Efficient oncolytic virus as well as composition and application thereof

Abstract

The invention discloses an efficient oncolytic virus as well as a composition and application thereof, specifically, the composition comprises an oncolytic virus and an immunoregulatory factor, and the immunoregulatory factor comprises CXCL16. The composition disclosed by the invention shows a better treatment effect in a tumor model, and can induce stronger anti-tumor immune response.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a highly effective oncolytic virus and a composition thereof, and applications thereof. Background Art

[0002] Oncolytic viruses can replicate specifically in tumor cells and kill tumor cells without damaging normal cells, making them a potential cancer treatment. In addition to direct oncolytic effects, oncolytic viruses can also induce immunogenic cell death and promote anti-tumor immune responses. Oncolytic viruses use a variety of mechanisms to enter host cells, including cell surface receptors overexpressed on cancer cells. Some viruses are able to enter cells through multiple receptors, while some receptors can promote the entry of multiple types of viruses. Oncolytic viruses enter cells through endocytosis, membrane fusion, and syncytium formation. Some oncolytic viruses are known to have the characteristic of preferentially targeting cancer cells, but their exact cell surface receptors have not yet been determined.

[0003] Oncolytic viruses currently used in clinical practice have a natural tropism for proteins abnormally expressed on the surface of cancer cells. For example, HSV-1 recognizes cell surface receptors including the herpes simplex virus entry mediator (HVEM) and certain selective lectins; measles virus, especially the Edmonston strain, uses the surface receptor CD46 to enter cells. CD46 is usually used by cells to evade the complement pathway for clearance by the immune system and is overexpressed in cancer cells; coxsackievirus can enter cells through intercellular adhesion molecule 1 (ICAM-1, also known as CD54) and decay accelerating factor (DAF, also known as CD55), which may be overexpressed in cancers such as multiple myeloma, melanoma and breast cancer; as a member of the enterovirus family, echovirus has a higher specificity for ovarian cancer cells because it uses the I domain of integrin α2β1 to enter cells. These receptors are overexpressed on ovarian cancer cells; poliovirus enhances its specificity for various cancers by targeting CD155, a receptor that may weaken the anti-tumor response of NK cells and is overexpressed in some cancer cells. In addition, oncolytic viruses can also be designed to directly target receptors uniquely expressed on the surface of cancer cells. For example, adenovirus Ad5 / 3-Δ24 is an oncolytic virus that has been modified to bind to integrins that are highly expressed on ovarian cancer cells and is currently being studied in clinical trials. In addition, measles virus has been designed to express a single-chain antibody that recognizes carcinoembryonic antigen (CEA), a tumor antigen that is selectively expressed only in certain adenocarcinomas.

[0004] Although oncolytic viruses are considered promising cancer immunotherapy, due to the heterogeneity of the tumor microenvironment, oncolytic virus therapy drugs have only produced lasting responses in a subset of cancer patients. In response to this situation, researchers have modified oncolytic viruses to enhance their anti-tumor effects. However, a large number of modifications to armed oncolytic viruses, such as which cytokines to add to oncolytic viruses, are simple combinations based on existing knowledge and do not have sufficient scientific support. In addition, there are a large number of cytokines that can be added, and strategies for combining these cytokines will lead to the problem of "combinatorial explosion." The production of a large number of modified oncolytic viruses does not necessarily produce the expected results. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a new method for treating tumors by combining oncolytic viruses and immunomodulatory factors.

[0006] In order to achieve this purpose, the present invention adopts the following technical solutions:

[0007] A first aspect of the present invention provides an oncolytic virus composition, wherein the oncolytic virus composition comprises an oncolytic virus and an immunomodulatory factor, wherein the immunomodulatory factor comprises CXCL16.

[0008] In some embodiments, the immunomodulatory factors further include CCL5, GM-CSF, IL-12 and / or IFN-γ.

[0009] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0010] As a preferred embodiment, the immunomodulatory factor is a combination of CXCL16, CCL5, GM-CSF, IL-12 and IFN-γ.

[0011] In some embodiments, the immunomodulatory factors further include CCL4, CXCL9, IL-2, IL-10 and / or IL-18.

[0012] In some embodiments, the oncolytic virus composition further comprises a FLT3 agonist, a CD40 agonist, a PD-1 antagonist, a PD-L1 antagonist and / or a CTLA-4 antagonist.

[0013] In some embodiments, the FLT3 agonist comprises a recombinant oncolytic virus encoding FLT3L.

[0014] In some embodiments, the CD40 agonist is a CD40 agonist antibody.

[0015] In some embodiments, the PD-1 antagonist is a PD-1 antagonist antibody.

[0016] In some embodiments, the PD-L1 antagonist is a PD-L1 antagonist antibody.

[0017] In some embodiments, the CTLA-4 antagonist is a CTLA-4 antagonist antibody.

[0018] In some embodiments, the oncolytic virus comprises herpes simplex virus (type 1 or type 2), adenovirus, vaccinia virus, measles virus, Newcastle disease virus, vesicular stomatitis virus, reovirus, coxsackievirus, poliovirus, alphavirus, yellow fever virus, West Nile virus.

[0019] As a preferred embodiment, the oncolytic virus is herpes simplex virus (type 1).

[0020] A second aspect of the present invention provides a recombinant oncolytic virus, wherein the recombinant oncolytic virus contains a nucleic acid sequence encoding an immunomodulatory factor, and the immunomodulatory factor includes CXCL16.

[0021] In some embodiments, the immunomodulatory factors further include CCL5, GM-CSF, IL-12 and / or IFN-γ.

[0022] In some embodiments, the immunomodulatory factors further include CCL4, CXCL9, IL-2, IL-10 and / or IL-18.

[0023] The third aspect of the present invention provides a method for preparing the recombinant oncolytic virus according to the second aspect of the present invention, the method comprising:

[0024] Introducing a nucleic acid sequence encoding an immunomodulatory factor into the oncolytic virus to be modified to obtain the recombinant oncolytic virus;

[0025] Wherein, the immune regulatory factors include CXCL16.

[0026] In some embodiments, the immunomodulatory factors further include CCL5, GM-CSF, IL-12 and / or IFN-γ;

[0027] In some embodiments, the immunomodulatory factors further include CCL4, CXCL9, IL-2, IL-10 and / or IL-18;

[0028] In some embodiments, the oncolytic virus to be modified includes herpes simplex virus (type 1 or type 2), adenovirus, vaccinia virus, measles virus, Newcastle disease virus, vesicular stomatitis virus, reovirus, coxsackievirus, poliovirus, alphavirus, yellow fever virus, and West Nile virus.

[0029] As a preferred embodiment, the oncolytic virus to be modified is herpes simplex virus (type 1).

[0030] In some embodiments, the nucleic acid sequence encoding the immunomodulatory factor is inserted into or replaces the ICP 34.5 locus of the oncolytic virus to be modified.

[0031] In some embodiments, the encoded immunomodulatory factors are a combination of CXCL16, CCL5, GM-CSF, IL-12 and IFN-γ.

[0032] As a preferred embodiment, the method comprises:

[0033] (1) introducing a nucleic acid sequence encoding CCL5 into the ICP 34.5 locus of the oncolytic virus to be modified;

[0034] (2) Nucleic acid sequences encoding IFN-γ, CXCL16, GM-CSF and IL-12 are sequentially inserted into the locus of the UL26-UL27 gene interval to obtain a recombinant oncolytic virus.

[0035] The fourth aspect of the present invention provides a drug combination, which comprises a first component and a second component, wherein the first component comprises the recombinant oncolytic virus described in the second aspect of the present invention, and the second component comprises a FLT3 agonist, a CD40 agonist, a PD-1 antagonist, a PD-L1 antagonist and / or a CTLA-4 antagonist.

[0036] In some embodiments, the FLT3 agonist comprises a recombinant oncolytic virus encoding FLT3L.

[0037] In some embodiments, the CD40 agonist is a CD40 agonist antibody.

[0038] In some embodiments, the PD-1 antagonist is a PD-1 antagonist antibody.

[0039] In some embodiments, the PD-L1 antagonist is a PD-L1 antagonist antibody.

[0040] In some embodiments, the CTLA-4 antagonist is a CTLA-4 antagonist antibody.

[0041] The fifth aspect of the present invention provides a pharmaceutical preparation, which comprises the oncolytic virus composition described in the first aspect of the present invention, the recombinant oncolytic virus described in the second aspect of the present invention, or the drug combination described in the fourth aspect of the present invention.

[0042] In some embodiments, the dosage form of the pharmaceutical preparation includes lyophilized powder injection, injection, tablet, capsule or drop.

[0043] The oncolytic virus composition described in the first aspect of the present invention, the recombinant oncolytic virus provided in the second aspect of the present invention, the drug combination provided in the fourth aspect of the present invention, or the drug preparation described in the fifth aspect of the present invention are used in the preparation of drugs for preventing or treating tumors.

[0044] In some embodiments, the tumor comprises a solid tumor. In the present invention, the solid tumor includes but is not limited to pancreatic cancer, colon cancer, melanoma, glioma, head and neck tumor, esophageal cancer, gastric cancer, liver cancer, breast cancer, sarcoma, prostate cancer, and lung cancer.

[0045] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier.

[0046] The sixth aspect of the present invention provides a method for screening immunomodulatory factors that can enhance the anti-tumor ability of oncolytic viruses, the method comprising: screening for differentially expressed immunomodulatory factors between tumor tissues that respond to oncolytic virus therapy and tumor tissues that do not respond to oncolytic virus therapy.

[0047] As a preferred embodiment, single-cell transcriptome sequencing of intratumoral immune cells and / or high-throughput detection of intratumoral cytokines are used to screen for differentially expressed immune regulatory factors between tumor tissues that respond to oncolytic virus therapy and tumor tissues that do not respond to oncolytic virus therapy.

[0048] As a preferred embodiment, the method further comprises constructing a Tet-On system to evaluate the differentially expressed immunomodulatory factors.

[0049] In the above methods, differential gene expression and Luminex multifactor immune detection are the basis for guiding the selection of candidate factors. By analyzing the proportion of immune cells, activation status, communication, cytokine expression, etc. in tumor tissues between the response group and the non-response group, the characteristics of the tumor immune microenvironment related to response treatment are obtained, and candidate immune factors that can promote oncolytic virus therapy are screened.

[0050] The Tet-On system is a system that can induce the expression of target proteins. It is based on the specific Tet resistance operator in the Escherichia coli Tn10 transposon. This system can change the conformation of regulatory proteins through inducers such as Tet, thereby controlling the expression of target proteins. Its working principle is as follows: rtTA is a fusion protein of rTetR and VP16. When Dox is not present, rtTA cannot bind to TRE, resulting in the shutdown of downstream gene expression; when Dox is present, the conformation of rtTA changes, and rtTA can bind to TRE, thereby starting the expression of downstream genes (such as Figure 6 Currently, this system has been widely used in the research of gene function and gene therapy.

[0051] The seventh aspect of the present invention provides a method for preventing or treating tumors, the method comprising administering to a subject a therapeutically effective amount of the oncolytic virus composition described in the first aspect, the recombinant oncolytic virus described in the second aspect of the present invention, the drug combination described in the fourth aspect of the present invention, or the drug preparation described in the fifth aspect of the present invention.

[0052] In some embodiments, the oncolytic virus composition described in the first aspect of the present invention, the recombinant oncolytic virus described in the second aspect of the present invention, the drug combination described in the fourth aspect of the present invention, or the drug preparation described in the fifth aspect of the present invention can be administered by intravenous, intraperitoneal or intratumoral injection.

[0053] Beneficial effects:

[0054] The present invention improves the response rate of the original oncolytic virus therapy by loading the screened immunomodulatory factors and antibody combination therapy on the oncolytic virus, and can provide a new treatment method for patients who do not respond to the original therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 A flow chart of oncolytic virus design strategies based on OV therapy tolerance mechanisms is shown;

[0056] Figure 2 The structural diagram of the oncolytic virus drug oHSV (also referred to herein as OV) is shown;

[0057] Figure 3 Tumor growth curves and tumor weights of the oHSV-treated Pan02-HVEM model are shown;

[0058] Figure 4 The differences between the selected specific immune cell types and ligand-receptor pairs in the response group and the non-response group are shown, wherein the upper bubble chart is the response group and the lower bubble chart is the non-response group;

[0059] Figure 5The cytokine expression in tumor tissues of the Pan02-HVEM pancreatic cancer model response group and non-response group is shown;

[0060] Figure 6 The working principle of the Tet-On system is shown;

[0061] Figure 7 The Tet-On system is shown to evaluate the efficacy of CCL family chemokines combined with OV;

[0062] Figure 8 The Tet-On system is shown to evaluate the efficacy of CXCL family chemokines combined with OV;

[0063] Fig. 9 The Tet-On system is shown to evaluate the efficacy of T / NK cell activation-related cytokines combined with OV;

[0064] Fig.10 The Tet-On system is shown to evaluate the efficacy of macrophage activation-related factors combined with OV;

[0065] Fig.11 The structural diagrams of 6 single-factor oncolytic viruses are shown;

[0066] Fig.12 The results of the anti-tumor ability test of single-factor oncolytic virus are shown;

[0067] Fig.13 The results of the first round of “OV-six” in vivo efficacy evaluation are shown;

[0068] Fig.14 The results of the second round of “OV-five” in vivo efficacy evaluation are shown;

[0069] Fig.15 A diagram showing the structure of the OV-5A virus;

[0070] Fig.16 The results of ELISA detection of the expression of CCL5, CXCL16, IFN-γ, IL-12 and GM-CSF cytokines in the local tumor are shown;

[0071] Fig.17 The results of OV-5A efficacy validation are shown;

[0072] Fig.18 The results of monitoring the body weight of mice showing the efficacy of OV-5A in treating pancreatic cancer;

[0073] Fig.19 The results of liver injury detection in pancreatic cancer-bearing mice treated with OV-5A are shown;

[0074] Fig. 20The results of H&E staining of liver, lung, kidney and spleen organs after OV-5A treatment of pancreatic tumors are shown;

[0075] Fig.21 The biodistribution detection of OV-5A virus is shown;

[0076] Fig. 22 OV-5A treatment of bilateral tumor model is shown;

[0077] Fig.23 The results showed that OV-5A treatment induced anti-tumor immune memory and prevented tumor recurrence.

[0078] Fig.24 The efficacy of OV-5A in various tumor models is shown;

[0079] Fig.25 The structure diagram of human single-factor oncolytic virus is shown;

[0080] Fig.26 The ELISA test of the cytokine expression capacity of human single-factor oncolytic virus is shown;

[0081] Fig. 27 The results of the killing experiment of tumor organoids by oncolytic virus and TIL co-culture are shown;

[0082] Fig.28 The results of the killing experiment of tumor organoids by co-culturing oncolytic viruses and macrophages are shown;

[0083] Fig.29 The experimental process of using PBMC-PDX model to verify the efficacy of OV-5A is shown;

[0084] Fig.30 The results of monitoring tumor growth in an experiment using a PBMC-PDX model to validate the efficacy of OV-5A are shown;

[0085] Fig.31 The figure shows the results of monitoring body weight changes in an experiment using a PBMC-PDX model to verify the efficacy of OV-5A;

[0086] Fig.32 The heat map of T cell immune checkpoint expression in the CR and PR groups after OV-5A treatment is shown;

[0087] Fig.33 The effect of OV-5A treatment on PD-L1 expression in tumor cells and myeloid cells in the tumor microenvironment is shown;

[0088] Fig.34 Shows the interaction between PD-L1 signaling pathway and immune cells;

[0089] Fig.35 The interaction relationship between immune cells in the CR and PR groups is shown;

[0090] Fig.36 A diagram showing the interaction of the CD40 signaling pathway among immune cells;

[0091] Fig.37 A diagram showing the interaction of the FLT3 signaling pathway among immune cells;

[0092] Fig.38 A diagram showing the structure of the OV-mFlt3L virus;

[0093] Fig.39 The pharmacodynamic results of OV-5A in combination with αCD40 antibody and OV-mFlt3L are shown;

[0094] Fig.40 The therapeutic effect of OV-5A combined with OV-mFlt3L, αCD40 and αPD-1 on pancreatic cancer in mice is shown;

[0095] Fig.41 The therapeutic effect of OV-5A in combination with OV-mFlt3L, αCD40 and αPD-1 on mouse colon cancer is shown. DETAILED DESCRIPTION

[0096] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0097] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0098] Unless otherwise specified, experimental methods, detection methods, and preparation methods not described in detail in the present invention all adopt conventional techniques in the technical field.

[0099] In order to further explore the drug resistance mechanism of oncolytic virus oHSV (hereinafter referred to as OV) treatment, the present invention performed scRNA-seq and intratumoral cytokine omics detection in a mouse pancreatic cancer model to clarify the gene expression, signaling pathways, and immune cell composition and functional changes of oHSV treatment resistance. The aim is to rationally design strategies for inserting different immunostimulatory factors and combinations into the oncolytic virus backbone through data mining of drug resistance mechanisms, so as to induce stronger anti-tumor immune responses.

[0100] Figure 1 The flow chart of the oncolytic virus design strategy based on the OV treatment tolerance mechanism of the present invention is shown. OV treated the tumors of the response group and the non-response group of Pan02-HVEM or MC38 tumor-bearing mice, and isolated immune cells (CD45 + ) for single-cell sequencing; at the same time, the proteins obtained from tumor tissue homogenate were subjected to proteomic detection (Luminex platform).

[0101] The screened factor combination included CCL5, CXCL16, GM-CSF, IL-12 and IFN-γ.

[0102] Combination therapy: Herpes simplex virus loaded with CCL5, CXCL16, GM-CSF, IL-12 and IFN-γ is used in combination with FLT3L, CD40 antibodies and PD-1 antibodies.

[0103] Example 1 Screening of immunomodulatory factors capable of enhancing the anti-tumor ability of oncolytic viruses

[0104] The OV used in the present invention is based on the wild-type HSV-1 strain as the basic skeleton and is modified by gene editing technology. The wild-type HSV-1 virus is isolated from a patient with oral herpes infection. After gene sequencing analysis, it is consistent with the HSV-1F strain sequence of ATCC (Cat. No.: VR-733). First, the CRISPR / Cas9 method is used to knock out the ICP 34.5 (double copy, encoding neurotoxic molecules) and ICP 47 (single copy, encoding viral proteins) sequences, and at the same time, the double copy of the ICP 34.5 site is replaced with a gene sequence encoding green fluorescent protein (GFP) to obtain the oncolytic virus oHSV ( Figure 2 ), similar to Delytact, an oncolytic virus drug developed by Japan's Daiichi Sankyo Co., which was approved by the FDA in 2021.

[0105] The mouse pancreatic cancer cell line Pan02-HVEM is a modified Pan02 cell line that stably expresses the HVEM receptor. Vero cells are African green monkey kidney cells.

[0106] 1. Establishment of a drug-resistant model of oncolytic virus therapy in subcutaneous tumor-bearing mice and division into responder and non-responder groups

[0107] Pan02-HVEM (mouse pancreatic cancer) cells were used to construct a tumor model by subcutaneous injection in the axilla on both sides of C57BL / 6J mice. The tumor volume reached 50-100mm 3 The mice were enrolled at 4 2 h and oHSV was administered intratumorally. The dosage of oHSV was 5 × 10 6 pfu, once every three days, for a total of three treatments. From the day of treatment, the tumor volume was measured every three days, and the length and width of the tumor were recorded until the volume of the mouse was greater than 2,000 mm 3 The animals were killed by cervical dislocation and recorded as death; the tumor volume was calculated by the formula: V (mm 3 )=(length×width 2 ) / 2. T RTV / C RTV The relative tumor proliferation rate, T RTV / C RTV % values ​​less than 40% are considered effective responses. RTV / C RTV % is calculated as follows: T RTV / C RTV % = T RTV / C RTV × 100%, where relative tumor volume (RTV) = tumor volume after treatment (Vt) / tumor volume before treatment (V0), T RTV The treatment group RTV, C RTV The control group RTV was used. By observing the response of the tumor, the treatment response and non-response groups were named R and NR, respectively.

[0108] Figure 3 Shown are the tumor growth curves and tumor weights of the OV-treated Pan02-HVEM model. Figure 3 A) OV treatment of Pan02-HVEM pancreatic cancer. Pan02-HVEM tumor-bearing mice had tumors growing to 50 mm 3 Around 2.5 hours after injection, 5×10 6 pfu OV (n=15), treated once every three days, and measured the tumor volume every three days. On the third day after three treatments, the tumor weights of the responder group (n=9) and the non-responder group (n=6) were weighed, and the mouse tumor growth curve was drawn ( Figure 3 Middle B), mouse tumor photograph ( Figure 3 C) and counted the tumor weight ( Figure 3 Middle D). **p<0.01.

[0109] like Figure 3As shown in the mouse Pan02-HVEM pancreatic cancer model, 40% (6 / 15) mice were insensitive to OV treatment and were recorded as the non-response group. On the contrary, 60% (9 / 15) mice responded well to OV treatment; the average tumor volume of the response group mice was 383mm 3 The average tumor weight was 0.49 g, which was significantly lower than the tumor volume of the non-responding group (average 1148 mm 3 The above results showed that pancreatic cancer in mice showed resistance to OV treatment, proving that the drug-resistant model of oHSV treatment was successfully established.

[0110] 2. Differences in interactions between immune cells in the responding and non-responding groups

[0111] Pan02-HVEM tumor-bearing mice were treated with 4% paraformaldehyde (Pan02) and 1% paraformaldehyde (HVEM) when the tumor volume grew to 50-100 mm. 3 The mice were treated with oHSV every two days. After three treatments, the tumor tissues of the three mice with the best treatment effect in the response group and the three mice with the worst treatment effect in the non-response group were selected to isolate CD45 + For immune cells, single-cell transcriptome sequencing samples were prepared using the 10×Genomics single-cell transcriptome sequencing platform and sequenced using an Illumina sequencer. After sequencing, data analysis was performed to determine the changes in immune cells and differential gene expression in tumor tissues of mice in the response group and the non-response group.

[0112] The tumor microenvironment is a complex environment in which tumor cells, immune cells, stromal cells and other cells are intricately interwoven. It is an important place for tumor cells to create immunosuppression and immune escape. Therefore, exploring the changes in the tumor microenvironment of the response group and the non-response group plays a key role in guiding the transformation of the new generation of oncolytic viruses. In the single-cell sequencing of this study, Cellchat was used to analyze the relationship between the cell-to-cell communication network. The basic principle is to calculate the possibility of interaction between ligand cells and receptor cells based on the average expression of the intercellular ligand-receptor pair genes.

[0113] Cellchat cell communication network analysis found that the predicted number of ligand-receptor pairs in the response group was 3,548, which was higher than the 2,596 in the non-response group. At the same time, the intensity of the ligand-receptor pairs in the response group was significantly stronger than that in the non-response group.

[0114] The present invention statistically analyzed the signal pathways and their expression conditions of the 22 subgroups with strong communication effects between the response group and the non-response group, and selected specific immune cells and specific ligand receptor pairs for detailed analysis as shown in the figure ( Figure 4 ).

[0115] 3. Differences in cytokine expression in tumor tissues

[0116] Cytokines inside tumor tissue are important mediators of the correlation between immune cells in TME. Traditional cytokine detection methods, such as ELISA, have low throughput and low detection efficiency. In order to detect the changes in local secretion of cytokines by high throughput and rapid detection, the present invention adopts Luminex multiple immune factor detection technology, the basic principle of which is that microspheres labeled with different fluorescent codes are bound to the antibodies of the object to be tested in a covalently cross-linked manner. After the object to be tested combines with the antibody to form a complex and reacts with fluorescein, the purpose of efficient quantification is achieved by detecting the fluorescence intensity, and the expression of each factor in the sample is realized by high throughput detection.

[0117] Figure 5 The cytokine expression in the tumor tissues of the Pan02-HVEM pancreatic cancer model response group and non-response group is shown. The tumor volume of C57BL / 6J mice bearing Pan02-HVEM cells reached about 50-100mm 3 Every three days, mice were injected intratumorally with PBS or 5×10 6 pfu OV, three treatments in total; on the third day after the last treatment, the tumor tissues of the response group (n=5) and the non-response group (n=5) were homogenized to obtain intratumoral proteins, and the Luminex multi-factor immunoassay platform was used to detect the expression of intratumoral cytokines, including interleukins, colony stimulating factors, interferons, and chemokines. The left picture includes interleukins, colony stimulating factors, and interferons, and the right picture is chemokines.

[0118] The results are as follows Figure 5 As shown in the figure, compared with the non-response group, the secretion of Th1 cytokines that promote cell-mediated immune response in the response group, including IFN-γ, IL-2, GM-CSF, IL-12p40, etc., was significantly increased, and the secretion of IL-12p70 showed an upward trend; except for the increase in the secretion of IL-10, there was no difference in the secretion of other Th2 cytokines such as IL-4, IL-5, IL-13, etc.; the secretion of IL-1β, a cytokine that promotes inflammatory response, was significantly increased, and the secretion of IL-17A, TNF-α, etc. showed an upward trend, and the secretion of IL-6 decreased. There was no difference in the secretion of other cytokines such as IL-3 and IL-1α.

[0119] Analysis of chemokine secretion revealed that compared with the non-response group, in the CCL subfamily of the response group, the secretion of CCL1, CCL4, and CCL5 increased significantly, and the secretion of CCL3, CCL12, CCL22, etc. showed an upward trend; in the CXC subfamily, the secretion of CXCL10, CXCL11, CXCL12, and CXCL16 showed an upward trend, and there was no difference in the secretion of CXCL1, CXCL5, and CXCL13; the secretion of CX3CL1 in the CX3C subfamily showed an upward trend.

[0120] Compared with the non-response group, the secretion of Th1 cytokines that promote cell-mediated immune response in the response group, including IFN-γ, IL-2, GM-CSF, IL-12p40, etc., was significantly increased, and the secretion of IL-12p70 showed an upward trend; except for the increase in the secretion of IL-10, the secretion of other Th2 cytokines such as IL-4, IL-5, IL-13, etc., showed no difference; the secretion of IL-1β, a cytokine that promotes inflammatory response, was significantly increased, the secretion of IL-17A, TNF-α, etc. showed an upward trend, and the secretion of IL-6 decreased. There was no difference in the secretion of other cytokines such as IL-3 and IL-1α ( Figure 5 ).

[0121] Analysis of chemokine secretion revealed that compared with the non-response group, in the CCL subfamily of the response group, the secretion of CCL1, CCL4, and CCL5 increased significantly, and the secretion of CCL3, CCL12, CCL22, etc. showed an upward trend; in the CXC subfamily, the secretion of CXCL10, CXCL11, CXCL12, and CXCL16 showed an upward trend, and there was no difference in the secretion of CXCL1, CXCL5, and CXCL13; the secretion of CX3CL1 in the CX3C subfamily showed an upward trend.

[0122] The above data show that cytokines including IL-2, IL-12, IL-10, IFN-γ, GM-CSF, and chemokines CCL4, CCL5, CCL22, CXCL10, CXCL11, CXCL12, CXCL16, etc. are candidate factors for combination therapy with OV. Example 2 Construction of Tet-On system and evaluation of candidate factors

[0123] 1. Construction of Tet-On inducible expression system cell line

[0124] The present invention first constructs the Tet-On inducible expression system into tumor cell lines to overexpress candidate cytokines for evaluating the in vivo efficacy of a single cytokine combined with an oncolytic virus. The working principle of the Tet-On system is as follows: Figure 6This can bypass the time-consuming, laborious and inefficient work of modifying more than a dozen oncolytic viruses, and well simulate the in vivo efficacy of single-factor oncolytic viruses. Specifically, the present invention needs to construct the gene sequence of cytokines into pLVX-TetOne-Puro or pLVX-TetOne-GFP, which mainly includes packaging the plasmid into a lentivirus and using the lentivirus to transfect tumor cells and screen them.

[0125] 2. Tet-On system to evaluate the in vivo efficacy of single cytokines combined with oncolytic viruses

[0126] (1) Tumor-bearing mice: C57BL / 6J mice were subcutaneously inoculated with 5×10 5 Tet-On-cytokine cell lines, tumors generally grow to 50-100 mm in size within 5-7 days 3 ;

[0127] (2) Treatment: On the day before the mice were enrolled, 2 mg / mL of Dox was added to the drinking water of the mice. After enrollment, 5×10 6 pfu basic oncolytic virus, oncolytic virus treatment once every three days, a total of three treatments, Dox supply started from the day before the first oncolytic virus treatment and stopped on the fourth day after the last treatment;

[0128] (3) Volume monitoring: Tumor volume was measured every three days, and the tumor growth curve and survival curve of mice were recorded and plotted; relative tumor proliferation rate T / C (%) = RTV of treatment group / RTV of PBS group × 100%, where RTV = tumor volume at enrollment / tumor volume at monitoring;

[0129] Figure 7 The Tet-On system is used to evaluate the efficacy of CCL family chemokines combined with OV. C57BL / 6J mice were inoculated with Tet-On-mCCL4, Tet-On-mCCL5 or Tet-On-mCCL22 tumor cell lines. When the tumor volume was about 50-80 mm 3 At 14:00, Dox was added to the drinking water, and OV was injected intratumorally 12 hours later; treatment was once every three days, for a total of three treatments; Dox was added from 12 hours before the first treatment to the fourth day after the last treatment; tumor volume was measured every three days and tumor growth curve, relative tumor proliferation rate (T / C) and mouse survival curve were drawn. n = 6 (each group). T / C value less than 40% represents effective inhibition of tumor growth. *p<0.05; **p<0.01; ***p<0.001: ****p<0.0001.

[0130] like Figure 7As shown, among the CCL family chemokines, on the basis of OV treatment, intratumoral overexpression of CCL5 can extend the median survival of mice from 22 days to 30.5 days; while in the combination group that induced intratumoral expression of CCL4 or CCL22, the median survival of mice was only extended by 5 days.

[0131] Figure 8 The Tet-On system is shown to evaluate the efficacy of CXCL family chemokines combined with OV. C57BL / 6J mice were inoculated with Tet-On-mCXCL9, Tet-On-mCXCL10 or Tet-On-mCXCL16 tumor cell lines, and the tumor volume was about 50-80mm. 3 Dox was added to drinking water at 12h, and OV was injected intratumorally 12h later; treatment was once every three days, for a total of three treatments; Dox addition time started from 12h before the first treatment to the fourth day after the last treatment; tumor volume was measured every three days and tumor growth curve, relative tumor proliferation rate (T / C) and mouse survival curve were drawn. n = 6 (each group). T / C value less than 40% represents effective inhibition of tumor growth. ns: not significant; *p<0.05; **p<0.01; ***p<0.001.

[0132] like Figure 8 As shown, among the CXCL family chemokines, compared with the OV treatment group, when CXCL16 was overexpressed in the tumor, the median survival of mice was extended from 22 days to 27 days; in the Tet-On-mCXCL9+OV and Tet-On-mCXCL10+OV groups, the median survival of mice was extended from 22 days to 26.5 days and 25 days, respectively; the relative tumor proliferation rates of the three single-factor combined OV groups were also lower than 40%.

[0133] Fig. 9 The Tet-On system is shown to evaluate the efficacy of T / NK cell activation-related cytokines combined with OV. C57BL / 6J mice were inoculated with Pan02-HVEM, Tet-On-mIL-2, Tet-On-mIL-10 or Tet-On-mIL-18 tumor cell lines, and when the tumor volume was about 50-80mm 3At 14:00 p.m., Dox was added to drinking water. OV was injected intratumorally 12 h later. The OV-mIL-12 group was injected intratumorally with the same dose of OV-mIL-12 in Pan02-HVEM tumor-bearing mice. Treatment was once every three days for a total of three treatments. Dox was added from 12 h before the first treatment to the fourth day after the last treatment. Tumor volume was measured every three days and tumor growth curves, relative tumor proliferation rate (T / C) and mouse survival curves were drawn. n = 6 (each group). T / C values ​​less than 40% represent effective inhibition of tumor growth. *p<0.05; ***p<0.001; ****p<0.0001.

[0134] like Fig. 9 As shown, among the cytokines related to T / NK cell activation, the median survival time of mice in the Tet-On-mIL-2 and OV-mIL-12 combination groups was extended from 22 days in the OV group to 26.5 days; followed by the Tet-On-mIL-10+OV combination group, and the Tet-On-mIL-18+OV combination group had the worst ability to inhibit tumor growth; the relative tumor proliferation rates of the three single factor combination OV groups were also lower than 40%.

[0135] Fig.10 The Tet-On system is shown to evaluate the efficacy of macrophage activation-related factors combined with OV. C57BL / 6J mice were inoculated with Pan02-HVEM, Tet-On-mM-CSF or Tet-On-mIFN-γ tumor cell lines, and when the tumor volume was about 50-80mm 3 At 12h, Dox was added to the drinking water, and OV was injected intratumorally 12h later. The OV-mGM-CSF group was injected intratumorally with the same dose of OV-mGM-CSF in Pan02-HVEM tumor-bearing mice. Treatment was once every three days, for a total of three treatments; Dox was added from 12 hours before the first treatment to the fourth day after the last treatment; the tumor volume was measured every three days and the tumor growth curve, relative tumor proliferation rate (T / C) and mouse survival curve were drawn. n = 6 (each group). T / C values ​​less than 40% represent effective inhibition of tumor growth. *p<0.05; ***p<0.001; ****p<0.0001.

[0136] like Fig.10 As shown in the figure, among the macrophage activation-related factors, the combination of Tet-On-mIFN-γ+OV, OV-mGM-CSF and R428+OV significantly inhibited tumor growth, and the survival period was extended to 32.5 days, 29 days and 28 days, respectively; followed by the Tet-On-mM-CSF+OV combination group; similarly, the relative tumor proliferation rates of the three single factors and the combination of R428 and OV were all lower than 40% ( Fig.10 ).

[0137] The above results show that local overexpression of chemokines CCL4, CCL5, CXCL9 and CXCL16 in tumors can significantly improve the anti-tumor effect of OV, and IL-2, IL-10, IL-12 and IL-18 related to T / NK cell activation combined with OV treatment can significantly inhibit tumor growth. In addition, IFN-γ and GM-CSF related to macrophage activation or polarization can also enhance the ability of OV to treat tumors.

[0138] Example 3 Screening and verification of the best immunomodulatory factor combination

[0139] One or two factors with the best efficacy were selected from each family, namely CCL5, CXCL16, IL-2, IL-12, IFN-γ, and GM-CSF. In order to evaluate the efficacy of these six factors in combination with OV and screen for the combination with the best efficacy and the least number of cytokines, the present invention first transformed six single-factor oncolytic viruses (such as Fig.11 Mouse CCL5, CXCL16, IL-12, IFN-γ or GM-CSF were inserted into the ICP 34.5 locus of the OV backbone (i.e., oHSV virus) to construct OV-mCCL5, OV-mCXCL16, OV-mIL-2, OV-mIL-12, OV-mIFN-γ and OV-mGM-CSF, respectively.

[0140] 1. Detection of anti-tumor ability of single factor oncolytic virus

[0141] In order to verify whether the anti-tumor effect of the modified single-factor oncolytic virus is consistent with the Tet-On system, the present invention injected 5×10 6 pfu of single-factor oncolytic virus, treated once every three days, for a total of three treatments; the tumor volume was measured every three days and the tumor growth curve was drawn.

[0142] like Fig.12 As shown, the mouse tumor growth curve showed that the viral treatment effects of OV-mCCL5, OV-mCXCL16, OV-mIL-2, OV-mIL-12, OV-mIFN-γ and OV-mGM-CSF were consistent with Tet-On-cytokine, further confirming the reliability of the in vivo efficacy screening results of the Tet-On system.

[0143] 2. The first round of “OV-six” in vivo efficacy evaluation

[0144] In order to screen for the combination with the least number of factors and the best anti-tumor effect, the present invention uses the "minus one" method and multiple rounds of in vivo efficacy evaluation to achieve the purpose of gradually reducing the number of combinations. 3 At 4 pm, mice were treated with the following: (1) PBS (control group); (2) unmodified OV; (3) a mixture containing all six single-factor modified OVs, namely “OV-six”; (3) a mixture containing five single-factor modified OVs, namely the “minus one” treatment group, including “OV-six-mCCL5”, “OV-six-mCXCL16”, “OV-six-mIL-2”, “OV-six-mIL-12”, “OV-six-mIFN-γ”, and “OV-six-mGM-CSF” treatment groups (the total amount of drug administered to each mouse was the same, all 5 × 10 6 pfu). The treatment was performed once every three days for a total of four times. The tumor volume was measured every three days and the tumor growth curve and mouse survival curve were drawn.

[0145] The results are as follows Fig.13 As shown. Compared with the OV group, both the "OV-six" and "minus one" groups can significantly inhibit tumor growth and prolong the survival of mice; specifically, the "OV-six" and "OV-six-mIL-2" treatment groups have the strongest and closest tumor growth inhibition effects: the median survival of mice in the "OV-six" treatment group was extended from 26.5 days to 52.5 days, and half of the mice were cured (complete response, CR); the median survival of mice in the "OV-six-mIL-2" treatment group was extended from 26.5 days to 51 days, and half of the mice were also cured; this shows that removing IL-2 from "OV-six" does not affect the anti-tumor efficacy, proving that "OV-six" does not rely on IL-2 to exert anti-tumor immune response. In contrast, the tumor inhibition in the “OV-six-mCCL5”, “OV-six-mCXCL16”, “OV-six-mIL-12”, “OV-six-mIFN-γ” and “OV-six-mGM-CSF” treatment groups was slightly weaker, especially the “OV-six-mIL-12” treatment group had the worst ability to inhibit tumor growth, indicating that IL-12 is a cytokine necessary for “OV-six” to kill tumors.

[0146] 3. The second round of “OV-five” in vivo efficacy evaluation

[0147] The first round of in vivo efficacy evaluation of "OV-six" proved that "OV-six" prolonged the survival of mice dependent on IL-12, not IL-2. Therefore, the present invention continued the second round of in vivo efficacy evaluation of "OV-five". The specific experimental steps refer to the treatment scheme in the first round of evaluation, which are as follows: C57BL / 6J mice bearing Pan02-HVEM cell line, when the tumor volume reached 50-100mm3, received the following treatments: (1) PBS (control group); (2) unmodified OV; (3) a mixture containing all five single-factor modified OVs, namely "OV-five"; (3) a mixture containing five single-factor modified OVs, namely the "minus one" treatment group, including "OV-five-mCCL5", "OV-five-mCXCL16", "OV-five-mIL-12", "OV-five-mIFN-γ" and "OV-five-mGM-CSF" treatment groups (the total dosage of each mouse was the same, all 5×10 6 pfu). The treatment was performed once every three days for a total of four times. The tumor volume was measured every three days and the tumor growth curve and mouse survival curve were drawn.

[0148] Pan02-bearing mice were treated intratumorally with PBS (control group), unmodified OV, and "OV-five" (an equal mixture of OV-mCCL5, OV-mCXCL16, OV-mIFN-γ, OV-mIL-12, and OV-mGM-CSF). At the same time, tumor-bearing mice were also treated with the "minus one" regimen (containing all OVs minus one single gene modified OV).

[0149] like Fig.14 As shown, 4 out of 8 mice achieved complete response (CR) after OV-five treatment. Removal of any cytokine or chemokine from “OV-five” significantly reduced the anti-tumor efficacy, indicating that all five cytokines are indispensable for enhancing the anti-tumor efficacy. Therefore, CCL5, CXCL16, IFN-γ, IL-12, and GM-CSF were used to modify OV to improve the anti-tumor efficacy.

[0150] Example 4 Modification of OV-5A oncolytic virus and evaluation of its anti-tumor efficacy and safety

[0151] It has been previously demonstrated that “OV-five” (an equal mixture of OV-mCCL5, OV-mCXCL16, OV-mIFN-γ, OV-mIL-12, and OV-mGM-CSF) has a strong anti-tumor effect. Next, in order to explore whether oncolytic viruses carrying these five cytokines simultaneously exert the same tumor inhibitory effect, the present invention constructed the OV-5A virus and demonstrated the anti-tumor efficacy, safety, remote anti-tumor effect, and immune memory of OV-5A through a series of experiments, and at the same time detected whether it has the same anti-tumor efficacy in other tumor models.

[0152] Cell lines

[0153] Mouse melanoma B16-F10-HVEM was constructed from B16-F10 and stably expressed HVEM (hereinafter referred to as B16-F10); mouse pancreatic cancer KPC (Krasem4 (LSL-G12D) Trp53em4 (R172H) Pdx1em1 (Avi-CreERT2)) cell line was purchased from Shanghai Southern Model Organisms Center; mouse glioblastoma GL261 cell line was purchased from Shanghai Fuheng Biotechnology Co., Ltd.

[0154] 1. Construction of OV-5A virus and validation of its efficacy

[0155] 1. Plasmid

[0156] pCDH-UL27-mIL-12-mIFN-γ-mCXCL16-mGMCSF-UL26 is used to construct homology arm sequences containing IL-12, IFN-γ, CXCL16 and GM-CSF; lentiCRISPR-sgRNA is a guide RNA plasmid targeting UL26 and UL27 sequences.

[0157] The OV-5A virus is based on OV-mCCL5, and the mouse IFN-γ, CXCL16, GM-CSF and IL-12 coding gene sequences are inserted into the UL26-UL27 gene locus of the viral genome. Fig.15 ).

[0158] 2. Modification of OV-5A virus

[0159] (1) Donor DNA construction: pCDH-UL27-mIL-12-mIFN-γ-mCXCL16-mGM-CSF-UL26 was obtained through molecular cloning steps. The primers involved are shown in Table 1:

[0160] Table 1

[0161]

[0162]

[0163] (2) The design of lentiCRISPR-sgRNA is shown in Table 2:

[0164] Table 2 Oligos between the sequences of UL26 and UL27

[0165]

[0166] (3) BsmBI was used for restriction digestion and ligation to finally obtain lentiCRISPR-sgRNA targeting the UL26-UL27 intermediate sequence;

[0167] (4) Obtaining OV-5A virus: After OV-mCCL5 virus infection, virus recombination, PCR identification, and ELISA identification steps, the OV-5A monoclonal oncolytic virus precipitate is finally obtained and stored in a -80°C refrigerator. The primers required for identification are shown in Table 3:

[0168] Table 3

[0169]

[0170] 3. Virus modification verification

[0171] Pan02-HVEM tumor-bearing mice were injected intratumorally with PBS, OV or OV-5A and treated once every three days. On the third day after three treatments, the tumor tissue was removed and homogenized with a tissue homogenizer to obtain a protein suspension. ELISA was used to detect the expression of CCL5, CXCL16, IFN-γ, IL-12 and GM-CSF cytokines in the local tumor.

[0172] like Fig.16 As shown, the modified OV-5A retains the ability to replicate in tumor cells while promoting the expression of cytokines encoded by OV-5A in infected tumor cells.

[0173] 4. Efficacy verification

[0174] Pan02-HVEM tumor-bearing mice received four intratumoral injections of OV-5A, with treatments every three days, and tumor volumes were monitored every two days. Fig.17 As shown, the tumor growth curve and mouse survival curve show that the anti-tumor effect of OV-5A is consistent with that of "OV-five".

[0175] 2. Safety Evaluation of OV-5A

[0176] 1. Body weight monitoring of pancreatic cancer-bearing mice treated with OV-5A

[0177] While studying the efficacy of OV-5A in treating pancreatic cancer, the present invention monitored the weight changes of mice. Pan02-HVEM tumor-bearing mice were injected with PBS, OV or OV-5A intratumorally, and treated once every three days for four times. The weight of the mice was weighed every two days, and the weight change curve of the mice was drawn. n = 6.

[0178] like Fig.18 As shown, no body weight loss was observed in the PBS, OV, and OV-5A groups during the study period.

[0179] 2. Detection of liver damage in pancreatic cancer-bearing mice treated with OV-5A

[0180] (1) Treatment of tumor-bearing mice: Pan02-HVEM tumor-bearing mice were intratumorally injected with PBS, OV, or OV-5A once every three days for a total of three treatments;

[0181] (2) Extraction of serum: 24 hours, 48 ​​hours and 72 hours after the three treatments, blood was collected from the ophthalmic vein using a capillary tube, transferred to an EP tube, and allowed to stand at 4°C for 2 hours; centrifuged at 5000 rpm at 4°C for 8 minutes, the serum was carefully aspirated and transferred to a new EP tube, and the centrifugation and serum transfer were repeated. The serum could be tested immediately or stored at -80°C;

[0182] (3) Detection of ALT and AST levels: According to the instructions of the ALT and AST detection kits, the activity levels in the serum of mice in different treatment groups were detected.

[0183] like Fig.19 As shown, compared with the PBS group, the levels of ALT and AST did not change after OV-5A treatment, indicating that OV-5A treatment did not cause liver tissue damage.

[0184] 3. Hematoxylin-eosin staining (H&E staining)

[0185] To investigate whether OV-5A treatment would cause damage to other tissues or organs, H&E staining was performed on the liver, lung, kidney, and spleen after OV-5A treatment of pancreatic tumors three times. Fig. 20 As shown, compared with the PBS and OV groups, OV-5A treatment did not cause significant inflammation and damage.

[0186] 4. Detection of the biological distribution of OV-5A virus

[0187] like Fig.21 As shown, the viral genome was only detected in the tumor, and the viral genome persisted in the tumor tissue for at least three days, while no virus was detected in the spleen, liver, kidney and lung.

[0188] 3. Evaluation of the efficacy of five-factor oncolytic virus

[0189] 1. Bilateral tumor-bearing model:

[0190] Fig. 22 The OV-5A treatment bilateral tumor model is shown. C57BL / 6J mice were inoculated subcutaneously with 5×10 6 Pan02-HVEM cells, and wait until the bilateral tumor volume grows to 50-100mm 3 The bilateral tumor volumes were measured and randomly divided into groups to ensure that the mean ipsilateral tumor volume of each group was consistent; PBS (n=7) and 5×10 6 pfu OV (n = 8) or 5 × 10 6 pfu OV-5A (n=9), recorded as the treated side, the right axillary tumor was not treated and recorded as the distal or non-treated side, treated once every three days for a total of three times; the tumor volume on both sides was measured every three days, and the tumor growth curve was drawn. When the tumor volume on either side was greater than 2,000mm 3 The mice were killed by cervical dislocation and the end point of death was recorded.

[0191] like Fig. 22 As shown, OV-5A could significantly inhibit the growth of tumors on both the treated and non-treated sides, indicating that OV-5A treatment could induce a systemic anti-tumor response.

[0192] 2.Rechallenge experiment:

[0193] Pan02-HVEM tumor-bearing mice were injected intratumorally with 5×10 6 pfu OV-5A, cured mice were re-inoculated with the same amount of pancreatic cancer cells on the 80th day after the first inoculation of Pan02-HVEM cells. Wild-type mice of similar age were used as the control group and inoculated with the same amount of pancreatic cancer cells. The tumor volume was monitored every three days, and the tumor growth curve and survival curve of the mice were drawn. When the volume reached 2,000mm 3 , recorded as death.

[0194] like Fig.23 As shown, when mice with Pan02-HVEM tumors cured by OV-5A were re-inoculated with Pan02-HVEM tumor cells, the cured mice showed complete rejection of the tumors, while all age-matched wild-type control mice grew tumors after inoculation, indicating that OV-5A treatment can establish long-lasting anti-tumor immune memory.

[0195] 3. Drug efficacy experiments in various tumor models:

[0196] C57BL / 6J mice were subcutaneously inoculated with 5×10 5 B16-F10 cells ( Fig.24 Middle A), 1×106 GL261 cells ( Fig.24 Medium B), 5×10 5 KPC cells ( Fig.24 Middle C), 1×10 6 MC38 cells ( Fig.24 Middle D), tumor volume reaches 50-100mm 3 PBS (n=6), 5×10 6 pfu OV (n = 6) or 5 × 10 6 pfu OV-5A (n=6-8), treated once every three days for a total of three times, the tumor volume and mouse weight were measured every three days, and the mouse tumor growth curve (left), mouse survival curve (middle) and mouse weight change curve (right) were plotted. When the volume reached 2,000mm 3 **p<0.01; ***p<0.001; ****p<0.0001.

[0197] like Fig.24 As shown in the results, in the “cold” tumor model, such as the melanoma B16-F10 model, the OV-5A group significantly inhibited tumor growth compared with the OV treatment group, and the median survival time was extended from 26 days to 41 days without causing weight loss in mice ( Fig.24 In the GL261 glioma model, the OV-5A group also significantly inhibited tumor growth, extending the median survival from 37 days to 43 days without causing weight loss in mice ( Fig.24 In the pancreatic cancer KPC model, the median survival time was increased from 23 days to 29 days ( Fig.24 Middle C). In the “hot” tumor MC38 colon cancer model, median survival was extended from 30 days to 44.5 days ( Fig.24 The above data show that OV-5A exhibits strong anti-tumor efficacy in both "hot" and "cold" tumor models.

[0198] Example 5 Evaluation of the anti-tumor effect of OV-5A in clinical samples

[0199] In view of the anti-tumor effect of murine OV-5A revealed by single-cell sequencing, flow cytometry and other techniques in vitro and in vivo, the present invention further studied the potential efficacy of OV on clinical samples, and thus constructed the following oncolytic viruses: OV-hGM-CSF, OV-hIL-12, OV-hIFN-γ, OV-hCCL5 and OV-hCXCL16.

[0200] The reagents and consumables used are shown in Table 4:

[0201] Table 4

[0202]

[0203]

[0204] 1. Construction and identification of human single-factor oncolytic virus

[0205] like Fig.25 As shown, the present invention first replaces the GFP at the double-copy ICP 34.5 site of the OV genome with the gene of a human cytokine or chemokine, and constructs an OV expressing human CXCL16, CCL5, IFN-γ, IL-12 or GM-CSF, which are OV-hCCL5, OV-hCXCL16, OV-hIL-2, OV-hIL-12, OV-hIFN-γ and OV-hGM-CSF, respectively. The principles and methods of some repeated experiments involved are detailed in the previous embodiments.

[0206] like Fig.26 As shown, the modified human single-factor oncolytic virus has the ability to express cytokines.

[0207] Effect of OV-5A on the Cytotoxic Activity of T Cells and Macrophages in Colon Cancer Organoids

[0208] Tumor tissue organoids are three-dimensional structures cultured in vitro that can simulate the growth characteristics of real tumors and provide a model closer to the in vivo environment for drug screening and personalized treatment. In order to evaluate the tumor killing effect of OV-5A on T cells and macrophages, organoids from two patients with colon cancer were infected with unmodified OV or OV-h5A (an equal mixture of human single-factor oncolytic viruses), co-cultured with CFSE pre-stained autologous tumor infiltrating lymphocytes (TILs) or THP-1-derived macrophages for 24 hours, and tumor killing was detected.

[0209] Clinical samples: Organoids from two patients with colon cancer and the corresponding TIL cells were provided by the research group of Professor Chen Quan from the School of Life Sciences of Nankai University.

[0210] 1. Tumor organoid and TIL co-culture experiment

[0211] like Fig. 27 As shown in the figure, in the co-culture experiment with T cells, OV can infect certain tumor cells and cause their death, while the TIL in the PBS group was blocked around the organoids and did not infiltrate into the interior. When OV or OV-h5A and TIL co-existed, the OV-h5A treatment group more effectively enhanced the infiltration and killing of CD8+T cells on tumor organ tissues than the OV group; after OV-h5A plus TIL treatment, the organoids disintegrated and almost all tumor cells were lysed. The statistical results also showed that the average fluorescence area of ​​local tumor cell death in the organoids was the largest.

[0212] 2. Co-culture of Tumor Organoids and Macrophages

[0213] like Fig.28 As shown, in the co-culture experiment with macrophages, OV-h5A treatment was more cytotoxic to organoids than the OV group.

[0214] The above experiments show that OV-h5A promotes T cell infiltration while enhancing the cytotoxicity and killing ability of T cells and macrophages.

[0215] 3. PBMC-PDX model verifies the efficacy of OV-5A

[0216] like Fig.29 As shown, the primary tumor samples were from patients with head and neck squamous cell carcinoma who underwent surgery at Tianjin Stomatological Hospital. All patients gave informed consent, and the research procedures were approved by the Institutional Ethics Committee of Tianjin Stomatological Hospital. Tumor tissues were stored in DMEM culture medium supplemented with 10% FBS, 1% penicillin-streptomycin solution, 2.5 mg / mL amphotericin B, and 50 mg / mL gentamicin. C-NKG mice were purchased from Cyagen Biosciences. To construct the PDX model, tumor samples from patients were first cut into 2-mm small pieces, and then three pieces of tumor tissue were mixed with matrix gel (Biocoat, 354234) and implanted subcutaneously on the left side of the mouse. After implantation, the tumor was measured three times a week. The first-generation PDX tumor was extracted and cut into 10-mm 3 Small pieces were implanted subcutaneously on the left side of the mouse to generate the second generation, and so on. In this experiment, the fourth generation of PDX tumors were transplanted into mice. When the PDX tumor volume reached 50-100mm 3 5×10 6 The next day, 1×10 PBMCs were injected into the tumor 6 pfu of OV or OV-h5A were injected three times every three days. Tumor size was measured every other day.

[0217] The results are as follows Fig.30 and Fig.31 As shown, the present invention monitored tumor growth and body weight changes for two weeks. Compared with OV treatment, OV-h5A can more effectively inhibit tumor growth, and there is no significant change in body weight after OV-5A treatment.

[0218] Example 6 Rational design of a combination to promote the anti-tumor efficacy of OV-5A based on single-cell sequencing data

[0219] OV-5A significantly delayed the growth of mouse tumors in the treatment of pancreatic cancer models, and nearly half of the tumor-bearing mice were completely cured, indicating that the data-driven oncolytic virus design strategy has been well verified. In order to further explore the potential of improving the efficacy of OV-5A, the present invention will once again adopt the "data-driven oncolytic virus design strategy" and combine the results of single-cell sequencing analysis to rationally design the combination of OV-5A.

[0220] The humanized CD40 antibody was a reported antibody (WANG Y, JIN R, SHEN B, et al. High-throughput functional screening for next-generation cancer immunotherapy using droplet-based microfluidics [J]. Sci Adv, 2021, 7 (24).). Humanized CD40 mice were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0221] The reagents and consumables used are shown in Table 5:

[0222] Table 5

[0223]

[0224] 1. Tolerance mechanism of OV-5A in the treatment of pancreatic cancer

[0225] The present invention utilizes single-cell sequencing and other methods, and OV-5A, which is rationally designed based on data such as drug resistance mechanisms, can activate innate and adaptive immune responses, significantly delay tumor growth in all treated mice, and about half of the mice are cured (complete response). In order to explore whether the efficacy of OV-5A can be further improved, the present invention performs scRNA-seq analysis on tumor-infiltrating immune cells of complete responders (CRs) and partial responders (PRs) after OV-5A treatment. Pan02 tumor-bearing mice were intratumorally injected with OV-5A, and mice with different responses were divided into CR group and PR group according to the size of the tumor, and the isolated immune cells (CD45+) were subjected to scRNA-seq. The analysis process has been described in detail above.

[0226] 1. Immune checkpoint expression analysis

[0227] During immunotherapy, immune checkpoint expression usually increases, significantly affecting the efficacy of the treatment. Therefore, the present invention conducted immune checkpoint expression analysis.

[0228] Fig.32The figure shows the heat map of T cell immune checkpoint expression in the CR group and the PR group after OV-5A treatment. The horizontal axis shows the name of the immune checkpoint, and the vertical axis represents different groups. The darker the red color, the higher the expression of the immune checkpoint in the complete response group; the darker the green color, the higher the expression of the immune checkpoint in the partial response group. The most obvious change in the immune checkpoint is the upregulation of exhaustion-related genes in CD8+T cells in the PR group, especially the expression of Pdcd1.

[0229] 2. Flow cytometry analysis and verification

[0230] To further verify the changes in the PD-1 / PD-L1 signaling axis, the present invention used flow cytometry to analyze the effects of OV-5A treatment on the expression of PD-1 or PD-L1 in tumor cells and myeloid cells in the tumor microenvironment.

[0231] like Fig.33 As shown, when tumor cells are infected with OV, the expression of PD-L1 in tumor cells is also upregulated; the present invention also detects that the expression of PD-L1 in myeloid cells is also significantly upregulated.

[0232] 3. Cellchat analyzes the communication network relationship between immune cells

[0233] In order to analyze the interaction between the PD-L1 signaling pathway and various immune cells, the present invention utilizes Cellchat analysis.

[0234] like Fig.34 As shown, the analysis results showed that the PD-L1 signaling pathway in the PR group was more active than that in the CR group, indicating that OV treatment would induce T cell exhaustion, thereby affecting the anti-tumor efficacy.

[0235] 2. Effects of FLT3L and CD40 signaling pathways combined with OV on DC maturation

[0236] 1. Cellchat analysis

[0237] In order to study the interaction between immune cells, the present invention uses Cellchat to analyze the communication network relationship between immune cells.

[0238] Fig.35 The figure shows the interaction between immune cells in the CR group and the PR group. The relative signal flow between the CR group and the PR group, red represents the CR group, green represents the PR group, and the vertical axis shows the name of the selected signal pathway of interest; the overall signal type of the CR group and the PR group, the left picture is the CR group, the right side is the PR group, the darker the color, the more active the signal pathway, and the horizontal axis shows the immune cell type. The results show that the intensity of the two signal interactions in the CR group is significantly stronger than that in the PR group, which highlights that DC maturation is crucial to the anti-tumor efficacy of OV.

[0239] Fig.36 and Fig.37 These are the interaction diagrams of CD40 signaling pathway and FLT3 signaling pathway between immune cells. The cells pointed by the arrows represent cells expressing receptors, and the cells from which the lines are emitted represent cells expressing ligands; the thicker the line, the more ligands there are. The results show the detailed interaction between CD40 and FLT3 signaling pathways between various immune cells, and it was found that the interaction strength of the two signals in the CR group was significantly stronger than that in the PR group.

[0240] 3. Verification of the role of CD40 and FLT3L

[0241] Based on the above signaling pathway analysis, the present invention evaluated whether CD40 agonist antibody (αCD40), mouse Flt3L-armed OV (OV-mFlt3L) or their combination (αCD40 / OV-mFlt3L) can promote DC maturation.

[0242] like Fig.38 As shown, the present invention replaces the GFP at site 34.5 of the OV viral genome with the mouse Flt3L gene sequence to construct an OV-mFlt3L virus expressing mouse mFlt3L.

[0243] 1. Oncolytic virus combination experiment

[0244] 1.1 Combination of human αCD40 antibody and OV-mFlt3L oncolytic virus

[0245] (1) Tumor-bearing mice: 5×10 tumor cells were subcutaneously inoculated in the axilla of CD40 humanized mice. 5 Pan02-HVEM cells, and wait until the tumor volume grows to 50-100mm 3 When, random grouping;

[0246] (2) Treatment: 150 μg of humanized CD40 antibody was injected intraperitoneally once every three days for a total of three injections; 100 μL of oncolytic virus containing 5×10 6 pfu, injected once every three days for a total of three injections;

[0247] (3) Monitoring tumor growth: Tumor volume was measured every three days and when the volume was greater than 2,000 mm 3 The animals were killed by cervical dislocation and recorded as death.

[0248] 1.2 Combination of OV-5A with αCD40 antibody, OV-mFlt3L and αPD-1

[0249] (1) Tumor-bearing mice: 5×10 tumor cells were subcutaneously inoculated in the axilla of CD40 humanized mice. 5Pan02-HVEM cells, and wait until the tumor volume grows to 50-100mm 3 When, random grouping;

[0250] (2) Treatment: 150 μg of CD40 humanized antibody was injected intraperitoneally once every three days for a total of three injections; 100 μL of oncolytic virus containing 6×10 6 pfu, once every three days for a total of three injections, of which 5×10 6 pfu OV-5A and 1 × 10 6 pfu OV-mFlt3L composition;

[0251] (3) Monitoring tumor growth: The tumor volume was measured every three days. When the volume was greater than 2,000 mm3, the mice were sacrificed by cervical dislocation and recorded as death.

[0252] Efficacy results such as Fig.39 As shown, the combined use of αCD40 and OV-mFlt3L could more effectively inhibit tumor growth than the use of CD40 agonist or OV-mFlt3L alone, and the median survival of mice was extended from 26 days in the OV group to 32 days.

[0253] 4. Antitumor effects of DC maturation and immune checkpoint inhibitors combined with OV-5A

[0254] To evaluate the antitumor efficacy of OV-5A combined with PD-1 antagonist antibody (αPD-1, BE0146, Bio X Cell, New Hampshire, USA), αCD40 and OV-Flt3L, Pan02-HVEM tumor-bearing mice were treated with 8 different combinations including PBS, αPD-1, αCD40+OV-Flt3L, αCD40+OV-Flt3L+αPD-1, OV-5A, OV-5A+αPD-1, OV-5A+αCD40+OV-Flt3L and OV-5A+αCD40+OV-Flt3L+αPD-1. The antitumor efficacy of different combinations was evaluated by tumor growth curves and mouse survival curves.

[0255] 3. Animal experiments based on Pan02-HVEM

[0256] Pan02-HVEM tumor-bearing mice received PBS, αPD-1 (ip, 100 μg / dose), OV-mFlt3L+αCD40 (ip, 150 μg / dose), OV-mFlt3L+αCD40+αPD-1, OV-5A, OV-5A+αPD-1, OV-5A+OV-mFlt3L+αCD40, and OV-5A+OV-mFlt3L+αCD40+αPD-1, a total of 8 groups, and were treated once every three days for a total of three treatments, in which the oncolytic virus treatment method and dose were intratumoral (it) and 5×10 6 pfu / dose, tumor volume was measured every three days, and tumor growth curve (upper) and mouse survival curve (lower) were plotted. n / 6: cured mice / total number of mice. *p<0.05.

[0257] like Fig.40 As shown, αPD-1 or αCD40 and OV-Flt3L can improve the efficacy of OV-5A; while the therapeutic effect of αPD-1, αCD40 and OV-Flt3L is relatively poor, and no mice with complete response were obtained. The combined treatment of OV-5A, αPD-1, αCD40 and OV-Flt3L was the most effective in inhibiting tumor growth and curing most mice, with 5 of 6 mice having complete response (about 83%).

[0258] 4. Animal experiments based on MC38

[0259] The present invention also evaluated the anti-tumor efficacy of the combination in the mouse MC38 colon cancer model. MC38 tumor-bearing mice received PBS, OV-mFlt3L+αCD40+αPD-1, OV-5A and OV-5A+OV-mFlt3L+αCD40+αPD-1, a total of 4 groups, and were treated once every three days for a total of three treatments. The tumor volume was measured every three days, and the tumor growth curve (upper) and mouse survival curve (lower) were drawn. n / 6: cured mice / total number of mice. ***p<0.001.

[0260] The results are as follows Fig.41 As shown, the combined treatment of OV-5A, αPD-1, αCD40 and OV-Flt3L can also significantly inhibit tumor growth and prolong the survival of mice. Four of the six mice in the OV-5A+OV-Flt3L+αCD40+αPD-1 combination group responded completely (about 66.7%). The above data show that under the guidance of single-cell sequencing data, the combined use of OV-5A, αPD-1, αCD40 and OV-Flt3L can further enhance the anti-tumor activity.

[0261] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. An oncolytic virus composition, comprising an oncolytic virus and an immunomodulatory factor, wherein: The immunomodulatory factors include CXCL16.

2. The oncolytic virus composition according to claim 1, wherein The immunomodulatory factors also include CCL5, GM-CSF, IL-12 and / or IFN-γ. Preferably, the immunomodulatory factors further include CCL4, CXCL9, IL-2, IL-10 and / or IL-18; Preferably, the oncolytic virus composition further comprises a FLT3 agonist, a CD40 agonist, a PD-1 antagonist, a PD-L1 antagonist and / or a CTLA-4 antagonist; Preferably, the oncolytic virus includes herpes simplex virus (type 1 or type 2), adenovirus, vaccinia virus, measles virus, Newcastle disease virus, vesicular stomatitis virus, reovirus, coxsackievirus, poliovirus, alphavirus, yellow fever virus, and West Nile virus.

3. A recombinant oncolytic virus, comprising a nucleic acid sequence encoding an immunomodulatory factor, wherein: The immunomodulatory factors include CXCL16.

4. The recombinant oncolytic virus according to claim 3, wherein The immunomodulatory factors also include CCL5, GM-CSF, IL-12 and / or IFN-γ; Preferably, the immunomodulatory factors further include CCL4, CXCL9, IL-2, IL-10 and / or IL-18.

5. A method for preparing the recombinant oncolytic virus according to claim 3 or 4, the method comprising: Introducing a nucleic acid sequence encoding an immunomodulatory factor into the oncolytic virus to be modified to obtain the recombinant oncolytic virus; Wherein, the immunomodulatory factors include CXCL16; Preferably, the immunomodulatory factors further include CCL5, GM-CSF, IL-12 and / or IFN-γ; Preferably, the immunomodulatory factors further include CCL4, CXCL9, IL-2, IL-10 and / or IL-18; Preferably, the oncolytic virus to be modified includes herpes simplex virus (type 1 or type 2), adenovirus, vaccinia virus, measles virus, Newcastle disease virus, vesicular stomatitis virus, reovirus, coxsackievirus, poliovirus, alphavirus, yellow fever virus, and West Nile virus.

6. A drug combination, comprising a first component and a second component, wherein: The first component comprises the recombinant oncolytic virus according to claim 3 or 4, and the second component comprises a FLT3 agonist, a CD40 agonist, a PD-1 antagonist, a PD-L1 antagonist and / or a CTLA-4 antagonist.

7. A pharmaceutical preparation, wherein The pharmaceutical preparation comprises the oncolytic virus composition of claim 1 or 2, the recombinant oncolytic virus of claim 3 or 4, or the drug combination of claim 6.

8. Use of the oncolytic virus composition of claim 1 or 2, the recombinant oncolytic virus of claim 3 or 4, the drug combination of claim 6 or the drug preparation of claim 7 in the preparation of a drug for preventing or treating tumors.

9. The use according to claim 8, wherein: The tumor includes a solid tumor, Preferably, the solid tumors include pancreatic cancer, colon cancer, melanoma, glioma, head and neck tumors, esophageal cancer, gastric cancer, liver cancer, breast cancer, sarcoma, prostate cancer, and lung cancer; Preferably, the drug further comprises a pharmaceutically acceptable carrier.

10. A method for screening immunomodulatory factors capable of enhancing the anti-tumor ability of oncolytic viruses, wherein: The method comprises: screening for differentially expressed immune regulatory factors between tumor tissues that respond to oncolytic virus therapy and tumor tissues that do not respond to oncolytic virus therapy; Preferably, single-cell transcriptome sequencing of intratumoral immune cells and / or high-throughput detection of intratumoral cytokines are used to screen for differentially expressed immune regulatory factors between tumor tissues that respond to oncolytic virus therapy and tumor tissues that do not respond to oncolytic virus therapy; Preferably, the method further comprises constructing a Tet-On system to evaluate the differentially expressed immunomodulatory factors.

Images