Il33-loaded oncolytic vaccinia virus and combination therapy thereof with immune checkpoint inhibitor

Abstract

Provided in the present invention are an IL33-loaded oncolytic vaccinia virus and a combination therapy thereof with an immune checkpoint inhibitor. Specifically, provided in the present invention are an oncolytic vaccinia virus loaded with a human or mouse IL33 gene, a construction method therefor and the use thereof with an immune checkpoint inhibitor. The oncolytic vaccinia virus of the present invention has a significant in vitro inhibitory effect on a variety of tumor cells such as ovarian cancer, bladder cancer and liver cancer cells. The use of the oncolytic vaccinia virus in combination with an immune checkpoint inhibitor has a significant in vivo therapeutic effect on tumors. The present invention provides a new method for virus treatment of tumors, and has clinical application prospects.

Description

Oncolytic vaccinia virus loaded with IL33 and its combination therapy with immune checkpoint inhibitors Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and specifically to a method for constructing a replicative oncolytic vaccinia virus expressing human or mouse IL33, and a combined treatment method thereof with an immune checkpoint protein inhibitor. Background Art

[0002] Normal tissue homeostasis is a highly regulated process of cell proliferation and cell death. Imbalances in cell proliferation or cell death can develop into cancer. Examples include cervical cancer, kidney cancer, lung cancer, pancreatic cancer, colorectal cancer, and brain cancer. According to the "2018 Global Cancer Statistics" report, there were approximately 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018. China accounted for 3.804 million new cases and 2.296 million deaths. Compared to other countries, my country ranks first in both cancer incidence and mortality.

[0003] The emergence of immune checkpoint inhibitors (ICIs) has rapidly changed the treatment paradigm for a variety of cancers, and PD-(L)1 inhibitors have become a standard treatment for more than 20 different cancer indications. However, with the exception of certain diseases (e.g., melanoma, Merkel cell, Hodgkin lymphoma, and MSI-H tumors), where patients have a high response rate (40%-70%) to single-agent PD-1 blockade, the response rate for most other cancer indications is only 10-25%, and even in patients who initially respond to ICIs, the disease may eventually progress.

[0004] Similarly, oncolytic viruses (OVs) are an emerging class of tumor therapeutics that can selectively replicate in tumor cells, deliver multiple therapeutic genes, induce immunogenic cell death (ICD), and promote anti-tumor immunity. Because OVs can break down the immunosuppressive tumor microenvironment (TME) and promote the recruitment of immune cells into the TME, OVs are believed to synergize with ICIs to improve the effectiveness of immune checkpoint blockade (ICB). Clinical trial studies have shown that the combination of oncolytic virus T-vec and PD-1 antibody increases the objective response rate of melanoma by 55% compared to single-agent PD-1 antibody, confirming that OVs are a new treatment approach to improve the response to immune checkpoint inhibitors. Currently, the herpes virus T-VEC has been approved by the FDA for the treatment of melanoma. Overall, oncolytic viruses have demonstrated considerable safety and efficacy in clinical trials.

[0005] Therefore, there is a need in this field to develop new oncolytic viruses and their combined treatment methods with immune checkpoint inhibitors. Summary of the Invention

[0006] The purpose of the present invention is to provide an oncolytic vaccinia virus loaded with IL33 and its combination therapy with immune checkpoint inhibitors.

[0007] In a first aspect of the present invention, a recombinant oncolytic virus is provided, wherein the recombinant oncolytic virus expresses interleukin 33 (IL33).

[0008] In another preferred embodiment, the recombinant oncolytic virus is selected from the following group: vaccinia virus, herpes simplex virus, and adenovirus.

[0009] In another preferred embodiment, the herpes simplex virus is herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2).

[0010] In another preferred embodiment, the vaccinia virus is selected from the following group: vaccinia virus Copenhagen strain, vaccinia virus Western Reserve strain, vaccinia virus Lister strain, vaccinia virus Wyeth strain or vaccinia virus Tiantan strain.

[0011] In another preferred embodiment, the IL33 is human or mouse IL33.

[0012] In another preferred embodiment, the DNA sequence encoding the human IL33 is shown as SEQ ID NO: 1.

[0013] In another preferred embodiment, the DNA sequence encoding the mouse IL33 is shown as SEQ ID NO: 2.

[0014] In another preferred embodiment, the recombinant oncolytic virus contains an exogenous human or mouse IL33 gene.

[0015] In another preferred embodiment, the functional vaccinia growth factor F3L region gene of the vaccinia virus is completely or partially deleted.

[0016] In another preferred embodiment, the vaccinia virus comprises functional F2L and / or F4L genes.

[0017] In a second aspect of the present invention, a method for preparing the recombinant oncolytic virus according to the first aspect of the present invention is provided, comprising the following steps:

[0018] (A) providing an oncolytic virus to be modified;

[0019] (B) Introducing the IL33 encoding nucleic acid into the oncolytic virus to be modified, thereby obtaining the recombinant oncolytic virus.

[0020] In another preferred embodiment, the IL33 is human or mouse IL33.

[0021] In another preferred embodiment, in step B, the IL33 encoding nucleic acid is introduced into the oncolytic virus to be modified via a recombinant vector, wherein the recombinant vector contains the IL33 encoding nucleic acid.

[0022] In another preferred embodiment, the oncolytic virus to be modified is vaccinia virus, preferably the Copenhagen strain of vaccinia virus.

[0023] In another preferred embodiment, the recombinant vector is a plasmid.

[0024] In another preferred embodiment, the recombinant vector is a pUC57-VV plasmid.

[0025] In another preferred embodiment, the IL33 encoding nucleic acid is inserted into the F3L region of the oncolytic virus.

[0026] In another preferred embodiment, the F3L region gene of the vaccinia virus is completely or partially deleted.

[0027] In a third aspect of the present invention, an F3L-deficient vaccinia virus is provided, wherein the functional vaccinia growth factor F3L region gene of the F3L-deficient vaccinia virus is completely or partially deleted.

[0028] In another preferred embodiment, the F3L region gene of the F3L-deficient vaccinia virus is deleted by at least 30%, preferably at least 50%, and more preferably at least 80% relative to the wild type.

[0029] In another preferred embodiment, the vaccinia virus is selected from the following group: vaccinia virus Copenhagen strain, vaccinia virus Western Reserve strain, vaccinia virus Lister strain, vaccinia virus Wyeth strain or vaccinia virus Tiantan strain.

[0030] In another preferred embodiment, the F3L-deficient vaccinia virus expresses interleukin 33 (IL33).

[0031] In another preferred embodiment, the IL33 is human or mouse IL33.

[0032] In another preferred embodiment, the DNA sequence encoding the human IL33 is shown as SEQ ID NO: 1.

[0033] In another preferred embodiment, the DNA sequence encoding the mouse IL33 is shown as SEQ ID NO: 2.

[0034] In another preferred embodiment, the vaccinia virus contains an exogenous human or mouse IL33 gene.

[0035] In another preferred embodiment, the vaccinia virus comprises functional F2L and / or F4L genes.

[0036] In a fourth aspect of the present invention, there is provided an active ingredient combination, comprising:

[0037] (M1) a first active ingredient, the first active ingredient being selected from the group consisting of the recombinant oncolytic virus according to the first aspect of the present invention, the F3L-deficient vaccinia virus according to the third aspect of the present invention, or a combination thereof; and

[0038] (M2) a second active ingredient, wherein the second active ingredient is an immune checkpoint inhibitor.

[0039] In another preferred embodiment, the immune checkpoint inhibitor is an antibody or a fragment thereof that specifically binds to the immune checkpoint protein, preferably a monoclonal antibody, a humanized antibody, a fully human antibody, or a fusion protein thereof, or a combination thereof.

[0040] In another preferred embodiment, the immune checkpoint inhibitor inhibits an immune checkpoint protein selected from the group consisting of cytotoxic T lymphocyte antigen 4 (CTLA4), programmed cell death protein 1 (PD-1), PD-L1, PD-L2, B7-H3, B7-H4, herpes virus entry mediator (HVEM), T cell membrane protein 3 (TIM3), galectin 9 (GAL9), lymphocyte activation gene 3 (LAG3), T cell activation inhibitor (VISTA) containing V domain immunoglobulin (Ig), killer cell immunoglobulin-like receptor (KIR), B and T lymphocyte attenuator (BTLA), T cell immunoreceptor with Ig and Π'IM domains (TIGIT), indoleamine 2,3-dioxygenase (IDO) or a combination thereof.

[0041] In another preferred embodiment, the immune checkpoint inhibitor is a monoclonal antibody that specifically binds to PD-1 or PD-L1.

[0042] In another preferred embodiment, the immune checkpoint inhibitor is selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, lambrolizumab, or a combination thereof.

[0043] In another preferred embodiment, the immune checkpoint inhibitor is a monoclonal antibody that specifically binds to CTLA4.

[0044] In another preferred embodiment, the immune checkpoint inhibitor is selected from the following group: ipilimumab, tremelimumab, or a combination thereof.

[0045] In another preferred embodiment, the second active ingredient includes one or more immune checkpoint inhibitors.

[0046] In another preferred embodiment, the active ingredient combination comprises:

[0047] (a) a CTLA4 inhibitor, a PD-1 inhibitor and the first active ingredient;

[0048] (b) a CTLA4 inhibitor, an IDO inhibitor and the first active ingredient;

[0049] (c) a PD-1 inhibitor, an IDO inhibitor, and the first active ingredient;

[0050] (d) a PD-1 inhibitor, a CTLA4 inhibitor, an IDO inhibitor, and the first active ingredient;

[0051] (e) a LAG3 inhibitor, a PD-1 inhibitor and the first active ingredient; or

[0052] (f) a TIGIT inhibitor, a PD-1 inhibitor and the first active ingredient.

[0053] In the fifth aspect of the present invention, provided is the use of the recombinant oncolytic virus as described in the first aspect of the present invention, the F3L-deficient vaccinia virus as described in the third aspect of the present invention, or the active ingredient combination as described in the fourth aspect of the present invention in the preparation of a pharmaceutical composition or a kit for treating tumors.

[0054] In another preferred embodiment, the tumor is selected from the group consisting of bladder cancer, ovarian cancer, liver cancer, pancreatic cancer, cervical cancer and osteosarcoma.

[0055] In another preferred embodiment, the tumor includes metastatic tumors and non-metastatic tumors.

[0056] In another preferred embodiment, the tumor is peritoneal metastatic bladder cancer.

[0057] In another preferred embodiment, the tumor does not express immune checkpoint proteins or expresses immune checkpoint proteins at relatively low levels.

[0058] In a sixth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0059] (1) a therapeutically effective amount of the recombinant oncolytic virus of the first aspect of the present invention, or the F3L-deficient vaccinia virus of the third aspect of the present invention, or the active ingredient combination of the fourth aspect of the present invention, or a combination thereof; and

[0060] (2) Pharmaceutically acceptable carrier.

[0061] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0062] In a seventh aspect of the present invention, a medicine kit is provided, comprising:

[0063] (a) a first preparation comprising a therapeutically effective amount of the recombinant oncolytic virus according to the first aspect of the present invention or the F3L-deficient vaccinia virus according to the third aspect of the present invention, and a pharmaceutically acceptable carrier; and

[0064] (b) a second formulation comprising an immune checkpoint inhibitor.

[0065] In another preferred embodiment, the drug kit further includes instructions, which describe a method for treating tumors by combining the recombinant oncolytic virus and an immune checkpoint inhibitor.

[0066] In another preferred embodiment, the first preparation and the second preparation are independent of each other.

[0067] In another preferred embodiment, the first preparation and the second preparation are freeze-dried preparations or liquid preparations, respectively.

[0068] In another preferred embodiment, the first preparation and the second preparation are injections.

[0069] In another preferred embodiment, the first preparation is administered before, during or after the administration of the second preparation.

[0070] In another preferred embodiment, the first preparation is administered by intratumoral injection.

[0071] In another preferred embodiment, the second preparation is administered by intratumoral, intravenous or intraperitoneal injection.

[0072] In the eighth aspect of the present invention, a method for treating a tumor in a subject in need is provided, comprising the steps of administering to a subject in need a therapeutically effective amount of the recombinant oncolytic virus as described in the first aspect of the present invention, or the F3L-deficient vaccinia virus as described in the third aspect of the present invention, or the active ingredient combination as described in the fourth aspect of the present invention, or the pharmaceutical composition as described in the sixth aspect of the present invention.

[0073] In another preferred embodiment, the subject in need thereof is a human or non-human mammal.

[0074] In another preferred embodiment, the non-human mammals include monkeys, gorillas, cows, pigs, dogs, sheep, rabbits or mice.

[0075] In another preferred embodiment, the subject in need is a mouse or a human.

[0076] In another preferred embodiment, the administration is intratumoral, intravenous or intraperitoneal administration.

[0077] In another preferred embodiment, the recombinant oncolytic virus is administered intratumorally.

[0078] In another preferred embodiment, the recombinant oncolytic virus is administered in an amount effective to induce the expression of immune checkpoint proteins in the tumor.

[0079] In another preferred example, before administering the recombinant oncolytic virus, the tumor does not express the immune checkpoint protein or expresses the immune checkpoint protein at a relatively low level.

[0080] In another preferred embodiment, multiple immune checkpoint inhibitors and the recombinant oncolytic virus are administered to the subject simultaneously.

[0081] In another preferred embodiment, the recombinant oncolytic virus is at about 104 to about 10 9 pfu, preferably about 10 7 pfu were administered.

[0082] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0084] Figure 1 shows the plasmid maps of pUC57-VV-mCherry and pUC57-VV-IL33.

[0085] FIG2 shows the ELISA and QRT-PCR identification results of the constructed oncolytic vaccinia virus OVV-hIL33.

[0086] Figure 3 shows the real-time cell analysis (RTCA) method for real-time monitoring of the half-inhibitory dose (MOI) of the oncolytic vaccinia virus OVV-hIL33 on human tumor cells SKOV3, ES-2, OVCAR-3, Caov-3, T24, UMUC3, BEL-7404 and MHCC97-H at different time periods. OVV-NC is an empty control virus.

[0087] Figure 4 shows the half-inhibitory dose (MOI) of oncolytic vaccinia virus OVV-mIL33 on mouse tumor cells ID8, MB49 and H22 detected by Realtime cell analysis (RTCA), and OVV-NC is the control virus.

[0088] Figure 5 shows that the oncolytic vaccinia virus OVV-NC significantly inhibits the inhibitory effect of mouse colorectal cancer cell CT26 homograft tumors compared to the vaccinia virus Copenhagen wild-type strain.

[0089] Figure 6 shows the inhibitory effect of oncolytic vaccinia virus OVV-hIL33 on human ovarian cancer cell SKOV3 xenografts in nude mice in vivo, with PBS and OVV-NC as controls.

[0090] FIG7 shows that the luciferase gene was successfully transfected into mouse tumor cells ID8 and MB49.

[0091] FIG8 shows the inhibitory effect of oncolytic vaccinia virus OVV-mIL33 on mouse ovarian cancer cell ID8 homograft tumors in vivo, with PBS and OVV-NC as controls.

[0092] FIG9 shows the inhibitory effect of oncolytic vaccinia virus OVV-mIL33 on mouse bladder cancer cell MB49 homografts in vivo, with PBS and OVV-NC as controls.

[0093] Figure 10 shows the inhibitory effects of oncolytic vaccinia virus alone or in combination with mouse PD-1 monoclonal antibody on mouse bladder cancer cell MB49 homograft tumors in vivo.

[0094] Figure 11 shows the changes in serum INF-γ levels during the treatment of mouse bladder cancer with oncolytic vaccinia virus alone or with mouse PD-1 monoclonal antibody alone or in combination.

[0095] Figure 12 shows the HE staining results of paraffin sections of mouse bladder cancer treated with oncolytic vaccinia virus alone or with mouse PD-1 monoclonal antibody alone or in combination.

[0096] Figure 13 shows the immunohistochemical detection of mIL33 expression in mouse bladder cancer tissues treated with oncolytic vaccinia virus alone or with mouse PD-1 monoclonal antibody alone or in combination.

[0097] Figure 14 shows the expression of Vaccinia virus / PD-L1 in bladder cancer tissues of mice treated with oncolytic vaccinia virus alone or with mouse PD-1 monoclonal antibody alone or in combination.

[0098] Figure 15 shows the expression of PD-1 in mouse bladder cancer tissues detected by mIHC using oncolytic vaccinia virus alone or in combination with mouse PD-1 monoclonal antibody.

[0099] Figure 16 shows the mIHC detection of ILC2 cell infiltration in mouse bladder cancer tissues treated with oncolytic vaccinia virus alone or with mouse PD-1 monoclonal antibody alone or in combination.

[0100] Figure 17 shows the mIHC detection of CD8+T lymphocyte infiltration in mouse bladder cancer tissues treated with oncolytic vaccinia virus alone or with mouse PD-1 monoclonal antibody alone or in combination. DETAILED DESCRIPTION

[0101] After extensive and in-depth research, the inventors have developed for the first time an oncolytic vaccinia virus loaded with the human or mouse IL33 gene and its combined use with immune checkpoint inhibitors. Experiments have confirmed that the oncolytic vaccinia viruses of the present invention, OVV-hIL33 and OVV-mIL33, not only have significant in vitro inhibitory effects on a variety of tumor cells, such as ovarian cancer, bladder cancer, and liver cancer cells, but also have significant therapeutic effects in animal models of tumors when combined with immune checkpoint inhibitors. The present invention can be used to prepare anti-tumor viral drugs, providing a new method for viral treatment of tumors. On this basis, the present invention was completed.

[0102] This invention provides a combination therapy of an IL33-loaded oncolytic vaccinia virus and ICIs. On the one hand, the "oncolytic" effect induces tumor cells to express PD-L1, recruiting T cell infiltration in the TIME. On the other hand, targeted in situ expression of IL33 in the tumor amplifies ILC2s and activates DCs, indirectly recruiting T cell infiltration. When PD-1 inhibition of T lymphocytes is relieved, the efficacy against "immune desert" and "immune exclusion" tumors is significantly enhanced.

[0103] As described in Examples 1 and 2, the oncolytic vaccinia virus OVV-hIL33 demonstrated significant in vitro inhibitory effects on human ovarian, bladder, and liver cancer cells, as determined by RTCA, in a dose- and time-dependent manner. Compared to the control virus, OVV-mIL33 demonstrated superior therapeutic efficacy in tumor-bearing mice, significantly prolonging their survival.

[0104] As described in Example 3, compared with the untreated control, OVV-hIL33 has a significant therapeutic effect on xenograft tumor-bearing mice.

[0105] As described in Examples 4 and 5, the combination of OVV-mIL33 and mouse PD-1 monoclonal antibody has a significant therapeutic effect on homologous transplanted tumor animal models, including inhibiting the growth of existing tumors or preventing metastasis, and significantly prolonging the survival time of tumor-bearing mice.

[0106] the term

[0107] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0108] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0109] As used herein, the terms “contain,” “have,” or “include” include “comprise,” “mainly consist of,” “substantially consist of,” and “consist of”; “mainly consist of,” “substantially consist of,” and “consist of” are subordinate concepts of “contain,” “have,” or “include.”

[0110] Recombinant oncolytic virus and construction method thereof

[0111] As used herein, the terms "recombinant oncolytic virus of the present invention" and "oncolytic virus of the present invention" refer to the oncolytic virus expressing IL33 described in the first aspect of the present invention.

[0112] IL33 is an IL-1 family protein secreted primarily by non-hematopoietic cells (fibroblasts, epithelial cells, endothelial cells, etc.). IL33 has been shown to regulate the activation of dendritic cells (DCs) through ST2 signaling, thereby activating anti-tumor immune responses. IL33 is also an essential factor for the growth of type II innate lymphoid cells (ILC2s). IL33-activated ILC2s can also produce apoptotic ligands and inhibit melanoma, lymphoma, and colon cancer. Existing technologies have revealed that IL33 stimulates the expansion of ILC2s in pancreatic cancer and secretes CCL5 to recruit CD103+ DCs, activating anti-tumor CD8+ T cells. Studies have shown that anti-tumor ILC2s are also inhibited by the PD-1 pathway. Blocking the PD-1 pathway in ILC2s can co-activate ILC2s and other anti-cancer lymphocytes, inhibiting pancreatic cancers that are poorly responsive to PD-1 monoclonal antibodies.

[0113] In one embodiment, the oncolytic virus of the present invention can be vaccinia virus. Vaccinia virus is a double-stranded DNA virus with an envelope. Compared with other oncolytic viruses, it has the following advantages: (1) It is unique among DNA viruses and replicates only in the cytoplasm, which can minimize the risk of integration into the host genome; (2) Vaccinia virus was first made into a vaccine to fight smallpox virus, and its safety is greatly guaranteed; (3) Vaccinia virus has strong cloning ability and can allow the insertion of gene fragments of larger lengths; (4) Vaccinia virus has shown the ability to target and aggregate to cancer tissues in both experimental animals and humans. Therefore, vaccinia virus has become an ideal viral vector for cancer treatment.

[0114] As used herein, the terms "vaccinia virus of the present invention," "oncolytic vaccinia virus of the present invention," "OVV-hIL33," and "OVV-mIL33" all refer to oncolytic vaccinia viruses constructed according to the present invention that express the IL33 gene. "OVV-hIL33" expresses the human IL33 gene (GenBank: NM_033439), while "OVV-mIL33" expresses the mouse IL33 gene (GenBank: NM_133775).

[0115] The DNA sequence of human IL33 gene is shown in SEQ ID NO.1:

[0116] SEQ ID NO:1 (human IL33)

[0117] The DNA sequence of the mouse IL33 gene is shown in SEQ ID NO. 2:

[0118] SEQ ID NO:2 (murine IL33)

[0119] The oncolytic vaccinia virus of the present invention can be a vaccinia virus Copenhagen strain, a vaccinia virus Western Reserve strain, a vaccinia virus Lister strain, a vaccinia virus Wyeth strain or a vaccinia virus Tiantan strain.

[0120] The present invention also provides a method for constructing the oncolytic virus of the present invention, comprising introducing an IL33 encoding nucleic acid into the oncolytic virus to be modified, thereby obtaining the oncolytic virus of the present invention.

[0121] Taking vaccinia virus as an example, in one embodiment, the construction method of the present invention includes the following three steps:

[0122] (A) Site-directed mutagenesis was used to introduce an Nhe I site between F2L and F3L_R in pUC57-VV-mCherry.

[0123] (B) The gene sequence of human IL33 (hIL33) or mouse IL33 (mIL33) was inserted into the pUC57-VV plasmid via the Sal I and Nhe I sites to obtain the pUC57-VV-hIL33 plasmid or the pUC57-VV-mIL33 plasmid;

[0124] (C) pUC57-VV-hIL33 plasmid or pUC57-VV-mIL33 plasmid and vaccinia virus were recombined in U-2OD cells. After screening and identification, oncolytic vaccinia viruses carrying the human or mouse IL33 gene were obtained.

[0125] In one embodiment, the OVV-hIL33 and OVV-mIL33 vaccinia viruses are obtained using the Copenhagen strain of vaccinia virus and the pUC57-VV-hIL33 plasmid or the pUC57-VV-mIL33 plasmid.

[0126] In one embodiment, in steps (B) and (C), during recombination, vaccinia virus Copenhagen strain Cop-mCherry (Cop-mCherry is recombined from wild-type Copenhagen and pUC57-VV-mCherry plasmid in U-2OS cells) and pUC57-VV-hIL33 plasmid or pUC57-VV-mIL33 plasmid are recombined in U-2OS cells, and cell transfection is performed according to the instructions of the kit (Lipofectamine 3000 transfection reagent name); during screening, FACS fluorescence (mCherry-) is used to screen the recombinant virus liquid, and the recombinant virus is isolated by plaque assay; the recombinant virus specifically expresses the human or mouse IL33 gene, and PCR and ELISA identification are performed to obtain purified oncolytic vaccinia viruses OVV-hIL33 and OVV-mIL33.

[0127] The present invention also provides an FL3-deficient vaccinia virus. Compared to the wild-type vaccinia virus, the FL3-deficient vaccinia virus of the present invention has a complete or partial deletion of the functional vaccinia growth factor F3L region gene.

[0128] Pharmaceutical composition and administration method

[0129] The present invention also provides a pharmaceutical composition, which can be used to inhibit tumor growth and / or metastasis.

[0130] The pharmaceutical composition of the present invention comprises: an effective amount of the oncolytic virus of the present invention, or a combination thereof with an active ingredient of an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier.

[0131] Typically, the oncolytic virus or active ingredient combination of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable carrier medium, wherein the pH is generally about 5-8, preferably, about pH 6-8.

[0132] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and / or cells and can be accepted by humans and / or animals.

[0133] As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), that is, substances with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, polysorbate, ethanol, and combinations thereof. Generally, pharmaceutical preparations should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be prepared into a sustained-release preparation.

[0134] In addition, the oncolytic virus or active ingredient combination of the present invention can also be used together with other therapeutic agents (such as anti-tumor agents or chemotherapeutic agents).

[0135] When using the pharmaceutical composition, a safe and effective amount of the oncolytic virus or active ingredient combination of the present invention is administered to a mammal.

[0136] It should be understood that the effective amount of each active ingredient (or its formulation) in the pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the tumor. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to, pharmacokinetic parameters such as bioavailability, metabolism, and half-life; tumor severity; patient weight; patient immune status; and route of administration.

[0137] Typically, for oncolytic viruses, such as vaccinia virus, the therapeutically effective dose is about 10 4 to about 10 9 pfu, preferably about 10 7 For immune checkpoint inhibitors, the safe and effective dose is generally at least about 1 mg / kg body weight, and in most cases no more than about 20 mg / kg body weight, preferably about 2 mg / kg body weight to 10 mg / kg body weight. Of course, the specific dose should also take into account factors such as the type of drug used, dosage form, route of administration, and patient health status, all of which are within the skill of a skilled physician.

[0138] There is no particular limitation on the administration method of the pharmaceutical composition of the present invention. Representative examples include (but are not limited to): intratumoral injection, intraperitoneal injection, and intravenous injection.

[0139] The main advantages of the present invention include:

[0140] 1) The OVV-IL33 oncolytic virus and FL3-deficient OOV-NC virus of the present invention can produce significantly stronger tumor inhibitory effects than wild-type or empty-type viruses in a variety of cancer types.

[0141] 2) When the OVV-IL33 oncolytic virus of the present invention is used in combination with immune checkpoint inhibitors (such as anti-PD-1 antibodies), it can produce a synergistic effect, significantly improving the tumor inhibition rate, and its tumor inhibition rate is further increased compared with the therapy of combining the empty virus OVV-NC with anti-PD-1 antibodies.

[0142] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0143] The following examples describe the construction and application of oncolytic vaccinia viruses using the recombination of the Copenhagen strain of vaccinia virus with the human IL33 gene as an example. However, the scope of protection of the present invention is not limited thereto and is also applicable to the recombination of other vaccinia virus strains with the IL33 gene.

[0144] Example 1 Construction and identification of vaccinia virus OVV-hIL33

[0145] 1. The human IL33 gene sequence (SEQ ID NO. 1) was inserted into the pUC57-VV-mCherry plasmid through the Sal I and Nhe I sites to obtain the pUC57-VV-hIL33 plasmid.

[0146] The map of the pUC57-VV-hIL33 plasmid is shown in Figure 1. ΔF3L and F2L are homologously recombined with the F3L region of the wild-type virus to insert the exogenous gene into F3L, while causing partial or complete deletion of F3L, thereby obtaining the selectivity of the recombinant vaccinia virus for specific amplification in tumor cells.

[0147] 2. Recombination of Copenhagen strain vaccinia virus Cop-mCherry and pUC57-VV-hIL33 plasmid was performed as follows:

[0148] (1) At 6cm 2 An appropriate number of U-2OS cells were seeded into a culture dish to allow them to grow to 80-90% of the total size on the next day.

[0149] (2) Discard the culture medium and add 1 mL of virus solution (0.05-0.1 MOI, diluted with culture medium containing 2% serum). Incubate the cells in a 37°C, 5% CO2 incubator for 1 hour, shaking them approximately every 15 minutes to prevent local cell death.

[0150] (3) Cell transfection was performed according to the instructions of the kit (Lifofectamine 3000), as follows:

[0151] Add 2 μg of pUC57-VV-hIL33 to 150 μL of OptiMEM, then add 4 μL of P3000 to each volume, invert and mix five times, and let stand at room temperature for 5 minutes. Add 4 μL of Lifofectamine 3000 to 150 μL of OptiMEM, shake for 3 seconds, and let stand at room temperature for 5 minutes. Combine the two volumes, invert and mix five times, and let stand at room temperature for 5-10 minutes.

[0152] At the same time, discard the virus solution in step 2, add 4 mL of fresh culture medium containing 10% FBS, and add the mixed transfection solution into them respectively. Then place the culture dish in a 37°C, 5% CO2 incubator and culture for 6 to 18 hours, then aspirate the culture medium, wash once with PBS, and add 5 mL of fresh culture medium to continue culture.

[0153] (4) After the cells are completely diseased, the virus solution is collected in a biosafety cabinet, divided into centrifuge tubes, labeled, and the centrifuge tubes are repeatedly frozen and thawed three times at -80°C and 37°C to completely lyse the cells and release the virus. The supernatant is collected by centrifugation at 2000 rpm for 5 minutes and placed in a -80°C ultra-low temperature refrigerator for storage.

[0154] 3. Screening of recombinant viruses: The steps are as follows:

[0155] (1) U-2OS cells in good growth condition were inoculated into a culture dish. The cell density could reach about 80%-90% the next day.

[0156] U-2OS cells with good growth status were seeded into 96-well cell culture plates. The cell density could reach about 50%-70% the next day.

[0157] (3) Carefully add 1 μL of the previously packaged virus solution to each culture dish in (1) along the side wall and culture in a 37°C, 5% CO2 incubator for 2-4 hours. After about 2-4 hours, discard the suspended virus solution and add 3 mL of fresh culture medium.

[0158] (4) After one day, observe the cell pathological changes. Most cells will show red fluorescence. Use a flow cytometer to place single cells without red fluorescence in a 96-well cell culture plate. After about two or three days, observe the 96-well cell culture plate. Select the wells with cell pathological changes but without red fluorescence. Collect all the pathological cell fluid in a biosafety cabinet, freeze and thaw repeatedly three times, and store in a -80°C ultra-low temperature freezer for later use.

[0159] 4. Virus plaque picking and identification

[0160] (1) Preparation of 5% low-melting-point glue: Weigh 0.25 g of low-melting-point glue and dissolve it in 5 mL of PBS. Sterilize it by high pressure at 121°C for 20 min and then store it in a refrigerator at 4°C until use.

[0161] (2) Inoculate U-2OS cells in good condition into a six-well plate. The next day, when the cell density reaches about 90%, add the virus solution at a rate of 10 -4 ~10 -6 After performing a series of gradient dilutions, discard the old culture medium in the six-well plate and add 1 mL of diluted virus solution to each well to allow virus adsorption. After incubation in an incubator for 2-4 hours, place the boiled low-melting-point gel in a 40°C water bath to maintain warmth. Next, place the plate in a clean bench and add three volumes of DMEM culture medium to a final concentration of 1.25%. Mix quickly with a pipette and quickly remove the suspended virus from the plate using a pipette. Then, add 2 mL of culture medium containing 1.25% low-melting-point gel along the sidewalls using a pipette. The plate is then incubated in a 37°C, 5% CO2 cell culture incubator.

[0162] (3) Observe the cell pathological changes under an inverted microscope every day. If isolated virus plaques appear, pick them and place them in a 12-well plate with U-2OS cells in advance. Label them and culture them in a cell culture incubator at 37°C and 5% CO2. After they are fully pathological, collect the virus liquid in a 1.5mL centrifuge tube in a safety cabinet and store it in a -80°C ultra-low temperature refrigerator for the next step of identification.

[0163] (4) The recombinant virus was used to specifically express the human IL33 gene and the PCR and ELISA were performed for identification. 4×10 6HeLa cells were seeded in 10 cm culture dishes and infected with either OVV-hIL33 or uninfected OVV-NC at an MOI of 0.05. The cells were cultured in a CO2 incubator at 37°C for 72 h, and the cells and culture supernatant were harvested. The human IL33 ELISA Kit (Biyuntian, PI631) was used to measure the human IL33 level in the cell culture supernatant. The results showed that the human IL33 level in uninfected HeLa cells was 1923.17 ± 97.85 pg / ml, while that in the OVV-NC and OVV-hIL33 groups was 1938.62 ± 67.28 pg / ml and 2386.67 ± 48.49 pg / ml, respectively. OVV-hIL33-infected HeLa cells exhibited significantly higher IL33 secretion levels than the negative control (HeLa group) and the control virus OVV-NC (Figure 2A).

[0164] Cellular RNA was extracted using the RNAiso Plus (TaKaRa, Code No. 9108) kit as described. RNA was reverse transcribed using the PrimeScript RT Reagent Kit (TaKaRa, Code No. RR036A). Quantitative real-time quantitative PCR (qRT-PCR) amplification was performed using TB Green Premix Ex Taq™ II (Code No. RR820A). The expression of human IL33 gene in cells infected with the virus was compared with that in cells uninfected with the virus. Relative quantification (RQ) = 2-ΔΔCt (ΔCt = Ct of target gene - Ct of reference gene, ΔΔCt = ΔCt of experimental group - ΔCt of control group). Human IL33 was the target gene, human GAPDH was the reference gene, and HeLa cells were uninfected with the virus. Two independent groups were analyzed using the independent-samples T test, and multiple groups were analyzed using one-way ANOVA. Compared to the uninfected HeLa cell control group, the RQ values ​​of human IL33 in the OVV-NC and OVV-hIL33 groups were 39.16±26.85 and 2548192.00±114907.00, respectively (Figure 2B). This indicates that JX-IL33 virus infection of cells can significantly increase the expression of the cellular IL33 gene. Purified oncolytic vaccinia virus OVV-hIL33 was obtained.

[0165] The empty virus OVV-NC was prepared by a similar method as described above. The OOV-NC lacked the F3L sequence and did not have the exogenous IL33 gene inserted.

[0166] Example 2 Realtime cell analysis (RTCA) method to detect the in vitro inhibitory effect of OVV-hIL33 on various human tumor cells

[0167] In this study, ovarian cancer cells SKOV3, ES-2, OVCAR-3, and Caov-3, bladder cancer cells T24, UMUC3, SW780, and RT4, and liver cancer cells BEL-7404 and MHCC97-H (all purchased from the National Laboratory Cell Resource Sharing Platform) were selected and cultured at 5×10 4 The cells were inoculated at a density of 100 μL / well into E-Plate 16 wells (ACEA Biosciences, USA) dedicated to the RCTA method. 100 μL of OVV-hIL33 and OVV-NC virus suspensions at MOIs of 0.1, 0.05, 0.01, 0.005, and 0.001 were added to each well. Three replicate wells were set up. The experimental control group consisted of cells without virus addition.

[0168] The E-Plate 16 plate was placed in an RTCA Station (ACEA Biosciences, USA) in an incubator and cultured at 37°C, 5% CO2 for 96 hours. RTCA Software 2.0 was used to observe the cell growth curve. The time point of virus addition was used as the starting point of the Normalize Cell Index (NCI). The curve was normalized to 1 at this point. The NCI value data for each well at 12 hours, 24 hours, 36 hours, 48 ​​hours, and 60 hours were derived. Cell viability and inhibition rate were calculated at different time points: cell viability = (NCI value experimental group - NCI value control group) / NCI value control group) × 100%, cell inhibition rate = (NCI value control group - NCI value experimental group) / NCI value control group) × 100%. The IC50 values ​​of virus killing different cells at each time point were calculated. The IC50 values ​​of virus killing cells were plotted as the ordinate and the time point as the abscissa.

[0169] As shown in Figure 3, OVV-hIL33 significantly inhibited the proliferation of various human tumor cells in vitro, with the effect being time- and dose-dependent. The IC50 (MOI) of OVV-hIL33 in inhibiting different human tumor cells from 12 to 60 hours was lower than that of the control virus OVV-NC, indicating that OVV-hIL33 has a better inhibitory effect on tumor cells in vitro than the control virus OVV-NC.

[0170] Example 3 Real-time cell analysis (RTCA) method to detect the inhibitory effect of OVV-mIL33 on mouse tumor cells in vitro

[0171] This experiment used mouse ovarian cancer cell ID8 (donated by Professor Cui Heng, Peking University Health Science Center), mouse bladder cancer cell MB49, and mouse liver cancer cell H22 (purchased from Shanghai Yingwan Biotechnology Co., Ltd.). The RTCA method described in Example 2 was used to monitor the killing effect of the virus OVV-mIL33 at different MOI values ​​on mouse tumor cells in real time.

[0172] As shown in Figure 4, starting from 12 hours after virus treatment, OVV-mIL33 significantly inhibited the proliferation of mouse tumor cells in vitro in a dose-dependent manner and was significantly better than the control virus OVV-NC.

[0173] Example 4 OVV-NC significantly inhibits the growth of mouse colorectal cancer cell homografts CT26 compared to vaccinia virus Copenhagen wild-type strain

[0174] The wild-type vaccinia virus Copenhagen strain used in this example has the genome of the wild-type Copenhagen virus.

[0175] Six-week-old female Balb / C mice (5 mice per group) were implanted with 3×10 5 About 15 days later, when the tumor size reached 100 mm, the 3 [(length × width 2 ) / 2], the mice were randomly divided into three groups: PBS control group, wild-type Copenhagen virus group and OVV-NC group, with 5 mice in each group. On days 15, 17 and 19, PBS, 1×10 7 PFU of wild-type Copenhagen virus or OVV-NC. Tumor size was measured and mouse survival was monitored.

[0176] The results are shown in Figure 5. The growth rate of tumors in mice treated with OVV-NC was significantly lower than that in the control group, and the survival time of mice after OVV-NC treatment was longer than that of wild-type Copenhagen virus.

[0177] Example 5 OVV-hIL33 significantly inhibits the growth of human ovarian cancer cell SKOV3 xenografts in nude mice

[0178] Subcutaneous xenografts were established in nude mice using human ovarian cancer cell lines (SKOV3). OVV-hIL33, OVV-NC, or an equal volume of PBS were injected intratumorally, and tumor volume was measured regularly. Animal experimental methods are as follows:

[0179] All animal experiments in this study were carried out in strict accordance with the NIH laboratory animal guidelines. Six-week-old female BALB / c nude mice were selected and injected with 1×10 6 SKOV3 cells were injected subcutaneously in the hind groin of the rats at a rate of 100 μL cells / 100 μL. The growth of tumor volume was observed every day after the injection of cells and measured with a micrometer to calculate the tumor volume ((mm 3 )=(length×width 2) / 2). When the tumors grew to 21 days, they were divided into 3 groups: PBS group, OVV-NC group and OVV-hIL33 group, with 6-8 nude mice in each group. After grouping, each nude mouse was injected intratumorally with 1×10 5 pfu / 100μL virus or equal volume of PBS, injected 5 times in total, and measured the tumor volume every three days. Two days after the completion of the 5 injections, the tumor size was measured and the animals were killed at the same time to remove the transplanted tumor. The tumor volume of nude mice (mm 3 ) as the vertical axis and time as the horizontal axis to draw the tumor growth curve.

[0180] Paraffin Embedding: Mouse tumor tissues were fixed overnight in 5% formalin and placed in embedding boxes. The tissues were labeled with the number and time, rinsed in running water for 30 minutes, dehydrated in 60% to 95% ethanol for 30 minutes each, and then dehydrated in absolute ethanol for 60 minutes. Finally, the tissues were completely immersed in xylene for 15 minutes, and the process was repeated twice. Finally, the tissues were embedded in a paraffin embedding machine.

[0181] Tissue Sectioning and HE Staining: Paraffin sections were 5 μm thick, dewaxed and dehydrated, and then stained with hematoxylin for 7 minutes and eosin for 30 seconds, then mounted with neutral gum. Slides were scanned at 200× using a TissueGnostics Panoramic Tissue Cell Quantification System (Austria) to assess the extent of viral destruction of tumor cells.

[0182] The results are shown in Figure 6. After 5 injections of oncolytic virus, the tumor volume of the OVV-hIL33 group and the OVV-NC group was significantly reduced compared with the PBS control group. HE staining showed that a large number of tumor cells in the PBS control group were densely arranged, with some nutrient blood vessels passing through them. In the tumor sections of the OVV-hIL33 intervention group, a large number of glial dead tumor cells without cell structure appeared (Figure 6A). Comparing the tumor growth curves of the three groups, the PBS control group showed rapid growth, while the tumor volume of the OVV-hIL33 group and the OVV-NC group hardly increased (Figure 6B), indicating that both OVV-hIL33 and OVV-NC can significantly kill SKOV3 nude mouse transplanted tumors.

[0183] Example 6 Oncolytic Vaccinia Virus OVV-mIL33 Significantly Prolongs the Survival Time of Mice Bearing Tumors with Homologous Transplantation of Mouse Ovarian Cancer Cells ID8 and Mouse Bladder Cancer Cells MB49

[0184] The luciferase gene was transfected into mouse ovarian cancer cell ID8 and mouse bladder cancer cell MB49 using a lentiviral system. 2.5×10 4 / well was inoculated in a 24-well cell culture plate, MOI = 20, Polybrene concentration was 10 μg / mL, Luciferase lentivirus (Suzhou Jima Biotechnology Co., Ltd.) suspension was prepared and added to the corresponding cell wells, 37 ° C, 5% CO2 cells were cultured to a cell density of about 50%, and 5 μg / ml puromycin was used for selection and culture for one week before removal. After screening, cells ID8-luc and MB49-luc were seeded in 96-well plates at 2000 / well, 4000 / well, 8000 / well, 16000 / well and 32000 / well, respectively. Three replicates were set up, and according to the operating instructions of the luciferase detection reagent, 100 μL / well of luciferase reagent was added. Cell fluorescence was detected on a full-wavelength microplate reader within 3 to 5 minutes, indicating that the luciferase gene was successfully transfected (Figure 7).

[0185] ID8-luc and MB49-luc were used to establish intraperitoneal homologous tumors in C57BL / 6 mice (6 weeks old), and each mouse was intraperitoneally injected with 1×10 6 The tumor cells / 100 μL were detected every 7 days using a small animal in vivo imaging system (PerkinElmer IVIS Lumina III, USA). Observation method: The mice were injected with 250 mg / kg of Luciferase. After 5 minutes of injection, the mice were anesthetized with isoflurane and then placed in an in vivo imaging system for imaging. All mice had basically formed tumors at 2 weeks. Mice transplanted with ID8-luc tumors were divided into two groups and injected with 2.5×10 6 , 5×10 6 and 1×10 7 pfu OVV-mIL33 group and ID8-luc cell lysate control group, MB49-luc transplanted tumor mice were divided into two groups: injection of 5×10 6 , 1×10 7 and 1.5×10 7 The pfu OVV-mIL33 group and the MB49-luc cell lysate control group were used for regular monitoring of intraperitoneal tumor growth in mice by small animal live imaging. Mice were eliminated when the fluorescent area of ​​the intraperitoneal tumor exceeded 50% of the mouse area, or when the weight of the mice decreased by more than 20% compared with that before virus treatment, or when they died naturally, and the time of death of the mice was recorded.

[0186] As shown in Figure 8: Compared with tumor cell lysate, 2.5×10 6 ~1.0×10 7 Intraperitoneal injection of pfu OVV-mIL33 in ovarian cancer-bearing mice significantly delayed tumor growth (Figure 8B) and prolonged the median survival time of mice (Figure 8C). For ID8 ovarian cancer cells, the optimal dose of OVV-mIL33 for in vivo treatment was 5×10 6 pfu.

[0187] As shown in Figure 9: compared with tumor cell lysate, 1.0-1.5×10 7 Intraperitoneal injection of 5.0×10 OVV-mIL33 in bladder cancer-bearing mice significantly delayed tumor growth (Figure 9B). 6 ~1.0×10 7 pfu of OVV-mIL33 significantly prolonged the survival time of mice (Figure 9C). For MB49 cells, the optimal dose of OVV-mIL33 for in vivo treatment was 1×10 7 pfu.

[0188] Example 7 evaluated the ability of the oncolytic vaccinia virus OVV-mIL33 and a checkpoint inhibitor to treat tumors in a mouse model of peritoneal metastatic bladder cancer.

[0189] The tumor model used in this example has a growth pattern that mimics the peritoneal metastasis pattern of adult bladder cancer, is resistant to anti-PD-1 immunotherapy, and is immunoreactive.

[0190] Tumor models and treatment options

[0191] MB49-luc was used to establish bladder cancer intraperitoneal transplantation tumors in C57BL / 6 mice (6 weeks old). Each mouse was intraperitoneally injected with 1×10 6 The mice were observed to have obvious tumor formation after 2 weeks using a small animal in vivo imaging system (PerkinElmer IVIS Lumina III, USA). After the tumor formation was basically established, the mice were randomly divided into 6 groups:

[0192] ①MB49-luc cell lysate control group (Control): 100ul MB49-luc cell lysate was injected intraperitoneally;

[0193] ② Mouse PD-1 monoclonal antibody group (Anti-mPD-1): Mouse PD-1 monoclonal antibody (RMP1-14) was purchased from abinvivo, catalog number: B76401, isotype: rat IgG2a, κ, anti-mPD-1 was injected intraperitoneally at a rate of 250 μg / mouse, once every 1 day, for a total of 5 times;

[0194] ③OVV-NC group: mice were injected with 3×10 OVV-NC oncolytic virus at one time 6 pfu / unit;

[0195] ④OVV-mIL33 group: mice were injected with 3×10 OVV-mIL33 oncolytic virus at one time 6 pfu / unit;

[0196] ⑤OVV-NC+anti-mPD-1 group, a single injection of OVV-NC oncolytic virus 3×10 6 pfu / mouse, anti-mPD-1 250 μg / mouse was injected starting from day 3, and then anti-mPD-1 was injected every 1 day for a total of 5 times;

[0197] ⑥OVV-mIL33+anti-mPD-1 group, OVV-mIL33 oncolytic virus 3×10 6 pfu / mouse, anti-mPD-1 250 μg / mouse was injected starting from day 3, and then anti-mPD-1 was injected every 1 day for a total of 5 times;

[0198] Each group consisted of 12 to 15 mice, half of which were male and half were female.

[0199] Twenty-four hours after the first injection of oncolytic virus or anti-mPD-1, five mice were randomly selected from each group, and 50 μl of blood was collected from their orbits every day until sacrifice. The mice were treated for seven days with the first dose, and peritoneal tumor regression was observed using intravital imaging. On day 12, the mice, after which intravital bleeding was obtained, were sacrificed, and the peritoneal xenografts were removed, frozen in liquid nitrogen, and embedded in paraffin. The remaining mice were maintained until the end of treatment, with weekly intravital imaging used to monitor tumor growth and recurrence.

[0200] Serum ELISA analysis: The ELISA kit for mouse serum INF-γ factor was purchased from Wuhan Huamei Bioengineering Co., Ltd. The operation was carried out according to the kit instructions, 10 μl of serum sample to be tested was added to each well, and repeated 3 times.

[0201] Histological Analysis: For immunohistochemical HRP staining, paraffin-embedded mouse tumor tissue was sectioned at 5 μm thickness. After deparaffinization and dehydration, high-pressure antigen retrieval was performed using 1 mM Tris-EDTA buffer (pH 9.0) for 18 minutes. The specimens were blocked with 5% goat serum in PBST (0.03% Triton X-100 in PBS) and incubated with anti-mIL33 antibody (rabbit, AFFINITY) for 2 hours at 37°C. The specimens were incubated with secondary antibody Goat Anti-Rabbit IgG H&L (HRP, Zhengneng Bio) for 40 minutes at 37°C. After washing several times, the tissues were dripped with DAB solution (Fujian Maixin) and allowed to stand at room temperature for 3-5 minutes. The reaction was terminated by rinsing in running water. An appropriate amount of hematoxylin staining solution (Shanghai Biyuntian) was added for counterstaining for 5 minutes. The sections were then rehydrated with 1% hydrochloric acid ethanol rapid differentiation solution (Shanghai Biyuntian) for 3 seconds and mounted with neutral gum. The TissueGnostics panoramic tissue cell quantitative analysis system scans the entire area of ​​tissue and obtains immunohistochemical images.

[0202] Immunohistochemistry results were evaluated by multiplying the percentage of positive cells by the staining intensity score. A score of ≤3 indicated low expression, and >3 indicated high expression. Scoring criteria included 0, 1, 2, 3, and 4, corresponding to positive expression percentages <1%, 1%-25%, 25%-50%, 50%-75%, and 75%-100%, respectively. Positive cell staining intensity was assigned 3, 2, 1, and 0, respectively, for brown, brownish-yellow, light yellow, and no staining. The total score was calculated by multiplying the staining intensity by the percentage of positive cells.

[0203] For multiplex immunofluorescence studies using tyramide signal amplification (TSA), paraffin-embedded mouse tumor tissue was sectioned at 5 μm thickness. After deparaffinization and dehydration, antigen retrieval was performed using 1 mM Tris-EDTA buffer (pH 9.0) at high pressure for 18 minutes. The samples were blocked with immunostaining antibody blocking buffer (Shanghai Bio-Tech, Cat: P0102). The appropriate amount of the first primary antibody was added dropwise, and the slides were incubated at 37°C for 2 hours. The slides were rinsed three times with PBS. The appropriate amount of the corresponding secondary antibody was added dropwise, and the slides were incubated at 37°C for 40 minutes. The slides were then washed again with PBS. A single-color TSA fluorescent dye (PPD520, Berno Panoramic) diluted 1:100 with TSA signal amplification buffer (Benno Panoramic) was added dropwise. Next, a second primary antibody, secondary antibody, and second fluorescent stain (PPD570 or PPD650, Berno Panoramic) were added to detect expression of CD3+ T cells / CD8+ T cells, Vaccinia virus / PD-L1, ST2 / CD90, and CD103 / CCL5, respectively. Following fluorescent staining, cell nuclei were stained with a 1:500 DAPI solution (Berno Panoramic) diluted in PBS for 10 minutes at room temperature. Slides were mounted with enhanced anti-fluorescence quenching mounting medium (Berno Panoramic). Full-area tissue slides were scanned using the TissueGnostics Panoramic Tissue Cell Quantification System. Fluorescence quantitative analysis was performed using the Panoramic Tissue Cell Quantification System's included analysis software, "StrataQuest App." A 0.75 × 0.75 region of interest (ROI) was selected. Three to eight ROIs were randomly selected from each section, and ROI parameters were adjusted. Statistical analysis was performed to calculate the percentage of positive expression of the corresponding protein in each ROI.

[0204] Statistical Analysis: Results are expressed as mean ± standard deviation. Two independent groups were analyzed using the independent sample t test, and multiple groups were analyzed using one-way ANOVA. Statistical significance was set at p < 0.05. Kaplan-Meier curves were used to depict survival changes. Differences in survival curves between two groups were compared using the log-rank test, and between multiple groups using the Wilcoxon test. Differences were considered statistically significant at p < 0.05.

[0205] result:

[0206] Antitumor effect of vaccinia oncolytic virus + anti-PD-1 combination therapy: Compared with the continued proliferation of tumors in mice in the control group, the tumors of mice regressed after one week of injection of mPD-1 monoclonal antibody, OVV-NC, OVV-mIL33 oncolytic virus monotherapy, and oncolytic virus combined with mPD-1 monoclonal antibody. However, about two weeks after treatment, tumors recurred in the four groups including mPD-1 monoclonal antibody, OVV-NC, OVV-mIL33 monotherapy, and OVV-NC + mPD-1 monoclonal antibody, and the tumor volume increased significantly. The OVV-mIL33 combined with mPD-1 monoclonal antibody group inhibited the growth of transplanted tumors more significantly than the other groups (P<0.05). After 17 days of medication, the tumors almost disappeared (Figure 10A).

[0207] Comparing the growth curves of mouse tumors, there was no significant difference in tumor growth rate between OVV-NC monotherapy and combined with mPD-1 monoclonal antibody therapy, but the tumor growth of the combination therapy of OVV-mIL33 and mPD-1 monoclonal antibody was significantly inhibited compared with OVV-NC oncolytic virus treatment (P<0.05, Figure 10B); compared with single drug treatment, the tumor inhibition effect of OVV-mIL33 combined with mPD-1 monoclonal antibody was more significant (P<0.05, Figure 10C); as shown in Figure 10D, 17 days after the mouse tumors were treated with mPD-1 monoclonal antibody or OVV-mIL33, the tumors recurred and the tumor volume was larger than the volume of the initial treatment, while the tumors of the mice treated with the combination of mPD-1 monoclonal antibody and OVV-mIL33 were still in a state of regression, and the tumor volume was smaller than before treatment.

[0208] In addition, survival analysis of mice in each group after treatment (Figure 10E) showed that the median survival time of the Control group (n=11) was 12 days, the median survival time of the Anti-mPD-1 group (n=7) was 18 days, the median survival time of the OVV-NC group (n=8) was 25.5 days, the median survival time of the OVV-NC+anti-mPD-1 group (n=7) was 33 days, the median survival time of the OVV-mIL33 group (n=8) was 23 days, and the median survival time of the OVV-mIL33+anti-mPD-1 group (n=8) was 43.5 days. The median survival time of mice treated with oncolytic virus OVV-mIL33 combined with mPD-1 monoclonal antibody inhibitor was prolonged compared with that of the other groups. The survival time of bladder cancer mice treated with OVV-mIL33 combined with mPD-1 monoclonal antibody was significantly better than that of each single-drug treatment group (P=0.001). The combination of OVV-mIL33 and mPD-1 monoclonal antibody was also better than the combination of control virus and mPD-1 monoclonal antibody (P=0.046).

[0209] Changes in INF-γ levels in mouse serum during vaccinia oncolytic virus + anti-PD-1 combined treatment: Compared with the control group, the serum INF-γ level of mice increased within 24 hours after treatment with vaccinia oncolytic virus and anti-mPD-1 alone or in combination (Figure 11A). After 7 days of treatment, the INF-γ levels in each treatment group were significantly higher than those in the control group (Figure 11B). Except for the OVV-mIL33 + anti-mPD-1 group, which continued to rise slowly, the INF-γ levels in the other groups peaked on the 7th day and then began to decline. By the 12th day of treatment, the OVV-mIL33 + anti-mPD-1 group was significantly higher than those in the other groups (P < 0.05) (Figure 11C).

[0210] Pathological analysis of vaccinia oncolytic virus + anti-PD-1 combination therapy: After 12 days of treatment, HE staining of paraffin sections of mouse peritoneal xenografts (Figure 12) revealed that microscopically, numerous densely packed tumor cells, with some microvascular infiltration, were observed in the control group. In the other groups, acellular, dead tumor tissue was observed, interspersed with a small number of residual tumor cells, with significant lymphatic infiltration. Tumor cell death was most pronounced in the OVV-mIL33 + anti-mPD-1 group, with fragmented tumor tissue.

[0211] Immunohistochemical staining showed that the expression of mIL33 was significantly increased in the OVV-mIL33 and OVV-mIL33+anti-mPD-1 groups compared with the other groups ( FIG13 ).

[0212] Multicolor immunofluorescence staining showed that after vaccinia oncolytic virus treatment, both OVV-NC and OVV-mIL33 monotherapy and combination with anti-mPD-1 upregulated the expression of mPD-L1 in mouse tumor tissues (P<0.05) (Figure 14).

[0213] Compared with the control group, the anti-mPD-1 monotherapy group, and the OVV-NC monotherapy group, the expression of PD-1 in tumor tissues of mice in the OVV-mIL33 monotherapy group was significantly increased (P<0.05). Moreover, after the combination of OVV-mIL33 and anti-mPD-1, the expression of PD-1 in tumor tissues was significantly decreased (P<0.05) (Figure 15).

[0214] ILC2 cells were defined by ST2+ / CD90+. Vaccinia oncolytic virus OVV-NC and OVV-mIL33 alone or in combination with anti-mPD-1 treatment significantly increased the number of ILC2 in mouse tumor tissues. In particular, OVV-mIL33 alone or in combination with anti-mPD-1 treatment significantly increased the number of ILC2 compared with other groups (P<0.001) (Figure 16).

[0215] In the anti-mPD-1 monotherapy group, the OVV-NC monotherapy group, and the combined therapy group, intratumoral CD8 T cell infiltration increased. The number of CD8 T cell infiltration in the OVV-mIL33 monotherapy group was not significantly increased compared with the control group, but the simultaneous administration of OVV-mIL33 and anti-mPD-1 significantly increased intratumoral CD8 T cell infiltration compared with the other groups (P < 0.05) (Figure 17).

[0216] During anti-PD-1 therapy, elevated tumor PD-L1 expression and increased CD8+ T cell infiltration into tumors reflect tumor sensitivity to anti-PD-1 therapy. This study demonstrates that OVV-mIL33 can enhance tumor sensitivity to anti-PD-1 therapy. In OVV-mIL33-treated mice, IL33 expression was significantly elevated in tumor tissue, promoting the expansion of ILC2s and activating anti-tumor CD8+ T cells.

[0217] The above results indicate that vaccinia oncolytic virus loaded with IL33 can directly kill tumors and amplify the cytokine IL33 in situ in the tumor to induce the tumor microenvironment from a cold state to a hot state, and combine with ICI to trigger strong anti-cancer immunity, thereby overcoming resistance to immunotherapy.

[0218] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A recombinant oncolytic virus, characterized in that: The recombinant oncolytic virus expresses interleukin 33 (IL33).

2. The recombinant oncolytic virus according to claim 1, characterized in that The recombinant oncolytic virus is selected from the following group: vaccinia virus, herpes simplex virus, and adenovirus.

3. The recombinant oncolytic virus according to claim 1, characterized in that The vaccinia virus is selected from the group consisting of vaccinia virus Copenhagen strain, vaccinia virus Western Reserve strain, vaccinia virus Lister strain, vaccinia virus Wyeth strain and vaccinia virus Tiantan strain.

4. The recombinant oncolytic virus according to claim 1, characterized in that The recombinant oncolytic virus contains an exogenous human or mouse IL33 gene.

5. The recombinant oncolytic virus according to claim 4, characterized in that The DNA sequence encoding the human IL33 is shown in SEQ ID NO:

1.

6. The recombinant oncolytic virus according to claim 4, characterized in that The DNA sequence encoding the mouse IL33 is shown in SEQ ID NO:

2.

7. The recombinant oncolytic virus according to claim 1, characterized in that The functional vaccinia growth factor F3L region gene of the vaccinia virus is completely or partially deleted.

8. A method for preparing a recombinant oncolytic virus according to claim 1, characterized in that: The steps include: (A) providing an oncolytic virus to be modified; (B) Introducing the IL33 encoding nucleic acid into the oncolytic virus to be modified, thereby obtaining the recombinant oncolytic virus.

9. The preparation method according to claim 8, characterized in that: The IL33 encoding nucleic acid is inserted into the oncolytic virus F3L region.

10. An active ingredient combination, characterized in that The active ingredient combination comprises: (M1) a first active ingredient, the first active ingredient being selected from the group consisting of: a recombinant oncolytic virus as described in claim 1; and (M2) a second active ingredient, wherein the second active ingredient is an immune checkpoint inhibitor.

11. The active ingredient combination according to claim 10, characterized in that The immune checkpoint inhibitor is a monoclonal antibody that specifically binds to PD-1 or PD-L1.

12. The active ingredient combination according to claim 10, characterized in that The immune checkpoint inhibitor is selected from the group consisting of BMS-936559, atezolizumab, durvalumab, avelumab, nivolumab, pembrolizumab, lambrolizumab, or a combination thereof.

13. Use of the recombinant oncolytic virus according to claim 1 or the active ingredient combination according to claim 10 in the preparation of a pharmaceutical composition for treating tumors.

14. A pharmaceutical composition, comprising: (1) a therapeutically effective amount of the recombinant oncolytic virus according to claim 1, or the active ingredient combination according to claim 10, or a combination thereof; and (2) A pharmaceutically acceptable carrier.

15. A method for treating a tumor in a subject in need thereof, comprising the step of administering to a subject in need thereof a therapeutically effective amount of the recombinant oncolytic virus of claim 1, or the active ingredient combination of claim 10, or the pharmaceutical composition of claim 14.