Gsdmb overexpression oncolytic virus construct and method for applying same to tumor treatment

Abstract

A Gasdermin B (GSDMB) overexpression oncolytic virus construct and a method for applying same to tumor treatment. Further provided is a tumor cell GSDMB activator, which is used for treating a PD-1 / PD-L1 inhibitor-resistant tumor, restoring the treatment sensitivity of a PD-1 / PD-L1 inhibitor, and / or preventing the resistance to the treatment with the PD-1 / PD-L1 inhibitor. The activator may comprise a GSDMB-expressing nucleic acid construct or recombinant oncolytic virus. Further provided are a pharmaceutical composition comprising the tumor cell GSDMB activator and a method for preparing the GSDMB activator.

Description

Construction of GSDMB-overexpressing oncolytic virus and its application in tumor therapy Technical Field

[0001] This invention belongs to the field of immune response and tumor biotherapy, and particularly relates to the construct of GSDMB for activating tumor cells and its application in tumor therapy. Background Technology

[0002] Malignant tumors are increasingly becoming a major threat to human health. According to the 2020 World Cancer Report, cancer has become the second leading cause of death among people aged 30-69 (accounting for 29.8%) in 134 countries worldwide. Current treatments for tumors, especially solid tumors, include surgical resection, chemotherapy, radiation therapy, immunotherapy, and combinations of these treatments. Chemotherapy and radiation therapy can both be accompanied by serious side effects such as decreased immune cells and anemia. Surgical treatment is not suitable for all tumors, such as triple-negative breast cancer, which is prone to metastasis and difficult to surgically remove, or intrahepatic cholangiocarcinoma, which has a recurrence rate as high as 65% after surgical resection. While tumor immunotherapy, including immune checkpoint inhibitors (ICBs), chimeric antigen receptor T-cell immunotherapy (CAR-T), and adoptive T-cell therapy (ACT), has greatly improved the survival of cancer patients, the overall response rate of immune checkpoint inhibitors, represented by PD-1 / PD-L1 inhibitors, is only about 20%–30% in cancer patients. The response rate is even lower in tumors with poor immune infiltration, such as triple-negative breast cancer, pancreatic cancer, intrahepatic cholangiocarcinoma, and liver cancer, ranging from only 5% to 20%. In melanoma and non-small cell lung cancer (lung adenocarcinoma, squamous cell carcinoma, and large cell carcinoma), the response rate of PD-1 / PD-L1 inhibitors is also only 20%–35%, posing a serious challenge to immunotherapy. There is still a need in this field for effective treatments of tumors, especially those resistant to PD-1 / PD-L1 inhibitors. Summary of the Invention

[0003] This invention discovers that by activating Gasdermin B (GSDMB) in tumor cells, tumors can be transformed from "cold tumors" with a predominantly immunosuppressive microenvironment to "hot tumors" that primarily promote adaptive immune responses. This can promote the formation of high endothelial veins within the tumor and tumor antigen-specific CD8. +T cells can enter the tumor parenchyma, thus becoming effective in treating tumors, especially tumors that were previously difficult to treat with PD-1 / PD-L1 inhibitors, making them sensitive to tumor drugs and thus treatable.

[0004] In some implementations, the invention described herein includes the following items.

[0005] 1. Tumor cell GSDMB activator, used to treat PD-1 / PD-L1 inhibitor resistant tumors, restore sensitivity to PD-1 / PD-L1 inhibitor therapy and / or prevent resistance to PD-1 / PD-L1 inhibitor therapy.

[0006] 2. The activator according to Item 1, wherein the activator comprises a nucleic acid construct, vector or recombinant oncolytic virus expressing GSDMB, such as any of the following recombinant oncolytic viruses: (1) single-stranded DNA virus, double-stranded DNA virus, single-stranded positive RNA virus, single-stranded negative RNA virus, double-stranded RNA virus; and (2) parvovirus, adenovirus, vaccinia virus, herpes virus, poliovirus, measles virus, Newcastle disease virus, respiratory enterovirus, such as the recombinant oncolytic virus including recombinant Newcastle disease virus NDV-GSDMB.

[0007] 3. The activator according to item 1 or 2, wherein the activator is used as the active ingredient of the drug, the drug comprising a systemic, intraperitoneal or intratumoral injection drug, preferably an intratumoral injection drug.

[0008] 4. An activator according to any one of items 1-3, wherein the activator is used as an active ingredient of a drug, the drug being used in combination with other anticancer therapies, wherein the other anticancer therapies include, for example, immune checkpoint blockade therapy, preferably PD-1 / PD-L1 immune checkpoint blockade therapy, such as the GSDMB activator being further used in combination with a PD-1 / PD-L1 inhibitor, optionally wherein the PD-1 / PD-L1 inhibitor comprises a PD-1 / PD-L1 antibody.

[0009] 5. The activator according to any one of items 1-4, wherein the PD-1 / PD-L1 inhibitor resistant tumors include tumors in cancer patients with an overall response rate to PD-1 / PD-L1 inhibitors of less than 50%, and / or tumors that have been treated with PD-1 / PD-L1 inhibitors and have been found to have a reduced treatment response, such as breast cancer such as triple-negative breast cancer, pancreatic cancer, cholangiocarcinoma such as intrahepatic cholangiocarcinoma, liver cancer, melanoma, colorectal cancer, glioma, renal cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer.

[0010] 6. The activator according to any one of items 1-5, wherein the GSDMB comprises the full-length GSDMB gene or a fragment thereof, such as a GSDMB gene fragment containing a GZMA cleavage site such as lysine 229 and / or 244, and optionally, wherein the expression comprises overexpression.

[0011] 7. A method for treating PD-1 / PD-L1 inhibitor-resistant tumors, restoring sensitivity to PD-1 / PD-L1 inhibitor therapy and / or preventing resistance to PD-1 / PD-L1 inhibitor therapy, comprising administering to an individual in need an effective amount of a tumor cell GSDMB activator as defined in any one of items 1-6.

[0012] 8. A pharmaceutical composition comprising a tumor cell GSDMB activator as defined in any one of items 1-6, optionally further comprising an immunosuppressant such as a PD-1 / PD-L1 inhibitor, an excipient, a pharmaceutical carrier, or a buffer.

[0013] 9. A kit comprising a tumor cell GSDMB activator as defined in any one of items 1-6, optionally further comprising an immunosuppressant such as a PD-1 / PD-L1 inhibitor, optionally for the treatment of PD-1 / PD-L1 inhibitor-resistant tumors.

[0014] 10. A method for preparing a tumor cell GSDMB activator as defined in any one of items 1-6, wherein the tumor cell GSDMB activator is a recombinant oncolytic virus expressing GSDMB, the method comprising introducing a nucleic acid molecule encoding GSDMB into the genome of the recombinant oncolytic virus to prepare a recombinant oncolytic virus expressing GSDMB.

[0015] 11. Use of tumor cell GSDMB activators as defined in any one of Items 1-6 in the preparation of medicaments for treating PD-1 / PD-L1 inhibitor resistant tumors, restoring sensitivity to PD-1 / PD-L1 inhibitor therapy and / or preventing resistance to PD-1 / PD-L1 inhibitor therapy.

[0016] In some aspects, the present invention provides tumor cell GSDMB activators, pharmaceutical compositions and kits containing tumor cell GSDMB activators, methods for preparing tumor cell GSDMB activators, uses of tumor cell GSDMB activators in the preparation of pharmaceuticals, and methods for treating diseases (such as tumors) by means of tumor cell GSDMB activators.

[0017] In this document, there are no particular limitations on the term "tumor cell GSDMB activator," which may include any agent that activates tumor cells to express GSDMB, transforms the tumor from a predominantly immunosuppressive microenvironment ("cold tumor") to a predominantly adaptive immune response ("hot tumor"), promotes the formation of high endothelial veins within the tumor, facilitates the entry of tumor antigen-specific CD8+ T cells into the tumor parenchyma, and / or induces tumor cell pyroptosis. In some embodiments, the tumor cell GSDMB activator may include a nucleic acid construct, vector, or recombinant oncolytic virus expressing GSDMB.

[0018] In this document, there are no particular limitations on the GSDMB gene that can be used, including, for example, the full-length GSDMB gene or any suitable fragment capable of inducing pyroptosis in tumor cells. In some embodiments, GSDMB may include various isoforms of GSDMB, such as GSDMB isoform 3 and GSDMB isoform 4, etc. In some embodiments, the GSDMB fragment may contain a GSDMB gene fragment with a GZMA cleavage site such as lysine 229 and / or 244.

[0019] In this document, PD-1 / PD-L1 inhibitor-resistant tumors (or cancers) can include tumors in cancer patients with an overall response rate of less than 50% to PD-1 and / or PD-L1 inhibitors (such as antibodies), including, for example, tumors with a response rate of less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, and no response. In this document, PD-1 / PD-L1 may suitably refer to PD-1 and / or PD-L1. For example, references to PD-1 / PD-L1 inhibitors herein include PD-1 and / or PD-L1, such as PD-1 and / or PD-L1 antibodies. In some embodiments, the tumor or cancer may include a tumor or cancer of an appropriate subject, such as a human or animal (e.g., a mouse or rat). In some embodiments, PD-1 / PD-L1 inhibitor-resistant tumors (or cancers) can be determined from a literature search or by evaluation of treatments known in the art. In some embodiments, PD-1 / PD-L1 inhibitor-resistant tumors include, for example, breast cancer such as triple-negative breast cancer, pancreatic cancer, cholangiocarcinoma such as intrahepatic cholangiocarcinoma, liver cancer, melanoma, colorectal cancer, glioma, renal cancer, and lung cancer such as small cell lung cancer and non-small cell lung cancer. In some embodiments, PD-1 / PD-L1 inhibitor-resistant tumors (or cancers) may include tumors that have been treated with PD-1 and / or PD-L1 inhibitors (such as antibodies) and optionally exhibit a reduced treatment response (e.g., resistance or tolerance to said treatment), such as tumors with a reduced response compared to initial PD-1 and / or PD-L1 inhibitor (such as antibody) treatment. In some embodiments, for example in tumors with a reduced response to PD-1 / PD-L1 inhibitor treatment, by administering the tumor cell GSDMB activator of the present invention (and / or further combined with a PD-1 / PD-L1 inhibitor), it is possible to restore the tumor's sensitivity to treatment and / or prevent the development of resistance, thereby achieving better therapeutic effects. In some embodiments, a reduction in treatment response can be determined by any method known in the art, such as by measuring tumor volume and / or the expression level of treatment biomarkers. In some embodiments, a reduction in treatment response (e.g., a reduction in tumor volume or a change in biomarker expression levels after a period of treatment, such as one or more cycles) can include, for example, a reduction of more than 5% compared to the initial response to PD-1 and / or PD-L1 inhibitor (e.g., antibody) treatment, including, for example, more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or no response. In some embodiments, prior PD-1 and / or PD-L1 inhibitor (e.g., antibody) treatment includes treatment discontinued after a period of treatment, such as one or more cycles, or treatment in progress, such as one or more cycles.In some embodiments, PD-1 / PD-L1 inhibitor-resistant tumors include, for example, those that have been treated with PD-1 and / or PD-L1 inhibitors (such as antibodies) and optionally exhibit a reduced treatment response, such as breast cancer (e.g., triple-negative breast cancer), pancreatic cancer, cholangiocarcinoma (e.g., intrahepatic cholangiocarcinoma), liver cancer, melanoma, colorectal cancer, glioma, renal cell carcinoma, and lung cancer (e.g., small cell lung cancer and non-small cell lung cancer) that have shown a reduced response compared to initial PD-1 and / or PD-L1 inhibitor (such as antibody) treatment. In some embodiments, the use of the tumor cell GSDMB activator of the present invention (and / or further combined with a PD-1 / PD-L1 inhibitor) includes restoring sensitivity to PD-1 and / or PD-L1 inhibitor (such as antibody) treatment and / or preventing the development of resistance to PD-1 and / or PD-L1 inhibitor (such as antibody) treatment.

[0020] In some embodiments, the present invention provides a combination of a tumor cell GSDMB activator and a PD-1 / PD-L1 inhibitor such as a PD-1 / PD-L1 antibody.

[0021] In this invention, it has been found that activating GSDMB can produce a significantly better tumor-suppressing effect than activating other Gasdermins such as GSDME, and that the combination of GSDMB activator and PD-1 / PD-L1 inhibitor can produce a significantly better tumor-suppressing effect than the combination of other Gasdermins such as GSDME activator and PD-1 / PD-L1 inhibitor.

[0022] In some embodiments, the present invention provides a nucleic acid construct that may comprise a nucleic acid sequence encoding GSDMB and a regulatory sequence operatively linked thereto, such as a promoter, such as a constitutive promoter or an overexpression promoter. In some embodiments, the nucleic acid construct may further comprise a nucleic acid sequence encoding an oncolytic viral protein, for example, a nucleic acid sequence encoding one or more proteins required for oncolytic viral replication.

[0023] In some embodiments, the present invention provides a vector comprising the above-described nucleic acid construct. In some embodiments, the present invention provides a recombinant oncolytic virus comprising a vector or a nucleic acid construct. In some embodiments, the present invention provides a host cell comprising a vector or a nucleic acid construct.

[0024] In some embodiments, the present invention provides pharmaceutical compositions or kits comprising the activator, combination, vector, nucleic acid construct, recombinant oncolytic virus, and / or host cells. In some embodiments, the present invention provides use of the activator, combination, vector, nucleic acid construct, recombinant oncolytic virus, host cells, pharmaceutical compositions, and / or kits, for example, for treating PD-1 / PD-L1 inhibitor-resistant tumors, restoring sensitivity to PD-1 / PD-L1 inhibitor therapy, and / or preventing resistance to PD-1 / PD-L1 inhibitor therapy. In some embodiments, the present invention provides a method of treating a disease (such as a tumor) using the activator, combination, vector, nucleic acid construct, recombinant oncolytic virus, host cells, pharmaceutical compositions, and / or kits, for example, treating PD-1 / PD-L1 inhibitor-resistant tumors, restoring sensitivity to PD-1 / PD-L1 inhibitor therapy, and / or preventing resistance to PD-1 / PD-L1 inhibitor therapy, which may include administering an effective amount of the activator, combination, vector, nucleic acid construct, recombinant oncolytic virus, host cells, pharmaceutical compositions, and / or kits to an individual in need of this treatment. In some embodiments, the individual may be a human. In some embodiments, the individual may be a non-human animal. In some embodiments, the activator, combination, vector, nucleic acid construct, recombinant oncolytic virus, host cell, pharmaceutical composition and / or kit is administered via systemic, intraperitoneal, or intratumoral injection, preferably intratumoral injection. The effective amount for treating tumors can be determined by any suitable method known in the art. In some embodiments, the pharmaceutical composition or kit may further comprise a PD-1 / PD-L1 inhibitor such as a PD-1 / PD-L1 antibody. In some embodiments, the pharmaceutical composition or kit can be used to treat tumors (preferably PD-1 / PD-L1 inhibitor-resistant tumors). In some embodiments, the pharmaceutical composition or kit may comprise the recombinant oncolytic virus described herein or a recombinant oncolytic virus prepared by the methods described herein. In some embodiments, the pharmaceutical composition may further comprise suitable excipients, pharmaceutical carriers, or buffers, which may be any suitable excipients, pharmaceutical carriers, or buffers known in the art, including, for example, lactose, sucrose, gelatin, starch, glucose, silica gel, sodium stearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, ethanol, etc. In one aspect, the present invention utilizes an oncolytic virus such as Newcastle disease virus (NDV) as a vector, inserting GSDMB between NDV viral genes such as P and M genes, performing exogenous expression, and constructing and rescuing a recombinant oncolytic virus. In some embodiments, the recombinant oncolytic virus of the present invention may include, for example, recombinant Newcastle disease virus NDV-GSDMB. In some embodiments, the present invention provides the use of recombinant oncolytic viruses (preferably in combination with PD-1 / PD-L1 inhibitors such as PD-1 / PD-L1 antibodies) for the treatment of tumors (preferably PD-1 / PD-L1 inhibitor-resistant tumors).

[0025] In some embodiments, the present invention provides methods for preparing vectors, nucleic acid constructs, recombinant oncolytic viruses and / or host cells, the methods including introducing a nucleic acid molecule encoding GSDMB into the genome of a recombinant oncolytic virus to prepare a nucleic acid construct or vector expressing GSDMB, and optionally transfecting host cells with the nucleic acid construct or vector to prepare host cells or recombinant oncolytic viruses expressing GSDMB.

[0026] This invention can effectively improve the efficacy of tumor immunotherapy by precisely and effectively inducing tumor cell pyroptosis through the expression of GSDMB in tumor cells. Attached Figure Description

[0027] Figure 1: Construction of NDV-GSDMB and NDV-GSDME.

[0028] Figure 2: NDV-GSDMB synergistically controls the growth of triple-negative breast cancer tumors (4T1) with anti-PD-L1. A is the experimental design diagram; B is the growth curve of mouse breast cancer tumors; and C is the survival curve of mice.

[0029] Figure 3: NDV-GSDMB synergistically promotes anti-PD-L1 to enhance tumor antigen-specific (gp70 tetramer positive) CD8 + T cells infiltrate the tumor microenvironment, where A represents tumor antigen-specific (gp70 tetramer positive) CD8. + Flow cytometry diagram of T cells in tumor draining lymph nodes (TdLN), peripheral blood mononuclear cells (PBMC), and tumor tissue infiltrating T cells (TIL); B represents tumor antigen-specific (gp70 tetramer positive) CD8. + T cells account for a significant portion of all activated CD8 cells. + T cells (CD44) + CD8 + The proportion of T cells; C represents tumor antigen-specific (gp70 tetramer positive) CD8. + The absolute number of T cells per million PBMCs and per gram of tumor tissue.

[0030] Figure 4: NDV-GSDMB synergistically improves the tumor microenvironment with anti-PD-L1, transforming it from a "cold tumor" dominated by macrophages, MDSCs, Tregs, and neutrophils into a tumor dominated by NK cells and T cells. H1. "Hot tumor" dominated by B cells and cDC1, where A is a flow cytometry diagram of NK cells; B is a statistical graph of the absolute number of NK cells per gram of tumor tissue; C is a flow cytometry diagram of B cells; D is a statistical graph of the absolute number of B cells per gram of tumor tissue; E is a flow cytometry diagram of dendritic cells (DCs); F is a statistical graph of the absolute number of DCs per gram of tumor tissue; G is a flow cytometry diagram of cDC1 and cDC2 cells; H is a statistical graph of the absolute number of cDC1 and cDC2 cells per gram of tumor tissue; I is a flow cytometry diagram of the expression of cDC1 MHCI, CD80, and CD86 in NDV-GSDMB, NDV-GSDMB+aPD-1, NDV-GSDME, and NDV-GSDME+aPD-1; J is a statistical graph of I; K is a flow cytometry diagram of CD4+. + Flow cytometry diagram of T cells; L represents CD4. + Statistical graph of the absolute number of T cells per gram of tumor tissue; M represents Treg CD4 + Flow cytometry diagram of T cells; N represents Treg CD4. + The graph shows the absolute number of T cells per gram of tumor tissue; O is a flow cytometry diagram of macrophages and MDSCs; PQ is a graph showing the absolute number of macrophages (P) or MDSCs (Q) per gram of tumor tissue.

[0031] Figure 5: NDV-GSDMB synergistically improves the tumor microenvironment with anti-PD-L1, promoting tumor antigen-specific CD8. + T cells enter the tumor microenvironment.

[0032] Figure 6: NDV-GSDMB synergistically controls intrahepatic cholangiocarcinoma growth with anti-PD-L1. A is the experimental design flowchart; B is the survival curve of mice bearing intrahepatic cholangiocarcinoma.

[0033] Figure 7: NDV-GSDMB synergistically controls lung adenocarcinoma growth with anti-PD-L1. A is the experimental design flowchart; B is the survival curve of lung adenocarcinoma-bearing mice.

[0034] Figure 8: NDV-GSDMB synergistically controls spontaneous metastatic breast cancer growth with anti-PD-L1. A is the experimental design flowchart; B is the survival curve of tumor-bearing mice.

[0035] Figure 9: NDV-GSDMB synergistically controls spontaneous pancreatic ductal adenocarcinoma growth with anti-PD-L1. A is the experimental design flowchart; B is the survival curve of tumor-bearing mice.

[0036] Figure 10: Anti-tumor immune circulation. Detailed Implementation

[0037] Studies have found that the tumor microenvironment (TME) has transformed from a suppressive immune microenvironment characterized by low immune infiltration to one characterized by high immune infiltration, such as cytotoxic CD8. + T cells (Cytotoxicity CD8) + An increased number of T cells (CTLs) and an enhanced effector immune microenvironment are associated with the efficacy of ICB therapy. Therefore, improving the tumor-suppressive immune microenvironment and increasing the tumor-specific CD8+ killing effect are crucial. + T cell tumor infiltration and effector function are key to controlling tumor growth.

[0038] Tumor antigen-specific CD8 + T cells are the most important effector cells controlling tumors. Tumor-specific CD8+ + T cell-mediated anti-tumor immune responses are the main pathway by which the body combats the occurrence and development of tumors. The anti-tumor immune cycle (The Cancer-Immunity Cycle, Figure 10, Daniel S. Chen et al., Immunity, 2013) begins when antigens (①) produced by tumor cells are recognized, taken up, and processed by antigen-presenting cells (APCs, ②), and then presented to antigen-specific CD8+ cells in the tumor draining lymph nodes (TdLNs). + T cells (③). Antigen-specific CD8 + After T cells bind to the antigen peptide-MHC I complex (p-MHC I), they proliferate and differentiate into cytotoxic T lymphocytes (CTLs, ③), enter the bloodstream (④), and then mainly migrate and infiltrate tumor tissues through blood vessels (⑤). In tumor tissues, antigen-specific CTLs recognize tumor cells (⑥) and directly kill tumor cells by secreting effector molecules such as granzymes (Gzms) and perforin. In addition, CTLs also secrete some cytokines, such as IFN-γ, TNF-α, and IL-2, further enhancing the immune cell response (⑦).

[0039] Oncolytic viruses are a novel class of immunotherapeutic drugs used to treat tumors due to their selective infection of tumor cells. The main mechanisms by which oncolytic viruses inhibit tumors in vivo include: ① Oncolytic viruses multiply within tumor cells, producing numerous viral particles that lead to tumor lysis; ② As viruses, oncolytic viruses can induce systemic and local immunity, activating T cells to target and kill tumor cells. Because of their specific ability to infect tumor cells, oncolytic viruses can also serve as vectors carrying genes into tumor cells.

[0040] The inventors discovered that the GSDMB gene can be carried by oncolytic viruses, for example, by injecting small amounts of oncolytic virus multiple times, thereby inducing tumor cells to express GSDMB protein and mediating pyroptosis. A recombinant NDV-GSDMB strain was obtained by overexpressing human GSDMB protein in NDV virus (Figure 5). Intratumoral injection for tumors such as triple-negative breast cancer in combination with anti-PD-L1 immune checkpoint inhibitors showed that it can improve the tumor microenvironment, making it more susceptible to M2 macrophages, myeloid suppressor cells, neutrophils, and regulatory CD4+ cells. + The previously T-cell (Treg) dominant suppressive immune microenvironment has transformed into one dominated by antigen-specific CD8+. + T cells, T H An inflammatory immune microenvironment dominated by M1 cells and M1 macrophages plays a long-term role in controlling tumor growth. The inventors discovered that activating GSDMB produces a significantly better tumor-suppressing effect than activating other gasdermins such as GSDME, and that the combination of GSDMB with PD-1 / PD-L1 inhibitors produces a significantly better tumor-suppressing effect than the combination of other gasdermins such as GSDME with PD-1 / PD-L1 inhibitors.

[0041] In some embodiments, the present invention provides a novel method for expressing GSDMB in tumor cells and its application. In some embodiments, the present invention utilizes the characteristic of oncolytic viruses readily infecting tumor cells, inserting the GSDMB gene into the genome of the oncolytic virus, and further treating the tumor by intratumoral injection in combination with PD-1 or PD-L1 blocking antibodies. In some embodiments, the present invention provides the application of the oncolytic virus with the inserted GSDMB gene in anti-tumor treatment, wherein the oncolytic virus is Newcastle disease virus (NDV-GSDMB) with the inserted GSDMB gene, and the tumor may include PD-1 / PD-L1 inhibitor-resistant tumors such as triple-negative breast cancer. In some embodiments, the present invention provides co-administration of the NDV-GSDMB with PD-1 / PD-L1 immune checkpoint blockade therapy, preferably, the PD-1 / PD-L1 immune checkpoint blockade therapy is an anti-PD-L1 or PD-1 antibody.

[0042] In some implementations, the method for preparing recombinant viruses and treating tumors may include the following steps:

[0043] (1) Insert GSDMB (such as GSDMB3) into oncolytic viruses, such as NDV;

[0044] (2) Amplification of NDV-GSDMB oncolytic virus;

[0045] (3) Inject NDV-GSDMB into the tumor;

[0046] (4) Administer PD-1 / PD-L1 immune checkpoint blockade therapy with intratumoral injection of NDV-GSDMB.

[0047] In some embodiments, the objective of the present invention can be achieved by the following technical solution: inserting GSDMB (such as GSDMB3) into an oncolytic virus using genetic technology, amplifying the oncolytic virus, and treating tumors by intratumoral injection or intravenous injection of the oncolytic virus alone or in combination with PD-1 / PD-L1 immune checkpoint blockade therapy.

[0048] In some embodiments, the method described in this invention can precisely and effectively induce GSDMB expression in a subset of tumor cells, thereby causing pyroptosis. In some embodiments, it demonstrates a significant synergistic effect with PD-1 / PD-L1 immune checkpoint blockade therapy. This invention effectively improves the efficacy of tumor immunotherapy and provides new directions and inspiration for related research in this field.

[0049] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and are not intended to limit the scope of this application. Various objects and advantages of this application will become apparent to those skilled in the art from the following detailed description of preferred embodiments.

[0050] Example 1: Construction of NDV strains overexpressing GSDMB and GSDME (Figure 1)

[0051] 1. Modification of wild-type aSG10 NDV virus: This example utilizes wild-type aSG10 NDV virus, modifying it into a vector capable of expressing exogenous proteins. Two restriction enzyme sites, Pme I and Eag I, were introduced into the 5'UTR region of the NDV P gene as insertion sites for the exogenous gene. Furthermore, a full-length genome plasmid pOK-aSG10 containing Pme I and Eag I restriction sites was constructed.

[0052] Full-length aSG10 NDV sequence (SEQ ID NO: 1-2)

[0053] SEQ ID NO:1:

[0054] SEQ ID NO:2 (continued from SEQ ID NO:1)

[0055] 2. Preparation of GSDMB and GSDME genes: In this example, the GSDMB isoform3, isoform4, uniprot and GSDME gene sequences were submitted to a nucleic acid synthesis company for synthesis. The restriction enzyme site Pme I, gene termination sequence (GE), gene spacer sequence (base T), gene initiation sequence (GS) and kozak sequence were introduced upstream of the GSDMB gene, and the restriction enzyme site Eag I was introduced downstream of the GSDMB gene.

[0056] GSDMB isoform3(SEQ ID NO:3):

[0057] GSDMB isoform4(SEQ ID NO:4):

[0058] GSDMB uniprot (SEQ ID NO:5):

[0059] GSDME (SEQ ID NO:6):

[0060] 3. Construction of pOK-aSG10-GSDMB or pOK-aSG10-GSDME plasmids: In this example, the pOK-aSG10-mB plasmid was digested into a linear structure using two restriction endonucleases, Pme I and Eag I. The synthesized GSDMB and GSDME genes, containing GE, T, GS, kozak sequences and Pme I and Eag I restriction sites, were inserted into the linear fragment of pOK-aSG10-mB to construct the recombinant plasmids pOK-aSG10-GSDMB and pOK-aSG10-GSDME. Next-generation sequencing confirmed the successful construction of these plasmids.

[0061] 4. Construction of NDV-GSDMB and NDV-GSDME viruses: 10 μg of pOK-aSG10-GSDMB plasmid or 10 μg of pOK-aSG10-GSDME plasmid was mixed with helper plasmids pCI-NP, pCI-P, and pCI-L at a ratio of 4:2:1:1 and co-transfected into BSR T7 / 5 cells stably expressing T7 RNA polymerase. 3-4 days after transfection, the supernatant was harvested and inoculated into 10-day-old specific pathogen-free SPF chicken embryos to amplify the recovered virus. These recovered recombinant viruses were then passaged 5 times in chicken embryos. The presence of the target gene was confirmed by RT-PCR and sequencing analysis. The virus was aliquoted and stored at -80°C.

[0062] Example 2: Establishment of an oncolytic virus intratumoral injection xenograft model (Figure 2A)

[0063] 1. Tumor cell line preparation: In this example, the triple-negative breast cancer cell line 4T1 was used to establish an orthotopic xenograft model. These cell lines were passaged and cultured in vitro using DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 2 mM L-glutamine.

[0064] 2. Tumor cell inoculation: When the 4T1 fusion density of mouse tumor cells reaches approximately 80%-90%, the cells are digested, counted, and then the concentration of the tumor cell suspension is adjusted to 2×10⁻⁶ using 1X PBS buffer. 6 Cells / mL, use 1mL BD insulin injection to draw 0.1mL of tumor cell suspension; subcutaneous inoculation in 4T1 mammary gland: inject 200mL of 1.25% aphthol anesthetic into the peritoneum. After anesthetizing the mouse, remove the hair from the right lower abdomen with depilatory cream, gently grasp the mammary pad with flat forceps and lift it up. Hold the syringe in the other hand and insert the needle horizontally (at an angle of about 0-15° with the plane of the mouse mammary gland) into the mouse mammary pad tissue. Pull back the negative pressure to inject the tumor cell suspension into the mammary pad, forming a small wheal. After the injection is completed, quickly withdraw the needle parallel to the surface to prevent the cell suspension from flowing out.

[0065] 3. Observe tumor growth 8-10 days after cell injection. The tumor usually grows to the size of a soybean around day 8. Afterward, measure and record the tumor size every two days using calipers (tumor volume calculation formula: tumor volume = 1 / 2 length x 2, unit: mm). 3 The observation endpoint should be determined according to specific experimental requirements (Figure 2A). From an ethical perspective, when the tumor volume reaches 2000 mmHg... 3 The mouse was immediately declared dead.

[0066] Example 3: Intratumoral injection of oncolytic virus (Figure 2A)

[0067] 1. After the tumor grows to the size of a soybean, the mice are anesthetized and given 50 μL of a drug containing 1 x 10^6 viruses. 6 TCID50 NDV-GSDMB or NDV-GSDME is injected at three points within the tumor.

[0068] 2. After three consecutive injections, two more injections are given after a one-day interval. This constitutes one round of treatment.

[0069] 3. If necessary, the next round of treatment can be carried out after an interval of 5 days.

[0070] 4. After the first dose of oncolytic virus, administer 150mg of anti-PD-1 / PD-L1 treatment, one injection every three days, for a total of 4 injections.

[0071] Example 4: NDV oncolytic virus overexpressing GSDMB, combined with anti-PD-1 / PD-L1, can effectively control tumor growth (Figures 2B and 2C).

[0072] The inventors waited until the tumor volume reached 100-150 mm on day 8 of tumor-bearing mice. 3 Treatment with NDV-GSDMB or NDV-GSDME was initiated. Intratumoral injections of NDV-GSDMB or NDV-GSDME, or control NDV, were administered on days 8, 9, 10, 12, and 13 after tumor inoculation. PD-L1 blocking antibody (BioXCell, Cat#BE0101) or control reagent (IgG) were administered on days 9, 12, 15, and 18. Results showed that intratumoral injection of NDV-GSDMB combined with anti-PD-L1 had a significantly higher antitumor effect than other groups.

[0073] Example 5: NDV oncolytic virus overexpressing GSDMB, combined with anti-PD-1 / PD-L1, significantly promoted tumor antigen-specific CD8. + T cells infiltrate into tumor tissue. (Figure 3)

[0074] 1. The inventors treated tumor-bearing mice with one round of NDV, NDV-GSDMB, and NDV-GSDME oncolytic viruses and administered three doses of anti-PD-L1 blocking antibody.

[0075] 2. Euthanize the mouse, disinfect the skin tissue on the surface of the tumor, cut open the skin with scissors, and bluntly separate the tumor tissue with forceps. After the tumor tissue is completely separated, cut it into small pieces with scissors and place it in 4 mL of RPMI 1640 medium containing 10% type II collagen fibrillase. Incubate in a shaker at 37°C for 45 minutes.

[0076] 3. After further grinding the digested tissue with a grinder, mix each gram of tissue with 8 mL of a solution containing 44% percol. Slowly add the mixed liquid to a solution containing 67% percol.

[0077] 4. Centrifuge at 2200 rpm at room temperature for 22 minutes using a slow incline and descent method.

[0078] 5. Carefully remove the middle layer of white, hazy liquid and place it in 50 mL of 1xPBS. Centrifuge at 1800 rpm for 5 minutes at room temperature.

[0079] 6. After discarding the supernatant, stain and detect antigen-specific CD8. + T cells (LiveCD45) + CD8 + CD44 + gp70tetramer + The ratio and number of 4T1 antigen-specific CD8+ were observed. It was found that NDV-GSDMB combined with anti-PD-L1 resulted in more 4T1 antigen-specific CD8+ compared to other groups. + T cells infiltrate the TME.

[0080] The results showed that intratumoral injection of NDV-GSDMB combined with anti-PD-L1 significantly enhanced tumor-specific CD8 compared to other groups. + The number of T-cell infiltrators within the tumor.

[0081] Example 6: NDV oncolytic virus overexpressing GSDMB, combined with anti-PD-1 / PD-L1, can transform "cold tumors" with an immunosuppressive microenvironment dominated by MDSCs and Tregs into tumors dominated by NK cells and T cells. H 1. "Hot tumors" primarily promoting adaptive immune responses, such as cDC1. (Figure 4)

[0082] The inventors further analyzed other CD45 in the TME. + Immune cells were found to show that the combination of NDV-GSDMB and anti-PD-L1 resulted in more NK cells (LiveNK1.1) compared to other groups. + CD45.2 + B (LiveCD4) + CD45.2 + CD4 (LiveCD19) + CD45.2 + cDC1 (LiveMHCII) + CD11C + XCR1 + CD172a - Cells that promote adaptive tumor immune responses, such as those that infiltrate the TME, can infiltrate the TME.

[0083] Example 7: NDV oncolytic virus overexpressing GSDMB combined with anti-PD-1 / PD-L1 promotes high endothelial vein (HEV) formation and tumor antigen-specific CD8. + T cells enter the tumor parenchyma. (Figure 5)

[0084] 1. The inventors treated tumor-bearing mice with one round of NDV, NDV-GSDMB, and NDV-GSDME oncolytic viruses and administered three doses of anti-PD-L1 blocking antibody.

[0085] 2. Euthanize the mouse, disinfect the skin tissue on the surface of the tumor, cut open the skin with scissors, and bluntly separate the tumor tissue with forceps. After the tumor tissue is completely separated, fix it overnight in 4% paraformaldehyde fixative.

[0086] 3. After dehydration, paraffin embedding, sectioning, dewaxing, antigen retrieval, and blocking of the fixed tissue, immunofluorescence staining for DAPI, MECA79, and CD8a revealed that NDV-GSDMB combined with anti-PD-L1 resulted in more CD8a compared to other groups. + T cells infiltrate the TME and promote HEV formation.

[0087] The results showed that intratumoral injection of NDV-GSDMB combined with anti-PD-L1 significantly increased the number of immune cell subsets related to promoting immune response (cDC1, M1, etc.) and significantly reduced the number of cell subsets related to immunosuppression (Treg, M2, MDSC, etc.) in the tumor compared with other groups, thereby improving the tumor microenvironment and transforming it from a "cold" tumor to a "hot" tumor.

[0088] Example 8: NDV oncolytic virus overexpressing GSDMB combined with anti-PD-L1 can effectively control the growth of intrahepatic cholangiocarcinoma (Figure 6).

[0089] Establish an intrahepatic cholangiocarcinoma (ICC) model.

[0090] 1. Mix the plasmid in 2 mL of 1xPBS in the following amounts: 10 mg pNICD1 + 4 mg pAKT + 1 mg pSB100.

[0091] 2. The above-mentioned mixed plasmid was rapidly injected into the microvein of wild-type C57 mice to induce a semi-shock state in the mice.

[0092] 3. On the 8th day after modeling, the success of the model establishment was detected by a mouse in vivo imaging system.

[0093] NDV / NDV-GSDMB / NDV-GSDME in combination with anti-PD-L1

[0094] 4. Mice that were successfully modeled were injected with NDV / NDV-GSDMB / NDV-GSDME via the tail vein at times 8, 10, 13, 16, and 19 after modeling. Simultaneously, anti-PD-L1 was injected intraperitoneally at times 10, 13, 16, and 19 after modeling.

[0095] 5. Detect mouse mortality.

[0096] The results showed that intratumoral injection of NDV-GSDMB combined with anti-PD-L1 had a significantly better effect on controlling the growth of intrahepatic cholangiocarcinoma than other groups.

[0097] Example 9: NDV oncolytic virus overexpressing GSDMB combined with anti-PD-L1 can effectively control the growth of lung adenocarcinoma (Figure 7).

[0098] Establish a lung adenocarcinoma (LUAD) model.

[0099] 1. Packaging lentiviral particles containing Cre enzyme. The plasmid Lenti-LucOS (containing Cre enzyme) was mixed with psPAX2 (gag / pol) and pMD2.G plasmid at a ratio of 5 mg, 3 mg, and 2 mg, and then transfected into HEK-293T cells. The supernatant was collected 48 hours after transfection, concentrated, and the titer was determined before storage.

[0100] 2. KrasLSL-G12D / +-Trp53, 8-12 weeks old fl / fl mice (KP-LUAD), each mouse was administered 5 x 10 via endotracheal intubation 6 Transducing unit (TU) virus.

[0101] NDV / NDV-GSDMB / NDV-GSDME in combination with anti-PD-L1

[0102] 3. NDV, NDV-GSDMB, and NDV-GSDME viruses were administered twice weekly via nasal drops for 6-9 weeks after modeling.

[0103] 4. Intraperitoneal injection of anti-PD-L1 monoclonal antibody was administered 7-9.5 weeks after modeling.

[0104] 5. Detect mouse mortality.

[0105] The results showed that intratumoral injection of NDV-GSDMB combined with anti-PD-L1 had a significantly better effect on controlling the growth of lung adenocarcinoma than other groups.

[0106] Example 10: NDV oncolytic virus overexpressing GSDMB combined with anti-PD-L1 can effectively control the growth of metastatic breast cancer (MMTV-PyMT) (Figure 8).

[0107] Establish a spontaneous metastatic breast cancer model (MMTV-PyMT).

[0108] 1. The mice were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. as 8-10 week old magnetic mice.

[0109] 2. In this mouse, the cDNA sequence-specific expression of polyomavirus middle T antigen was driven by the long terminal repeat sequence of mouse mammary tumor virus (MMTV). The expression of polyomavirus middle T antigen led to widespread transformation of mammary epithelium and rapid development of multifocal breast adenocarcinoma.

[0110] 3. Approximately 50% of transgenic female mice develop palpable mammary tumors at 13 weeks of age, with the tumorigenesis rate increasing to around 80% by 19 weeks of age. Adenocarcinomas are more common in females; they are well-differentiated, multifocal, and eventually involve the entire mammary fat pad. The intermediate T antigen acts as a potent oncogene in mammary epithelial cells, and cells expressing it have enhanced metastatic potential, and can also develop into secondary metastatic tumors in the lungs.

[0111] NDV / NDV-GSDMB / NDV-GSDME in combination with anti-PD-L1

[0112] 4. NDV, NDV-GSDMB, and NDV-GSDME viruses are injected intratumorally every four days from 14 to 20 weeks after birth.

[0113] 5. Administer anti-PD-L1 monoclonal antibody intraperitoneally every five days from 15 to 18 weeks of age, for a total of four times.

[0114] 6. Detect mouse mortality.

[0115] The results showed that intratumoral injection of NDV-GSDMB combined with anti-PD-L1 had a significantly better effect on controlling the growth of metastatic breast cancer than other groups.

[0116] Example 11: NDV oncolytic virus overexpressing GSDMB, combined with anti-PD-L1, can effectively control the growth of spontaneous pancreatic ductal adenocarcinoma (PDAC) (Figure 9).

[0117] Establish a spontaneous pancreatic ductal adenocarcinoma model (PDAC).

[0118] Kras and Trp53 are key factors in the development of pancreatic cancer. Inactivation of the Kras gene combined with inactivation of the Trp53 tumor suppressor gene accelerates the development of pancreatic cancer in the model and promotes its metastasis to other sites. This model carries three genotypes: LSL-Kras G12D, LSL-Trp53 R172H, and Pdx1-Cre.

[0119] 1. First, LSL-Kras G12D(KI / +) mice were crossed with LSL-Trp53 R172H(KI / +) mice to produce LSL-Kras G12D(KI / +) and LSL-Trp53 R172H(KI / +) mice, i.e., KP mice. Then, KP mice were crossed with Pdx1-Cre mice to produce mice with three genotypes: LSL-Kras G12D(KI / +), LSL-Trp53 R172H(KI / +), and Pdx1-Cre(TG / +), i.e., KPC mice.

[0120] 2. At 8-10 weeks of age, KPC mice develop precursor lesions or pancreatic intraepithelial neoplasia (PanIN) in the pancreas; by 16 weeks of age, most KPC mice have developed locally invasive pancreatic ductal adenocarcinoma (PDAC) with intense fibroproliferative response.

[0121] NDV / NDV-GSDMB / NDV-GSDME in combination with anti-PD-L1

[0122] 3. NDV, NDV-GSDMB, and NDV-GSDME viruses are injected intratumorally every four days from 14 to 20 weeks after birth.

[0123] 4. Administer anti-PD-L1 monoclonal antibody intraperitoneally every five days from 15 to 18 weeks of age, for a total of four times.

[0124] 5. Detect mouse mortality.

[0125] The results showed that intratumoral injection of NDV-GSDMB combined with anti-PD-L1 had a significantly better effect on controlling PDAC growth than other groups.

[0126] References

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[0142] 16.Zhou Z,He H,Wang K,et al.Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells.Science 2020;368(6494).

[0143] 17.Zhang Z,Zhang Y,Xia S,et al.Gasdermin E suppresses tumour growth by activating anti-tumour immunity.Nature 2020;579(7799):415-20.

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Claims

1. A tumor cell GSDMB activator for use in treating PD-1 / PD-L1 inhibitor resistant tumors, restoring sensitivity to PD-1 / PD-L1 inhibitor treatment, and / or preventing resistance to PD-1 / PD-L1 inhibitor treatment.

2. The activator of claim 1, wherein the activator comprises a nucleic acid construct, a vector, or a recombinant oncolytic virus expressing GSDMB, such as a recombinant oncolytic virus of any one of (1) single-stranded DNA viruses, double-stranded DNA viruses, single-stranded positive-sense RNA viruses, single-stranded negative-sense RNA viruses, double-stranded RNA viruses; and (2) parvoviruses, adenoviruses, vaccinia viruses, herpesviruses, polioviruses, measles viruses, Newcastle disease viruses, and reoviruses, such as the recombinant oncolytic virus comprising a recombinant Newcastle disease virus NDV-GSDMB.

3. The activator of claim 1 or 2, wherein the activator is used as an active ingredient of a medicament, such as a medicament for systemic, intraperitoneal, or intratumoral injection, preferably intratumoral injection.

4. The activator of any one of claims 1-3, wherein the activator is used as an active ingredient of a medicament, such as a medicament used in combination with an additional anti-cancer therapy, wherein the additional anti-cancer therapy comprises, for example, an immune checkpoint blockade therapy, preferably a PD-1 / PD-L1 immune checkpoint blockade therapy, such as the GSDMB activator is further used in combination with a PD-1 / PD-L1 inhibitor, optionally wherein the PD-1 / PD-L1 inhibitor comprises a PD-1 / PD-L1 antibody.

5. The activator of any one of claims 1-4, wherein the PD-1 / PD-L1 inhibitor resistant tumors comprise tumors in a tumor patient with an overall response rate to a PD-1 / PD-L1 inhibitor of less than 50%, and / or tumors that have undergone PD-1 / PD-L1 inhibitor treatment and found to have a reduced treatment response, such as breast cancer, such as triple-negative breast cancer, pancreatic cancer, cholangiocarcinoma, such as intrahepatic cholangiocarcinoma, hepatocarcinoma, melanoma, colorectal cancer, brain glioma, renal cancer, lung cancer, such as small cell lung cancer and non-small cell lung cancer.

6. The activator of any one of claims 1-5, wherein the GSDMB comprises a GSDMB full-length gene or a fragment thereof, such as a GSDMB gene fragment comprising granzyme A (GZMA) cleavage sites, such as lysine at positions 229 and / or 244, optionally wherein the expression comprises overexpression.

7. A method for treating PD-1 / PD-L1 inhibitor resistant tumors, restoring sensitivity to PD-1 / PD-L1 inhibitor treatment, and / or preventing resistance to PD-1 / PD-L1 inhibitor treatment, comprising administering to an individual in need thereof an effective amount of a tumor cell GSDMB activator as defined in any one of claims 1-6.

8. A pharmaceutical composition comprising a tumor cell GSDMB activator as defined in any one of claims 1-6, optionally, the pharmaceutical composition further comprises an immunosuppressive agent, e.g., a PD-1 / PD-L1 inhibitor, an excipient, a pharmaceutical carrier or a buffer.

9. A kit comprising a tumor cell GSDMB activator as defined in any one of claims 1-6, optionally, the kit further comprises an immunosuppressive agent, e.g., a PD-1 / PD-L1 inhibitor, optionally, it is used for treating a PD-1 / PD-L1 inhibitor resistant tumor.

10. A method of making a tumor cell GSDMB activator as defined in any one of claims 1-6, wherein the tumor cell GSDMB activator is a recombinant oncolytic virus expressing GSDMB, the method comprising introducing a nucleic acid molecule encoding GSDMB in the genome of a recombinant oncolytic virus to make a recombinant oncolytic virus expressing GSDMB.

11. Use of a tumor cell GSDMB activator as defined in any one of claims 1-6 for the manufacture of a medicament for treating a PD-1 / PD-L1 inhibitor resistant tumor, restoring sensitivity to PD-1 / PD-L1 inhibitor treatment and / or preventing resistance to PD-1 / PD-L1 inhibitor treatment.

Images