司Tianjin is Kaitong Chemical Reagent Co., Ltd.
0.65μm囊式滤器0.65μm capsule filter	德国赛多利斯公司Sartorius
0.1μm中空纤维柱0.1μm hollow fiber column	美国GE公司American GE
蠕动泵Peristaltic pump	保定雷弗流体科技有限公司Baoding Refu Fluid Technology Co., Ltd.
荧光倒置显微镜Fluorescence inverted microscope	日本奥林巴斯公司Olympus Japan
二氧化碳培养箱Carbon dioxide incubator	日本三洋仪器公司Sanyo Instruments Corporation
生物安全柜Biological safety cabinet	上海力申科学仪器有限公司Shanghai Lishen Scientific Instrument Co., Ltd.

The primer sequences used in Example 1 are shown in Table 1-2:

Table 1-2

The herpes simplex virus HSV-1 strain used in the examples is derived from clinical isolates. Specifically, the HSV-1 genome was recombined into a bacterial artificial chromosome (BAC) to construct a plasmid BAC-HSV-1 that can shuttle between prokaryotic cells and eukaryotic cells, and through the use of BAC-HSV-1 The gene was modified and a recombinant HSV-1 strain was quickly obtained.

1.1 Construction of BAC-HSV-1 plasmid

The HSV-1 virus genome was used as a template to PCR amplify homologous arm fragments, and the amplified fragments were ligated and cloned into the bacterial artificial chromosome BAC C223 plasmid with the red reporter gene DsRed. Subsequently, the plasmid and the genomic DNA of the HSV-1 virus strain were co-transfected into Vero cells for homologous recombination by liposome transfection method, and the BAC-HSV-1 recombinant virus was purified by fluorescent reporter gene screening and extracted by Hirt method. The circular DNA of the virus was transferred to DH10B competent cells by electroporation, and the plasmid was extracted by shaking bacteria to obtain the BAC-HSV-1 plasmid.

1.2 Construction of recombinant HSV-1 virus

After obtaining the BAC-HSV-1 plasmid, two copies of the γ34.5 gene were deleted using RED recombination technology to obtain the BAC-HSV-1-del34.5 plasmid. The BAC-HSV-1-del34.5 plasmid was transfected into Vero cells by lipofection. After the cells showed disease, the Cre/loxP recombination system was used to delete the BAC sequence. After plaque purification, purified deletion two were obtained. A virus strain that copies the γ34.5 gene.

The oncolytic virus used in the embodiment of the present invention is an oncolytic virus obtained by the above method or an oncolytic virus in which different exogenous gene expression cassettes are recombined on the basis thereof. Specifically, in the present invention, the oncolytic viruses used in the embodiments of the present invention include:

(1) Oncolytic virus H1, a recombinant virus of human herpes simplex virus HSV-1 with a double copy of γ34.5 gene deleted, and its structure is shown in Figure 1A.

(2) Oncolytic virus H1-DsRed, the red fluorescent reporter gene DsRed expression cassette is recombined into the H1 oncolytic virus genome. The structure of the recombinant virus is shown in Figure 1B. The virus can be used to express red fluorescent protein in infected cells to detect the infection efficiency of oncolytic viruses on different cells.

(3) Oncolytic virus H1-IAA, any single immune activator gene expression cassette can be recombined into the H1 oncolytic virus genome. The expression cassette is composed of a strong CMV promoter, an IAA coding sequence, and a terminator. The structure of the recombinant virus is shown in Figure 1C.

(4) Recombinate the de-agonistic OX40scFv-Fc antibody gene expression cassette into the H1 oncolytic virus genome to obtain the oncolytic virus H1-OX40scFV-Fc. The structure of the recombinant virus is shown in Figure 1D, where the right side of the arrow is a schematic diagram of the expressed OX40scFv-Fc antibody.

(5) Recombined into the OX40mab full antibody sequence gene expression cassette in the H1 oncolytic virus genome to obtain oncolytic virus H1-OX40mab. The structure of the recombinant virus is shown in Figure 1E, and the right side of the arrow is a schematic diagram of the expressed OX40mab whole antibody.

(6) Recombinate the OX40L-Fc fusion protein gene expression cassette in the H1 oncolytic virus genome to obtain the oncolytic virus H1-OX40L-Fc. The structure of the recombinant virus is shown in Figure 1F, and the right side of the arrow is a schematic diagram of the structure of the expressed OX40L-Fc fusion protein. (OX40L is the polypeptide sequence of the extramembrane region of OX40 ligand)

1.3 Preparation of oncolytic virus

The Vero cells were cultured in a 37°C incubator with a cell culture spinner flask at 10 rpm. When the cell confluence reaches 80%-90%, discard the culture medium, add serum-free culture medium, and inoculate the oncolytic virus at MOI=0.005. The temperature of the incubator was adjusted to 33°C, the rotation speed was 10 rpm, the virus was adsorbed for 2 hours, and FBS with a final concentration of 2% was added to continue the culture. After 48-72 hours of oncolytic virus inoculation, observe the cytopathic effect (CPE). When all the cells are diseased and 1/3 are floating, the virus supernatant is harvested. Shake the flask to detach the cells from the flask wall. The cells were collected in a sterile container and stored in a -80°C refrigerator after repeated freezing and thawing three times.

1.4 Purification of oncolytic virus

The H1 virus stock solution after centrifugation was subjected to microfiltration with a 0.65 μm filter, and then to a 0.1 μm hollow fiber column for ultrafiltration. After ultrafiltration, 10% final concentration of glycerol was added as a protective agent, and the virus titer was measured using the TCID50 method. Adjust the titer of H1 virus solution to 1×10 ⁸ pfu/mL, then aliquot and store at -80°C.

1.5 Determination of oncolytic virus titer

The titer of oncolytic virus was determined by TCID50 method. Specifically, the density of Vero cells was adjusted to 1×10 ⁵ cells/mL, 100 μL/well were seeded on a 96-well cell culture plate, and cultured overnight in a 37° C., 5% CO ₂ incubator. Take the oncolytic virus solution, and use serum-free DMEM medium to make a gradient dilution of the virus to 10 ^-8 dilution. When the Vero cells reach about 80% (24 hours), discard the culture medium. Select 10 ^-4 to 10 ^-8 dilutions to infect cells, 100μL virus solution/well, set 6 replicate wells for each dilution, and set 6 wells as negative control (no virus, only 100μL serum-free DMEM) ). Place the cells at 37°C and 5% CO _{2 and} continue to culture for 48-72 hours. Observe the cytopathic condition of each well under a microscope, count the ratio of the number of positive wells at each dilution (the presence of CPE is positive), and calculate the virus titer.

Example 2 Infection and killing efficiency of oncolytic viruses to different tumor cells in vitro

The materials used in Example 2 are shown in Table 2-1:

项目project	来源source
MTT溶液MTT solution	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.
台盼蓝染色液Trypan Blue Staining Solution	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.
24孔细胞培养板24-well cell culture plate	广州洁特生物过滤股份有限公司Guangzhou Jiete Biological Filtration Co., Ltd.
1640培养基1640 medium	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.

DMEM/F12培养基DMEM/F12 medium	北京索莱宝科技有限公司Beijing Soleibao Technology Co., Ltd.
MccoY’S5A培养基MccoY’S5A medium	美国hyclone公司American hyclone company

Cultivate different tumor cells under the conditions of 5% CO2 and 37℃: mouse colon adenocarcinoma cell CT-26, mouse B cell lymphoma cell A20, mouse kidney cancer cell Renca, mouse breast cancer cell 4T1, small Murine lung cancer cell LLC; human ovarian cancer cell SKOV-3, human pancreatic cancer cell PANC-1, human nasopharyngeal cancer cell CNE, human prostate cancer cell PC-3, human melanoma cell A375. At the same time, African green monkey kidney cells Vero were cultured as a control. The types of media used are shown in Table 2-2.

肿瘤细胞类型Tumor cell type	培养条件Culture condition
小鼠结肠癌CT-26Mouse colon cancer CT-26	DMEM培养基、10％FBSDMEM medium, 10% FBS
小鼠淋巴瘤A20Mouse lymphoma A20	1640培养基、10％FBS1640 medium, 10% FBS
小鼠肾癌RencaMouse Renca Renca	1640培养基、10％FBS1640 medium, 10% FBS
小鼠乳腺癌4T1Mouse breast cancer 4T1	1640培养基、10％FBS1640 medium, 10% FBS
小鼠肺癌LLCMouse Lung Cancer LLC	1640培养基、10％FBS1640 medium, 10% FBS
人胰腺癌细胞PANC-1Human pancreatic cancer cell PANC-1	DMEM培养基、10％FBSDMEM medium, 10% FBS
人卵巢癌细胞SKOV-3Human ovarian cancer cell SKOV-3	MccoY’S5A培养基、10％FBSMccoY’S5A medium, 10% FBS
人鼻咽癌细胞CNEHuman nasopharyngeal carcinoma cell CNE	1640培养基、10％FBS1640 medium, 10% FBS
人黑色素瘤细胞A375Human melanoma cell A375	DMEM培养基、10％FBSDMEM medium, 10% FBS
人前列腺癌细胞PC-3Human prostate cancer cell PC-3	DMEM/F12培养基、10％FBSDMEM/F12 medium, 10% FBS
非洲绿猴肾细胞VeroAfrican Green Monkey Kidney Cell Vero	DMEM培养基、10％FBSDMEM medium, 10% FBS

2.1 Determination of oncolytic virus infection of different tumor cells in vitro

The efficiency of oncolytic virus infection on different mouse and human tumor cells in vitro was verified by fluorescence method. Tumor cells were seeded in 24-well plates, each well of 5x10 ⁴ cells were inoculated. Cultivate for 24 hours, when the cell confluence reaches 90%, randomly select three wells on each plate to count the cells, and use the average of the cell counts in the three wells as the number of cells per well on the plate. The medium was discarded, and the H1-DsRed virus was inoculated with MOI values of 0, 1, 5, and 10 respectively. After 2 hours of infection, each well was supplemented with 2% FBS medium to 500 μL, and placed in a 37°C incubator for culture. Set 3 parallels for each MOI value. Observe the pathological changes and fluorescence of each cell after the virus is infected for 48 hours, and take pictures under high magnification (200X-400X).

Figures 2A and 2B show the infection efficiency of the oncolytic virus H1 used in the present invention on different mouse and human tumor cells. According to Figures 2A and 2B, the oncolytic virus used in this application can infect a variety of tumor cells in vitro.

2.2 MTT method to detect the ability of oncolytic virus to kill mouse tumor cells in vitro

Tumor cells were collected from mice CT-26, A20, Renca, ID8, MFC, cells were seeded in 96-well plates, ¹⁰⁴ cells per well. The cells were cultured until they were basically fused, the culture medium was discarded, and the H1 virus infected the cells with the MOI value of 5. After 2 hours, 2% FBS medium was added to 100 μL, and 6 parallel wells were set for each cell. At the same time, set cell wells without virus as negative control wells, and culture wells with medium and no cells as blank control wells. At the end of the time point, add 20μL of MTT (5mg/mL) to each well and continue to incubate for 4 hours; discard the culture supernatant, add 150μL of DMSO to each well, incubate in a 37℃ incubator for 10 minutes, then shake with a shaker at low speed 2 Minutes to fully dissolve the crystals. Use a microplate reader to detect the absorbance value of each well at 490nm. The absorbance value reflects the number of living cells. The greater the absorbance value, the greater the number of live cells, and vice versa, the fewer live cells, in order to reflect the killing ability of the oncolytic virus to cells.

The in vitro killing ability of oncolytic viruses on different mouse tumor cells is shown in Figure 2C. The figure shows that the oncolytic virus in the patent of the present invention has extremely significant (P<0.01) killing ability on the above five mouse tumor cells in vitro.

3. Trypan blue staining method to detect the ability of oncolytic virus to kill human tumor cells in vitro

Take human tumor cells CNE, SKOV-3, PANC-1, MDA-MB-435S, and A549, respectively, and add them to a 24-well cell culture plate with 8×10 ⁴ cells per well, and 6 wells for each cell inoculation. Incubate for about 24 hours, and when the cell confluence reaches about 90%, randomly select three wells on the plate for digestion and count the cells. The average of the cell counts in the three wells is the number of cells per well. The culture medium was discarded, and the cells were inoculated according to the virus MOI of 1. Each cell was connected to 3 wells, and the remaining three wells of this kind of cells were control wells. Two hours after virus infection, 500 μL of DMEM medium containing 2% FBS was added, and the culture was continued at 37° C. and 5% CO2. Digest the cells in each well at 144h, pipette 90μL into a new EP tube, add 10μL 0.4% trypan blue staining solution, mix well and count with a cell counter. Blue are dead cells, and colorless are living cells. Staining and counting are all completed within 3 minutes, including:

The ratio of live cells = (total number of cells-number of dead cells)/total number of cells.

The in vitro killing ability of oncolytic viruses on different human tumor cells is shown in Figure 2D, which shows that the oncolytic virus of the present invention has different in vitro killing abilities on different human tumor cells, and has different effects on CNE, SKOV-3 and PANC-1. , MDA-MB-435S cells have extremely significant (P<0.01) in vitro killing ability, but the in vitro killing ability of A549 is not obvious.

Example 3 Anti-tumor immune effect of intratumor injection of oncolytic virus H1 and TLR7/8 and 9 agonist composition in nude mice

The source of materials is shown in Table 3:

3.1 Experimental method:

1) Establishment of mouse solid tumor tumor model

Female BALB/c nude mice aged 5-6 weeks were injected subcutaneously to establish a solid tumor tumor model in mice. Specifically, after disinfecting the right armpit of the mouse with 75% alcohol, the SK-OV-3 cell suspension was sucked with a 1 mL sterile syringe, and 200 μL was injected subcutaneously. The number of injected cells was 1×10 ⁷ /mouse.

2) Experimental grouping and administration

After the tumor model was established, randomly divided into groups and selected different immune activators for pharmacodynamic test. Among them, physiological saline (NS) was used as a negative control, and a combination of oncolytic virus H1 and granulocyte macrophage colony stimulating factor GM-CSF As a positive control (because GM-CSF and HSV-1 oncolytic virus T-VEC expressing GM-CSF have been proven to effectively activate anti-tumor immunity), with oncolytic virus H1 and R848 (Resiquimod, TLR7/8 agonist) Or a combination with CPG ODN (agonist of TLR9, refer to Invivogen CpG ODN class 3 sequence and thio modification) as the test product. Specifically, 4-6 days after transplantation of tumor cells, mice with tumor diameters of about 5-7 mm were selected for the test, and each group of 6 mice was divided into 4 groups, and each group of mice was labeled. Before administration, the mouse body weight and tumor size were measured. The first dose was given when the tumor size was about 5mm. Administration route: intratumoral injection, divided into 3 to 4 points (the syringe enters the lesion area through a single injection point, the injection point is the center of the tumor tissue and the edge of the tumor, a total of 3 to 4 points). Dosing frequency and time: once every other day, 3 times in total. Dosage: 50μL/only.

The composition of the applied composition in each group is shown in Table 4:

组别Group	供试品名称Name of test product	病毒浓度Virus concentration		药物浓度Drug concentration
11	生理盐水(NS)Normal saline (NS)	——	——
22	H1+GM-CSF(HG)H1+GM-CSF(HG)	8.5×10 ⁶pfu/只 8.5×10 ⁶ pfu/piece	0.1ug/只0.1ug/only
33	H1+R848(HR)H1+R848(HR)	8.5×10 ⁶pfu/只 8.5×10 ⁶ pfu/piece	2ug/只2ug/only
44	H1+CpG ODN(HC)H1+CpG ODN(HC)	8.5×10 ⁶pfu/只 8.5×10 ⁶ pfu/piece	100ug/只100ug/only

3) Tumor volume measurement

Every 3-4 days after administration, the long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper, the tumor volume was calculated according to V=(ab ² )/2, and the tumor growth curve was drawn. Relative tumor volume RTV: RTV=Vt/V0, Vt: tumor volume obtained by measuring tumor every day, V0: initial tumor volume (before administration). Relative tumor growth rate T/C%=average RTV of the administration group/average RTV of the negative control group×100%.

4) Efficacy evaluation

According to tumor volume and tumor weight, the drug effect was evaluated. Use SPSS17.0 statistical software to perform statistical analysis on the results. The data were compared by one-way analysis of variance, and P<0.05 was considered statistically significant.

The standard for evaluating the efficacy based on tumor volume is: if the relative tumor proliferation rate T/C% ≤ 40%, and the administration group RTV and the negative control group RTV are statistically processed P<0.05, then the composition can inhibit tumor proliferation Effect; if T/C%>40%, it has no inhibitory effect on tumor proliferation.

The standard for evaluating the efficacy based on tumor weight is: comparing the difference in tumor weight between groups to further calculate the tumor weight inhibition rate IRTW%, taking IRTW%>60% as the effectiveness reference index, the calculation formula is as follows:

IRTW(%)=(W _{negative control group-} W _{administration group} )/W _{negative control group} ×100%

3.2 Experimental results:

As shown in Figures 3A-3C, compared with the PBS group, the composition of H1+TLR agonist R848 or CpG ODN significantly inhibited tumor growth (p values were 0.002 and 0.002, respectively; relative tumor proliferation was 16.97% and 10.34%, respectively) ; Tumor weight inhibition rates were 82.28% and 90.90%). The anti-tumor effect is greater than the positive control H1 and GM-CSF combination.

Example 4 The anti-tumor immune effect of subcutaneous injection of HOC (oncolytic virus H1+OX40mab+ homologous tumor cells) composition in mice

The source of materials is shown in Chart 5:

溶瘤病毒H1Oncolytic virus H1	见实施例1和2See Examples 1 and 2
OX40mab单克隆抗体OX86OX40mab monoclonal antibody OX86	英国Absolute Antibody公司Absolute Antibody, UK
BALB/c小鼠BALB/c mice	河南省实验动物中心Henan Experimental Animal Center
小鼠结肠癌细胞CT-26Mouse colon cancer cell CT-26	北京昭衍新药研究中心股份有限公司Beijing Zhaoyan New Drug Research Center Co., Ltd.

4.1 Experimental method:

The experimental method of this example is different from that of Example 3: The mouse is a BALB/c mouse, the tumor cell is a mouse colon cancer cell CT-26, the injection volume is 100 μL, and the number of injected cells is 5×10 ⁵ / only. The experiment was divided into 2 groups, the administration site was under the left axilla as shown in Fig. 4A, and the administration dose was 100 μL/head. The rest of the content is the same as in Example 3. The composition of the applied composition in each group is shown in Table 6:

4.2 Experimental results:

As shown in Figures 4A and 4B, compared with the PBS group, the H1+OX40mab monoclonal antibody composition significantly inhibited tumor growth (p<0.05), the relative tumor proliferation rate was 21.73%, and the tumor suppression rate was 78.82%. .

Example 5 Anti-tumor immune effect of HOR (H1+OX40mab+R848) composition intratumor injection in mice

The source of materials is shown in Table 7:

溶瘤病毒H1Oncolytic virus H1	见实施例1和2See Examples 1 and 2
OX40mab单克隆抗体OX86OX40mab monoclonal antibody OX86	英国Absolute Antibody公司Absolute Antibody, UK
Resiquimod R848Resiquimod R848	Tocris BioscienceTocris Bioscience
BALB/c小鼠BALB/c mice	河南省实验动物中心Henan Experimental Animal Center
小鼠结肠癌细胞CT-26Mouse colon cancer cell CT-26	北京昭衍新药研究中心股份有限公司Beijing Zhaoyan New Drug Research Center Co., Ltd.

5.1 Experimental method

The mouse used in this example is a BALB/c mouse, the tumor cell is a mouse colon cancer cell CT-26, the grafting site is the left axillary subcutaneous and the right axillary subcutaneous, the injection volume is 100 μL, and the number of cells injected is 5 ×10 ⁵ /only. The experiment was divided into 2 groups, the administration site was inside the left axillary tumor, and the administration dose was 100 μL/mouse. The operation is the same as in Example 3. The right axillary tumor was injected with PBS as the control group PBS(I), the left axillary was not injected with PBS as the control group PBS(N), the right axillary tumor was injected with HOR as the experimental group HOR(I), and the left axillary was not injected with the tumor. Injection of HOR is the experimental group HOR(N).

The composition of the applied composition in each group is shown in Table 8:

5.2 Experimental results

As shown in Figures 5A-5D, compared with the PBS group, intratumoral injection of the HOR composition (oncolytic virus H1+OX40mab monoclonal antibody+R848) can completely clear the tumor on the injection side, and can eliminate the contralateral side that has not been injected. Tumor (p value = 0.000), the relative tumor proliferation rate is 0, and the tumor suppression rate is 100%.

Example 6 The anti-tumor immune effect of subcutaneous injection of OR (OX40mab monoclonal antibody + R848) + homologous tumor tissue lysate (TL) composition in mice

The main materials and instrument sources are shown in Table 9:

OX40mab单克隆抗体OX86OX40mab monoclonal antibody OX86	英国Absolute Antibody公司Absolute Antibody, UK
R848R848	Tocris BioscienceTocris Bioscience
BALB/c小鼠BALB/c mice	河南省实验动物中心Henan Experimental Animal Center
小鼠结肠癌细胞CT-26Mouse colon cancer cell CT-26	北京昭衍新药研究中心股份有限公司Beijing Zhaoyan New Drug Research Center Co., Ltd.
匀浆器Homogenizer	上海净信实业发展有限公司Shanghai Jingxin Industrial Development Co., Ltd.
超声波粉碎仪Ultrasonic crusher	宁波新芝生物科技股份有限公司Ningbo Xinzhi Biological Technology Co., Ltd.

6.1 Experimental method:

In this example, OR (OX40mab monoclonal antibody + R848) + homologous tumor tissue lysate composition was used, and the remaining operations were the same as in Example 4.

Homogenizer processing method of tumor tissue lysate: The homologous tumor tissue lysate is taken from mouse tumor cell CT-26 transplantation and the tumor tissue is processed by the homogenizer. Specifically, the tumor tissue successfully transplanted with mouse tumor cells CT-26 was weighed and cut with surgical scissors to a size of about 1 mm ³ , and PBS was added in an amount of 3 mL/g. Transfer the tumor tissue slurry to a bacterial culture tube and place it in an ice bath for 10 minutes. Use a homogenizer to homogenize the cut tumor tissue several times at a speed of 30,000 rpm, and stop for 2 minutes to cool down every 1 minute of homogenization for a total of 5 minutes.

Tumor tissue lysate processing method: add appropriate amount of PBS to the tumor tissue lysate processed by the homogenizer to adjust to a final concentration of 5mL/g, and ultrasonically disrupt the tissue homogenate after constant volume with an ultrasonic cell disruptor (Working time is 2 seconds, intermittently 3 seconds, a total of 12 times that is 1 minute, the culture tube containing tissue slurry has been kept in an ice bath). The tumor tissue lysate with completely disrupted cells was aliquoted and stored at -80°C for later use.

The composition of the applied composition in each group is shown in Table 10:

6.2 Experimental results:

As shown in Figures 6A-6D, compared with the PBS group, subcutaneous injection of the CT-26 cell lysate+OX40mab+R848 composition can completely eliminate the transplanted CT-26 tumor (p value is 0.000, p<0.001), and inhibit The tumor rate can reach 100%.

Example 7. The anti-tumor immune effect of subcutaneous injection of OR(OX40mab+R848)+ homologous mouse lymphoma A20 lysate composition

Except that the tumor cells are mouse B-cell lymphoma cells A20, the rest are the same as in Example 6. The sources of materials and equipment are shown in Table 11:

OX40mab单克隆抗体OX86OX40mab monoclonal antibody OX86	英国Absolute Antibody公司Absolute Antibody, UK
R848R848	Tocris BioscienceTocris Bioscience
BALB/c小鼠BALB/c mice	河南省实验动物中心Henan Experimental Animal Center
小鼠B细胞淋巴瘤细胞A20Mouse B cell lymphoma cell A20	北京昭衍新药研究中心股份有限公司Beijing Zhaoyan New Drug Research Center Co., Ltd.
匀浆器Homogenizer	上海净信实业发展有限公司Shanghai Jingxin Industrial Development Co., Ltd.
超声波粉碎仪Ultrasonic crusher	宁波新芝生物科技股份有限公司Ningbo Xinzhi Biological Technology Co., Ltd.

7.1 Experimental method: The experimental method of this example is different from Example 6 in that the tumor cells are mouse B-cell lymphoma cells A20, and the other contents are the same as in Example 6.

The composition of the applied composition in each group is shown in Table 12:

7.2 Experimental results:

As shown in Figures 7A-7D, compared with the PBS group, subcutaneous injection of the A20 cell lysate+OX40mab+R848 composition significantly inhibited tumor growth (p value is 0.000, p<0.001), and the relative tumor proliferation rate was 10.68 %, the tumor suppressor rate was 88.43%.

Example 8 The anti-tumor immune effect of subcutaneous injection of OR (OX40mab+R848) and homologous mouse lung cancer LLC lysate composition

Except that the tumor cells are mouse lung cancer LLC, the rest are the same as in Examples 6 and 7. The sources of materials and equipment are shown in Table 13:

OX40mab单克隆抗体OX86OX40mab monoclonal antibody OX86	英国Absolute Antibody公司Absolute Antibody, UK
R848R848	Tocris BioscienceTocris Bioscience
BALB/c小鼠BALB/c mice	河南省实验动物中心Henan Experimental Animal Center
小鼠肺癌细胞LLCMouse Lung Cancer Cell LLC	北京昭衍新药研究中心股份有限公司Beijing Zhaoyan New Drug Research Center Co., Ltd.
匀浆器Homogenizer	上海净信实业发展有限公司Shanghai Jingxin Industrial Development Co., Ltd.
超声波粉碎仪Ultrasonic crusher	宁波新芝生物科技股份有限公司Ningbo Xinzhi Biological Technology Co., Ltd.

8.1 Experimental method:

The experimental method of this example is different from Example 6 in that the mouse is a C57BL/6 mouse, and the tumor cell is a mouse lung cancer cell LLC, and the rest is the same as in Example 6.

The composition of the applied composition in each group is shown in Table 14:

8.2 Experimental results:

As shown in Figures 8A-8D, compared with the PBS group, the subcutaneous injection of LLC cell lysate + OX40mab + R848 significantly inhibited tumor growth (p value 0.001), the relative tumor proliferation rate was 30.00%, and the tumor weight The tumor inhibition rate was 90.27%).

Example 9 Long-acting immune effect of subcutaneous injection of OR(OX40mab+R848)+ homologous tumor cell lysate composition

Source of materials and equipment: same as in Example 6

9.1 Experimental method

First, use 3 immunizations to completely remove the tumor, and then re-graft CT-26 tumor cells, 5× ^{10 5} cells per mouse, and observe the tumor formation rate of the transplanted tumor to study the prevention of the transplanted tumor metastasis caused by the immune memory caused by the composition of the application effect. The specific operation was the same as in Example 6, and the mice were pre-immunized three times and then transplanted with tumors in the same way.

The composition of the composition used in each group is shown in Table 16:

9.2 Experimental results

As shown in Figure 9, after three subcutaneous injections of the CT-26 cell lysate+OX40mab+R848 composition, the success rate of re-transplanting tumors was 28.57%, while that of the control group was 100%. In addition, normal mice were pre-immunized three times and then grafted with CT-26 tumor cells and still obtained similar results (data not shown here). The results show that OX40mab+R848+ autologous tumor lysate can produce long-term anti-tumor immune memory after immunization, thereby preventing tumor metastasis.

Example 10. Comparison of the anti-tumor immune effect of subcutaneous injection of OR(OX40mab+R848)+ fresh homologous tumor cell lysate composition or formalin-fixed homologous tumor cell lysate composition

The materials and equipment were the same as in Example 6.

10.1 Experimental method:

The experimental method of this example is different from Example 6 in that the experiment is divided into 3 groups, and the cell lysate used in the third group is derived from CT-26 tumor tissue fixed in formalin for 24 to 48 hours. Specifically, the tumor tissue fixed in formalin was cut into small pieces with a size of about 1 mm ³ with surgical scissors, and washed with PBS to remove residual formalin. The homogenizer and ultrasonic disrupter were then processed to obtain tumor cell lysates. The specific operation content is similar to Embodiment 6. The composition of the composition used in each group is shown in Table 18:

10.2 Experimental results

As shown in Figure 10, compared with the PBS control group, the subcutaneous injection of the lysate composition of OX40mab+R848+formalin-fixed/unfixed CT-26 tumor tissue significantly inhibited tumor growth (p<0.05), And there is no significant difference between the two (p>0.05).

Claims

A composition for inducing immune cell activity in subjects in need, which comprises:

(1) A tumor cell lysate autologous to the subject,

(2) OX40 agonist, and

(3) TLR agonists.
The composition of claim 1, wherein the OX40 agonist comprises OX40 antibody, OX40 ligand fusion protein, and any combination thereof.
The composition according to claim 1 or 2, wherein the TLR agonist comprises CpG oligonucleotides (oligodeoxynucleotides, CpG ODN), Resiquimod (Resiquimod, R848), Imiquimod (Imiquimod, R837) , DsRNA and combinations thereof.
The composition according to any one of claims 1 to 3, wherein the immune cell activity is selected from: proliferation of immune cells; differentiation, dedifferentiation or transdifferentiation of immune cells; release of immune cell cytokines; Cytotoxicity; movement and/or transportation of immune cells; exhaustion of immune cells and combinations thereof.
The composition according to any one of claims 1-4, wherein the immune cells are lymphocytes.
The composition according to any one of claims 1-5, wherein the lymphocytes include T cells and NK cells.
The composition according to any one of claims 1 to 6, wherein the subject's autologous tumor cell lysate is derived from the subject's autologous tumor cell or its progeny cells.
The composition according to any one of claims 1 to 6, wherein the subject's autologous tumor cell lysate is derived from autologous tumor cells of one or more lesions of the subject or Progeny cells.
The composition according to any one of claims 1 to 6, wherein the subject's autologous tumor cell lysate is derived from autologous tumor cells of one or more lesions of the subject or Progeny cells.
The composition according to any one of claims 1-6, wherein the subject's autologous tumor cell lysate is derived from autologous tumor cells of one or more tissues of the subject or Its progeny cells.
The composition of any one of claims 1-10, wherein the subject's autologous tumor cell lysate is derived from 1×10 4 to 1 × 10 tumor cells.
The composition of any one of claims 1-11, wherein the composition is administered to the subject in situ or ex situ.
The composition according to claim 12, wherein the ex situ administration includes subcutaneous injection, intramuscular injection, intraperitoneal injection, thoracic injection, intravenous injection, arterial injection, and combinations thereof.
The composition according to any one of claims 1-13, wherein the lysate of tumor cells is obtained by a method selected from the group consisting of homogizing, sonication or a combination of the two.
The composition according to any one of claims 1-14, wherein the lysate of tumor cells is derived from fresh tumor tissue or frozen tumor tissue or tumor tissue fixed with fixative.
The composition according to claim 15, wherein the fixing solution comprises formalin, formaldehyde, paraformaldehyde, glutaraldehyde, ethanol, or any combination thereof.
The composition according to any one of claims 1-16, wherein the composition further comprises one or more immunomodulators.
The composition according to claim 17, wherein the immunomodulatory comprises agonists of CD27, CD28, CD40, CD122, CD137 and GITR, A2AR, CD276, VTCN1, BTLA, CTLA-4, IDO, LAG3, KIR, NOX2 , PD-1, TIM-3, VISTA, SIGLEC7 and SIGLEC9 antagonists, cytokines IL-2, IL-12, IL15, IL6, IL18, IFN-ɑ, TNF-β, IFN-γ, GM-CSF, M-CSF, and any combination thereof.
A method for treating tumors by inducing immune cell activity in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the composition of any one of claims 1-18.
The method according to claim 19, wherein the treatment comprises regressing the tumor, inhibiting tumor progression and/or metastasis, preventing its recurrence and/or metastasis.
The method according to claim 19 or 20, wherein the method is used to treat a condition in which the subject is selected from the group consisting of lung cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colon cancer, rectal cancer , Colorectal cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, kidney cancer, leukemia, lymphocytic leukemia, myeloid leukemia, mixed cell leukemia, multiple myeloma, liver cancer, gallbladder cancer, lymphoma, melanoma, Mesothelioma, medulloblastoma, oral cancer, nasopharyngeal cancer, laryngeal cancer, thyroid cancer, mediastinal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, tracheal cancer, and vulvar cancer.
A kit for inducing immune cell activity in a subject in need, comprising the composition according to any one of claims 1-21 and instructions, wherein the instructions indicate to obtain from the subject and /Or a method for preparing autologous tumor cells, preparing a lysate of the tumor cell, and co-administering the tumor cell lysate with an OX40 agonist and a TLR agonist to the subject.
A composition for inducing immune cell activity in subjects in need, which comprises:

(1) Oncolytic virus,

(2) OX40 agonist, and

(3) TLR agonist; or

(1) Oncolytic viruses expressing OX40 agonists, and

(2) TLR agonists.
23. The composition of claim 23, wherein the oncolytic virus is a modified virus.
The composition according to claim 24, wherein the oncolytic virus is Herpes simplex virus.
The composition according to claim 25, wherein the herpes simplex virus is selected from HSV-1 or HSV-2.
The composition according to claim 26, wherein the oncolytic virus is herpes simplex virus HSV-1 modified to lack the function of γ34.5.
The composition of any one of claims 23-27, wherein the oncolytic virus is modified to encode the OX40 agonist or the TLR agonist or a combination thereof.
The composition according to any one of claims 23-28, wherein the amount of oncolytic virus in the composition is 1×10 5 -1× 10 9 pfu.
The composition of any one of claims 23-29, wherein the composition is administered to the subject in situ.
The composition of claim 30, wherein the in situ administration comprises intratumoral injection, peritumoral injection, and combinations thereof.
A composition for inducing immune cell activity in subjects in need, which comprises:

(1) Tumor cells autologous to the subject,

(2) Oncolytic virus, and

(3) One or more immune activators.
A method for treating tumors by inducing immune cell activity in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of the composition according to any one of claims 23-32.
The method according to claim 33, wherein the treatment comprises regressing the tumor, inhibiting tumor progression and/or metastasis, preventing its recurrence and/or metastasis.
A kit for inducing immune cell activity in a subject in need, comprising the composition according to any one of claims 23-32 and instructions, wherein the instructions instruct to administer the composition to the Subject to induce immune activity.