Cancer immunotherapy strategy targeting virus-specific bystander t cell within tumor

Abstract

Provided in the present invention is a recombinant oncolytic virus expressing a target virus antigen. The target virus antigen is identified by a bystander T cell within a tumor. The recombinant oncolytic virus targets the bystander T cell within tumor and can be used for the treatment of tumors.

Description

Cancer immunotherapy strategies targeting virus-specific bystander T cells within tumors Technical Field

[0001] The present invention belongs to the field of tumor immunotherapy and oncolytic viruses, and in particular relates to a novel cancer immunotherapy strategy targeting virus-specific bystander T cells in tumors. Background Art

[0002] Tumor antigen-specific CD8 + T cells recognize tumor antigen peptides / MHC-I class molecule complexes expressed on the surface of tumor cells through their surface T cell receptors (T cell receptors, TCR), thereby specifically killing tumor cells, which is the key mechanism of the body's anti-tumor. However, tumor cells can escape the body's immune surveillance through immune editing, rapidly proliferate and establish an immunosuppressive tumor microenvironment. In this environment, tumor antigens and various immunosuppressive molecules promote tumor antigen-specific CD8 through TCR and co-inhibitory signaling pathways. + T cells are continuously activated and eventually enter a state of functional exhaustion, so they are called exhausted T cells (Tex). The concept corresponding to Tex cells is memory CD8 + T (Memory T, Tmem) cells differentiate and form after the pathogens or cancerous cells they identify are completely eliminated, maintain a dormant state for a long time, and rapidly proliferate when the body encounters the same pathogens or cancerous cells again, exerting a highly efficient killing function to quickly eliminate pathogens or cancerous cells; on the contrary, Tex cells show a gradual loss of the function of secreting functional cytokines (IFN-γ, TNF-α, IL-2, etc.), reduced target cell killing ability, weakened proliferation ability, and susceptibility to apoptosis.

[0003] Tex cells are typically characterized by persistent, high expression of a series of inhibitory immune checkpoint molecules, including PD-1, CTLA-4, and LAG-3. The PD-1 receptor on the surface of Tex cells binds with high affinity to the PD-L1 ligand on the surface of tumor cells, thereby antagonizing a series of signaling pathways downstream of the TCR and CD28 on Tex cells, including the PI3K-AKT axis and the ZAP70-RAS-ERK-AP1 axis, thereby negatively regulating the effector function, proliferation, and survival of Tex cells. Based on this, immune checkpoint blockade (ICB) therapy, typified by monoclonal antibodies targeting PD-1 / PD-L1, can, to a certain extent, reverse the exhaustion of tumor-specific Tex cells, enhance their effector function and proliferation, and thus control tumor progression. This has revolutionized the treatment of various cancer types, including advanced melanoma, non-small cell lung cancer, and Hodgkin's lymphoma. However, a large number of clinical trials have shown that patients with various tumor types (such as gliomas and pancreatic cancer) are still largely unresponsive to PD-1 / PD-L1 ICB therapy. Even among responsive tumor types, the overall response rate of tumor patients is only 20-30%. In addition, most tumor patients who respond to PD-1 / PD-L1 ICB therapy in the initial treatment phase will also develop drug resistance in the later stages, and ultimately only less than 5% of tumor patients can be clinically cured. A possible reason for limiting the effectiveness of PD-1 / PD-L1 ICB therapy is that blocking the PD-1 / PD-L1 signaling pathway fails to change the functional exhaustion characteristics of Tex cells at the epigenetic level. Tex cells cannot acquire effector functions and memory potential similar to Tmem cells, and thus cannot effectively control tumors in the long term. Therefore, the exhaustion characteristics fixed in tumor antigen-specific Tex cells make them lose the ability to control tumor progression and greatly limit the clinical effectiveness of tumor immunity strategies targeting this group of cells.

[0004] Oncolytic viruses are a new type of tumor therapeutic drug that can selectively replicate and proliferate in tumor cells and induce an anti-tumor immune response. At present, a wide range of oncolytic viruses have been reported, including single-stranded DNA viruses (parvoviruses), double-stranded DNA viruses (adenoviruses, vaccinia viruses, herpes viruses), single-stranded positive-sense RNA viruses (polioviruses), single-stranded negative-sense RNA viruses (measles virus, Newcastle disease virus) and double-stranded RNA viruses (respiratory enteroviruses). As a new tumor treatment strategy, the anti-tumor effect of oncolytic viruses not only relies on the virus itself to infect and destroy tumor cells, but also relies mainly on the ability of oncolytic viruses to selectively deliver "payloads" to tumor cells, that is, the carrier characteristics of selectively targeting tumor cells. A large number of studies have found that oncolytic viruses can cooperate with the body's anti-tumor function by carrying "payload" genes such as immune checkpoint inhibitors, cytokines, T cell stimulating molecules, tumor-associated antigens, microRNAs, etc., to achieve more efficient anti-tumor effects. It is reported that there are nearly 100 clinical trials based on oncolytic virus therapy for tumor patients. Among them, HSV-1, an oncolytic virus that can encode granulocyte-macrophage colony-stimulating factor, has been shown to have good anti-tumor effects in clinical trials and was approved by the FDA for local treatment of advanced melanoma in 2016 (Journal for immunotherapy of cancer, 2016, 4, 53). However, the success of the above-mentioned oncolytic virus strategies depends largely on the tumor antigen-specific CD8 + The ultimate killing effect of T cells on tumor cells cannot circumvent the tumor antigen-specific CD8 + The problem of T cell functional exhaustion makes it difficult to maximize the anti-tumor effect of oncolytic viruses. Summary of the Invention

[0005] The present invention provides a novel oncolytic virus strategy targeting virus-specific Tbys cells in tumors. This strategy uses oncolytic viruses to effectively deliver antigenic epitopes recognized by Tbys cells into tumor cells for expression, mediating the recognition and killing of tumor cells by Tbys cells, thereby achieving the purpose of treating tumors.

[0006] Studies have shown that CD8 + T cells account for only a small number, while the majority of CD8 +T cells do not directly recognize tumor antigens, so the latter are called bystander T cells (abbreviated as Tbys cells). Studies have suggested that virus-specific Tbys cells in tumors are derived from virus-specific Tmem cells formed after the patient was previously infected with the corresponding virus. Therefore, Tbys cells retain certain Tmem cell characteristics and are more ideal tumor immunotherapy targets than Tex cells in tumors. Compared with functionally impaired and limited tumor-specific Tex cells, Tbys cells with complete functions and large numbers are more ideal tumor immunotherapy targets. In some aspects, the present invention provides a novel tumor immunotherapy strategy targeting Tbys cells in tumors. In some aspects, the present invention provides a novel oncolytic virus strategy targeting functional Tbys cells, which has important clinical value.

[0007] In some embodiments, the present invention relates to an activator targeting bystander T cells (Tbys cells) in tumors, which is used to treat tumors. In some embodiments, the present invention relates to the use of an activator targeting bystander T cells (Tbys cells) in tumors in the preparation of a drug for treating tumors. In this article, the activator targeting bystander T cells (Tbys cells) in tumors is not particularly limited and can include any agent that activates the recognition and killing function of bystander T cells in tumors to achieve the purpose of treating tumors, such as any agent that can effectively deliver antigen epitopes recognized by Tbys cells to tumor cells for expression. In this article, bystander T cells (Tbys cells) in tumors can include CD8 + Tbys cells and / or CD4 + Tbys cells.

[0008] In some embodiments, the present invention also provides recombinant oncolytic viruses. In some embodiments, the activator of the bystander T cells (Tbys cells) in the targeting tumor can include a recombinant oncolytic virus. In some embodiments, the recombinant oncolytic virus can include a nucleic acid molecule encoding an antigen of the target virus recognized by the Tbys cells. In some embodiments, the present invention provides polynucleotides, which include nucleic acid molecules and oncolytic virus genes encoding the antigen of the target virus recognized by the Tbys cells. In some embodiments, provided herein are vectors (such as viral vectors) and host cells comprising the polynucleotides. In some embodiments, the recombinant oncolytic virus includes a recombinant oncolytic virus capable of effectively delivering an antigenic epitope recognized by Tbys cells to tumor cells. In some embodiments, the oncolytic virus used in the present invention is not particularly limited, as long as the antigenic epitope recognized by Tbys cells can be delivered to tumor cells, bystander T cell function in the tumor is activated, and the oncolytic virus that mediates Tbys cells to recognize and kill tumor cells can be used. In some embodiments, the oncolytic virus used in the present invention may include any of the following viruses: (1) single-stranded DNA virus, double-stranded DNA virus, single-stranded positive-sense RNA virus, single-stranded negative-sense RNA virus, double-stranded RNA virus, etc.; (2) parvovirus, adenovirus, vaccinia virus, herpes virus, poliovirus, measles virus, Newcastle disease virus, respiratory enterovirus, etc.

[0009] In some embodiments, the antigens of the target virus used in the present invention that can be recognized by Tbys cells can include any target virus antigen that activates the recognition and killing function of tumor cells by bystander T cells in the tumor. In some embodiments, the target virus can include appropriate epidemic viruses, such as coronaviruses, such as SARS-CoV-2 viruses, influenza viruses such as influenza A, B, C or D viruses, Epstein-Barr viruses and cytomegaloviruses. In some embodiments, the target virus can include any appropriate subtype, for example, influenza A virus can include any of 18 different hemagglutinin (H) subtypes and 11 different neuraminidase (N) subtypes (H1-18 and N1-11), such as H1N1, H3N2 subtypes, etc. In some embodiments, the target virus is a virus that infects tumor patients and produces immune memory. In some embodiments, the target virus is not a virus that causes tumors or cancer. In some embodiments, the target virus antigen is not a tumor antigen or tumor-associated antigen (TAA). In some embodiments, the antigen of the target virus can include immunogenic proteins or nucleic acid molecules or fragments thereof of the target virus. As known to those skilled in the art, methods for determining whether a protein or nucleic acid molecule is immunogenic are known in the art. In some embodiments, known viral antigens, such as SARS-CoV-2 proteins (e.g., one or more of the S protein, N protein, M protein, E protein) or fragments thereof, can be used. In some embodiments, the antigen can be, for example, an RBD antigen of SARS-CoV-2, for example, an influenza virus antigen, such as an influenza virus HA antigen.

[0010] In some embodiments, the antigen of the target virus can be inserted into the genome of the recombinant oncolytic virus. In some embodiments, for example, Newcastle disease virus can be used as an oncolytic virus, wherein the antigen of the target virus can be inserted into the genome of the Newcastle disease virus, for example, between the P and M genes of NDV or other appropriate positions. In some embodiments, optionally, the recombinant oncolytic virus can also include nucleic acid molecules encoding one or more payload genes, such as nucleic acid molecules encoding immune checkpoint inhibitors, cytokines, T cell stimulating molecules, tumor-associated antigens and / or microRNA. In some embodiments, the payload gene and the antigen of the target virus can be constructed in the same construct or in different constructs.

[0011] In some embodiments, given the widespread presence of bystander T cells in various tumors, the tumors that can be treated by the strategies of the present invention are not particularly limited. In some embodiments, the tumors that can be treated by the strategies of the present invention may include melanoma, lung cancer, bladder cancer, ovarian cancer, brain cancer, pancreatic cancer, prostate cancer, sarcoma, breast cancer, cervical cancer, bone cancer, liver cancer, head and neck cancer, gastric cancer, kidney cancer, lymphoma, leukemia, thyroid cancer, colon cancer, and rectal cancer.

[0012] In some embodiments, the dosage form of the drug or pharmaceutical composition prepared by the present invention is not particularly limited and can be any dosage form suitable for activating the recognition and killing function of tumor cells by bystander T cells in the tumor. In some embodiments, the drug or pharmaceutical composition of the present invention includes a form suitable for systemic, intraperitoneal or intratumoral injection, preferably intratumoral injection of the drug. In some embodiments, preferably, the drug is used in combination with another anti-cancer therapy. In some embodiments, the other anti-cancer therapy includes, for example, immune checkpoint blockade therapy, preferably PD-1 / PD-L1 immune checkpoint blockade therapy.

[0013] In some embodiments, the present invention also provides recombinant oncolytic viruses. In some embodiments, the recombinant oncolytic virus may include a nucleic acid molecule encoding an antigen of the target virus recognized by the Tbys cells. In some embodiments, the present invention provides polynucleotides, vectors and / or hosts, which include nucleic acid molecules and oncolytic virus genes encoding antigens of the target virus recognized by the Tbys cells. In some embodiments, the antigen of the target virus can be inserted into the genome of the recombinant oncolytic virus. In some embodiments, for example, Newcastle disease virus can be used as an oncolytic virus, wherein the antigen of the target virus can be inserted into the genome of the Newcastle disease virus, for example, between the P and M genes of NDV or other appropriate positions. In some embodiments, optionally, the recombinant oncolytic virus may also include nucleic acid molecules encoding one or more payload genes, such as nucleic acid molecules encoding immune checkpoint inhibitors, cytokines, T cell stimulating molecules, tumor-associated antigens and / or microRNAs. In some embodiments, the payload gene and the antigen of the target virus can be constructed in the same construct or in different constructs.

[0014] In some embodiments, given the widespread presence of bystander T cells in various tumors, the tumors that can be treated by the strategies of the present invention are not particularly limited. In some embodiments, the tumors that can be treated by the strategies of the present invention may include melanoma, lung cancer, bladder cancer, ovarian cancer, brain cancer, pancreatic cancer, prostate cancer, sarcoma, breast cancer, cervical cancer, bone cancer, liver cancer, head and neck cancer, gastric cancer, kidney cancer, lymphoma, leukemia, thyroid cancer, colon cancer, and rectal cancer.

[0015] In some embodiments, the dosage form of the drug or pharmaceutical composition prepared by the present invention is not particularly limited and can be any dosage form suitable for activating the recognition and killing function of tumor cells by bystander T cells in the tumor. In some embodiments, the drug or pharmaceutical composition of the present invention includes a form suitable for systemic, intraperitoneal or intratumoral injection, preferably intratumoral injection of the drug. In some embodiments, preferably, the drug is used in combination with another anti-cancer therapy. In some embodiments, the other anti-cancer therapy includes, for example, immune checkpoint blockade therapy, preferably PD-1 / PD-L1 immune checkpoint blockade therapy.

[0016] In some embodiments, the present invention provides a recombinant oncolytic virus described herein.

[0017] In some embodiments, the present invention provides a method for preparing a recombinant oncolytic virus, wherein the method comprises introducing a nucleic acid molecule encoding an antigen of the target virus into the genome of the recombinant oncolytic virus.

[0018] In some embodiments, the present invention provides a pharmaceutical composition comprising a recombinant oncolytic virus described herein or a recombinant oncolytic virus prepared by the method described herein. In some embodiments, the pharmaceutical composition may also include a suitable excipient, a pharmaceutical carrier or a buffer, which may be any suitable excipient, a pharmaceutical carrier or a buffer 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 oncolytic viruses such as Newcastle disease virus (NDV) (eg, SG10 strain) as a carrier, inserts common viral antigens between NDV viral genes such as P and M genes, such as new coronavirus RBD (SARS-CoV-2 RBD), H1N1 influenza virus NP (H1N1 NP), exogenous expression is performed, and recombinant oncolytic viruses (eg, NDV-SARS-CoV-2 RBD and NDV-H1N1 NP) are constructed and rescued. In some embodiments, the recombinant oncolytic viruses of the present invention may include, for example, Newcastle disease virus NDV-SARS-CoV-2 RBD recombinant strain and Newcastle disease virus NDV-H1N1 NP recombinant strain.

[0019] In another aspect, the virus-specific Tbys cell population targeted by the present invention is characterized in that the cell population is derived from virus-specific Tmem cells formed after an individual has been previously infected with a common epidemic virus (such as the new coronavirus, H1N1 influenza virus) or has been previously immunized with a viral vaccine. Therefore, Tbys cells have the multifunctionality of Tmem cells. Tbys cells do not participate in the recognition of tumor cell-associated antigens and do not have the functional exhaustion characteristics of Tex cells.

[0020] In yet another aspect, the present invention provides an application of an oncolytic virus targeting viral-specific Tbys cells in tumor treatment. In a specific embodiment, the present invention provides an oncolytic virus (such as NDV) carrying a viral antigen (such as a novel coronavirus antigen) to treat tumors (such as melanoma) with viral-specific (such as novel coronavirus-specific) Tbys cells of the purpose. In another specific embodiment, the present invention provides an oncolytic virus (such as NDV) carrying a viral antigen (such as an influenza virus antigen) to treat tumors (such as melanoma) with viral-specific (such as influenza virus-specific) Tbys cells of the purpose. In another specific embodiment, the present invention provides an oncolytic virus (such as NDV) carrying a viral antigen (such as an influenza virus antigen) to treat tumors (such as melanoma) with viral-specific (such as influenza virus-specific) Tbys cells of the purpose. In another specific embodiment, the present invention provides an oncolytic virus co-administered with PD-1 / PD-L1 immune checkpoint blockade therapy to treat tumors (such as breast cancer).

[0021] In some embodiments, the present invention provides a method for treating tumors, comprising administering an effective amount of an activator, oncolytic virus, or pharmaceutical composition described herein to an individual in need thereof. In some embodiments, the individual is a human. In some embodiments, in some embodiments, the activator, oncolytic virus, or pharmaceutical composition is administered systemically, intraperitoneally, or intratumorally, preferably intratumorally. In some embodiments, preferably, the activator, oncolytic virus, or pharmaceutical composition is used in combination with another anticancer therapy. In some embodiments, the other anticancer therapy includes, for example, immune checkpoint blockade therapy, preferably PD-1 / PD-L1 immune checkpoint blockade therapy. The effective amount for treating a tumor can be determined by any appropriate method known in the art.

[0022] In some embodiments, the purpose of the present invention can be achieved by the following technical solutions:

[0023] (1) Using reverse genetics technology to construct and rescue recombinant oncolytic virus strains (such as NDV strains NDV-SARS-CoV2-RBD and NDV-H1N1-NP) carrying target viral antigens (such as new coronaviruses, influenza viruses);

[0024] (2) using flow cytometry to analyze whether Tbys cells specific for the target viral antigen are present in the body of the individual to be treated;

[0025] (3) Individuals with Tbys cells specific for the corresponding viral antigens in (2) are treated by injection (e.g., intratumoral injection) of an oncolytic virus (such as NDV) in (1) or in combination with PD-1 / PD-L1 immune checkpoint blockade therapy.

[0026] In some embodiments, the method described in the present invention utilizes an oncolytic virus as a vector to selectively deliver Tbys cell epitopes to tumor cells. These epitopes are processed within the tumor cells and presented to the cell surface, thereby mediating Tbys cells to recognize and kill tumor cells presenting the corresponding epitopes, thereby achieving the purpose of treating tumors. This novel oncolytic virus strategy can circumvent the poor drug effect caused by T cell exhaustion in traditional tumor immunotherapy regimens (including PD-1 / PD-L1 antibody blocking therapy and adoptive T cell therapy), is an important supplement to tumor immunotherapy, and provides new directions and inspiration for related research in this field. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1. Schematic diagram of the full-length plasmid of the NDV oncolytic virus (NDV-SARS-CoV-2 RBD) carrying the novel coronavirus antigen in the present invention.

[0028] Figure 2 is a schematic diagram of the full-length plasmid of the NDV oncolytic virus (NDV-H1N1 NP) carrying influenza virus antigens of the present invention.

[0029] Figure 3. Sequencing results of allantoic fluid of NDV-SARS-CoV-2 RBD rescued strains P1 and P5.

[0030] Figure 4. Sequencing results of allantoic fluid of NDV-H1N1 NP rescued strains P1 and P5.

[0031] Figure 5. Expression of exogenous proteins in Vero cells infected with allantoic fluid of P1 and P5 generations of NDV-SARS-CoV-2 RBD rescue virus.

[0032] Figure 6. Expression of exogenous proteins in Vero cells infected with allantoic fluid of passages P1 and P5 of NDV-H1N1 NP rescue virus.

[0033] Figure 7. NDV-SARS-CoV-2 RBD treats humanized mouse melanoma models with SARS-CoV-2-specific T cells. Figure 7A shows the specific CD8 T cells against SARS-CoV-2 RBD protein in uninfected and recovered COVID-19 patients (COVID-19 donor 1, COVID-19 donor 2). + T cells and CD4 + Figure 7B is a schematic diagram of the humanized mouse melanoma model with SARS-CoV-2 RBD treatment of novel coronavirus-specific T cells; Figure 7C is a tumor growth curve of humanized mouse melanoma with novel coronavirus-specific T cells treated with NDV-SARS-CoV-2 RBD or NDV-WT.

[0034] Figure 8. NDV-H1N1 NP treats a humanized mouse melanoma model with influenza virus-specific T cells. Figure 8A shows the specific CD8 T cells of a patient who recovered from H1N1 influenza infection that target the H1N1 NP protein. + Figure 8B shows the flow cytometry of T cells; Figure 8B shows the specific CD4 T cells against H1N1 NP protein in patients who recovered from H1N1 influenza infection. + Figure 8C is a schematic diagram of the humanized mouse melanoma model with influenza virus-specific T cells treated with NDV-H1N1 NP; Figure 8D is a tumor growth curve of humanized mouse melanoma with influenza virus-specific T cells treated with NDV-H1N1, NDV-WT, or PBS.

[0035] Figure 9. NDV-SARS-CoV-2 RBD combined with PD-L1 antibody treatment of a breast cancer model in mice immunized with the COVID-19 vaccine. Figure 9A is a schematic diagram of a breast cancer model in mice immunized with the COVID-19 vaccine using NDV-SARS-CoV-2 RBD combined with PD-L1 antibody treatment; Figure 9B is a graph showing the tumor growth curve of a breast cancer model in mice harboring COVID-19-specific T cells treated with NDV-SARS-CoV-2 RBD combined with PD-L1 antibody treatment. DETAILED DESCRIPTION

[0036] Example 1: Rescue of NDV-SARS-CoV-2 RBD and NDV-H1N1 NP.

[0037] 1. Test method

[0038] 1.1 Construction of full-length cDNAs of NDV-SARS-CoV-2 RBD and NDV-H1N1 NP genomes

[0039] Because the exogenous target fragment is inserted between the P and M genes of NDV, we introduced a unique restriction site, Pme1, in the intergenic region between the P and M genes when constructing the full-length vector pOK-SG10 (its structure is shown in Figures 1 and 2), and digested the full-length plasmid with enzymes. Primers were designed (Table 1) to amplify the SARS-CoV-2 RBD fragment and the H1N1 NP gene. The target gene was constructed between the P and M genes of the full-length genomic plasmid using homologous recombination. At the same time, the translation elements GE+GS+KOZAK sequence were introduced before the target fragment to construct the full-length genomic cDNA of NDV-SARS-CoV-2 RBD (Figure 1) and NDV-H1N1 NP (Figure 2).

[0040] Table 1 Homologous primers used to amplify target genes

[0041] 1.2 Rescue of NDV-SARS-CoV-2 RBD and NDV-H1N1 NP recombinant strains

[0042] The constructed full-length genome plasmids pOK-NDV-SARS-CoV-2 RBD and pOK-NDV-H1N1 NP were co-transfected into BSR-T7 / 5 cells with the helper plasmids pCI-NP, pCI-P, and pCI-L, respectively. After 6 hours, the culture medium was changed to DMEM supplemented with 2% serum and incubated for another 3-4 days. The culture medium was harvested and inoculated into 9-11-day-old SPF chicken embryos. After 4 days of inoculation, allantoic fluid was collected for hemagglutination (HA) and hemagglutination inhibition (HI) assays. Positive viral fluids were selected for RNA extraction and sequence verification. The rescued viruses were named NDV-SARS-CoV-2 RBD and NDV-H1N1 NP.

[0043] 1.3 Genetic stability and exogenous protein expression of NDV-SARS-CoV-2 RBD and NDV-H1N1 NP rescue viruses

[0044] Based on the location of the inserted exogenous protein within the full-length cDNA, specific primers SG10-WY-F and SG10-WY-R were designed for sequencing verification (Table 2). The resulting NDV-SARS-CoV-2 RBD and NDV-H1N1 NP recombinant strains were serially passaged five times in 9-11-day-old SPF chicken embryos. The first and fifth generation viruses were selected for sequence verification of the insertion site and to test the stability of the rescued exogenous gene. Vero cells were infected with the first and fifth generation viruses to extract proteins, and the expression of the target proteins was detected by Western blot.

[0045] Table 2 NDV-specific detection primers

[0046] 1.4 Reproductive characteristics of NDV-SARS-CoV-2 RBD and NDV-H1N1 NP recombinant strains

[0047] The 5th generation recombinant virus chicken embryo allantoic fluid was selected and its TCID was determined using Vero cells. 50 The parental strain was used as a control.

[0048] 2. Test results

[0049] 2.1 Rescue of NDV-SARS-CoV-2 RBD and NDV-H1N1 NP recombinant strains

[0050] The present invention successfully rescued the NDV-SARS-CoV-2 RBD and NDV-H1N1 NP recombinant strains using reverse genetic manipulation technology. All chicken embryos died within 60 hours after inoculation of the cell solution, and their HA titers were measured to be 6log2 and 7log2, respectively.

[0051] 2.2 Genetic stability of NDV-SARS-CoV-2 RBD and NDV-H1N1 NP recombinant strains

[0052] The first and fifth allantoic fluids of the NDV-SARS-CoV-2 RBD and NDV-H1N1 NP recombinant strains were selected, RNA was extracted, reverse transcribed, and the inserted exogenous fragments were amplified by PCR using specific primers SG10-WY-F and SG10-WY-R, and sequenced. The results showed that the insertion position and sequence of the exogenous genes in the first and fifth generation viruses were correct, without mutations, and could be stably inherited (Figures 3 and 4). Western blot was used to detect the expression of the exogenous proteins RBD and NP in Vero cells infected with the allantoic fluid of the P1 and P5 generations, indicating that the constructed recombinant virus can effectively express the exogenous proteins (Figures 5 and 6).

[0053] 2.3 Reproduction characteristics of NDV-SARS-CoV-2 RBD and NDV-H1N1 NP recombinant strains in chicken embryos

[0054] The reproduction titers of the recombinant strains in the chicken embryo allantoic fluid were determined to be: TCID of NDV-SARS-CoV-2 RBD 50 is 10 7.0 / 0.1mL, TCID of NDV-H1N1 NP 50 is 10 7.25 / mL; TCID of parental strain NDV-WT 50 is 10 7.0 / 0.1mL. The results showed that the rescued recombinant virus had the same reproductive characteristics as the parental strain.

[0055] Example 2: NDV-SARS-CoV-2 RBD treats a humanized mouse melanoma model with SARS-CoV-2-specific T cells.

[0056] 1. Construct a humanized mouse melanoma model with new coronavirus-specific T cells: Collect anticoagulated peripheral blood from recovered infected patients containing new coronavirus-specific T cells (Figure 7A), dilute and mix with PBS at a ratio of 1:1, and dispense the mixture into 15mL centrifuge tubes according to 6mL. Then, insert an open Baxter tube into the bottom of the centrifuge tube, add 3mL of Ficoll solution through the Baxter tube, and centrifuge at 2200rpm / 25 minutes (the speed is zero). After the centrifugation is completed, remove the centrifuge tube, use a pipette to absorb the middle cell layer, centrifuge and discard the supernatant, resuspend and obtain peripheral blood PBMC cells. Then, 5×10 6 PBMC cells were reinfused into NOD / ShiLtJGpt-Prkdc cells via tail vein em6Cd52 Il2rg em26Cd22 / Gpt (i.e., NCG) mice (purchased from Jicui Yaokang Biotechnology Co., Ltd.) were injected and the degree of humanization (hCD45:mCD45 ratio) was tested weekly. When the degree of humanization of the mice was appropriate (hCD45:mCD45 ratio greater than 1 / 5), the mice were subcutaneously inoculated with 5×10 6 A375 human melanoma cells ( FIG7B ).

[0057] 2. NDV-SARS-CoV-2 RBD treatment of humanized mouse melanoma model: On days 7, 8, 9, 11, and 12 after mice were tumor-bearing in step 1, NDV-SARS-CoV-2 RBD oncolytic virus was diluted to 2×10 8 PFU / mL and place on ice until use. Tumor-bearing mice were intratumorally injected with 50 μL of NDV-SARS-CoV-2 RBD virus dilution using an insulin needle. After the injection, the insulin needle was removed and the needle port was pressed with a sterile cotton swab for 20 seconds. Control mice were intratumorally injected with 50 μL of the same concentration of NDV-WT (Figure 7B).

[0058] 3. Tumor growth curve recording: Observe tumor growth 5-6 days after tumor cell injection. Usually, the tumor grows to the size of soybeans in about 5 days. Then, use vernier calipers to measure and record the tumor size every two days (the formula for calculating tumor volume is: tumor volume = 1 / 2 length × width 2 The results showed that compared with the NDV-WT treatment group, the NDV-SARS-CoV-2 RBD significantly suppressed the growth of melanoma tumors in humanized mice with SARS-CoV-2-specific T cells (Figure 7C).

[0059] Example 3: NDV-H1N1 NPs treat a humanized mouse melanoma model with influenza virus-specific T cells.

[0060] 1. Construct a humanized mouse melanoma model with influenza virus-specific T cells: Collect anticoagulated peripheral blood from recovered patients containing influenza virus-specific T cells (Figure 8A, Figure 8B), dilute and mix with PBS at a ratio of 1:1, and dispense the mixture into 15mL centrifuge tubes in 6mL portions. Then, insert an open Baxter tube into the bottom of the centrifuge tube, add 3mL of Ficoll solution through the Baxter tube, and centrifuge at 2200rpm / 25 minutes (the speed is zero). After the centrifugation is completed, remove the centrifuge tube, use a pipette to absorb the middle cell layer, centrifuge and discard the supernatant, and resuspend to obtain peripheral blood PBMC cells. Then, 5×10 6 PBMC cells were reinfused into NOD / ShiLtJGpt-Prkdc cells via tail vein em6Cd52 Il2rg em26Cd22 / Gpt (i.e., NCG) mice, and then the degree of humanization (hCD45:mCD45 ratio) was tested once a week. When the degree of humanization of the mice was appropriate (hCD45:mCD45 ratio was greater than 1 / 5), the mice were subcutaneously inoculated with 5×10 6 A375 human melanoma cells ( FIG8C ).

[0061] 2. NDV-H1N1 NP treatment of humanized mouse melanoma model: On days 7, 8, 9, 11, and 12 after mice were tumor-bearing in step 1, NDV-H1N1 NP oncolytic virus was diluted to 2×10 8 PFU / mL and place on ice until use. Tumor-bearing mice were intratumorally injected with 50 μL of NDV-H1N1 NP virus dilution using an insulin needle. After injection, the insulin needle was removed and the needle entry site was pressed with a sterile cotton swab for 20 seconds. A control group of mice was intratumorally injected with 50 μL of the same concentration of NDV-WT (Figure 8C).

[0062] 3. Tumor growth curve recording: Observe tumor growth 5-6 days after tumor cell injection. Usually, the tumor grows to the size of soybeans in about 5 days. Then, use vernier calipers to measure and record the tumor size every two days (the formula for calculating tumor volume is: tumor volume = 1 / 2 length × width 2 The results showed that NDV-H1N1 NP significantly inhibited melanoma tumor growth in humanized mice harboring SARS-CoV-2-specific T cells compared to the NDV-WT treatment group (Figure 8D).

[0063] Example 4: NDV-SARS-CoV-2 RBD combined with PD-L1 antibody treatment of breast cancer model in mice immunized with the new coronavirus vaccine.

[0064] 1. Establishment of a mouse SARS-CoV-2 RBD protein immunization model: Female BALB / c mice were injected intramuscularly with 100 μL of a SARS-CoV-2 RBD protein / CpG adjuvant mixture (containing 10 μg of SARS-CoV-2 RBD protein and 10 μg of CpG adjuvant) into the tibialis muscle. On day 14 after the initial immunization, mice were re-injected with the SARS-CoV-2 RBD protein / CpG adjuvant mixture using the same protocol.

[0065] 2. Establishment of mouse orthotopic breast cancer model: 4T1 breast cancer cells were cultured in DMEM + 10% FBS. When the 4T1 cells grew to about 80% of the culture dish, the tumor cells were digested with 1% trypsin and prepared into 2×10 6 After the tumor-bearing mice (60 days after the initial immunization in step 1 above) were anesthetized, the abdomen was shaved and the skin was prepared to expose the nipples. After the needle was inserted subcutaneously, it was pushed into the mammary fat pad of the mouse and 100 μL of tumor cell suspension (i.e., 0.2×10 6 4T1 cells).

[0066] 3. NDV-SARS-CoV-2 RBD combined with PD-L1 antibody treatment of mouse breast cancer model: On the 7th, 8th, 9th, 11th, and 12th days after the mice were tumor-bearing in step 2, NDV-SARS-CoV-2 RBD oncolytic virus was diluted to 2×10 8 PFU / mL, placed on ice until use. Use an insulin needle to inject 50 μL of NDV-SARS-CoV-2 RBD virus dilution into the tumor of tumor-bearing mice. After the injection, remove the insulin needle and press the needle port with a sterile cotton swab for 20 seconds. The control group mice were injected intratumorally with 50 μL of NDV-WT or PBS of the same concentration (Figure 9A). On the 10th, 13th, and 16th days after the mice were tumor-bearing in step 2, mice injected with PBS or NDV-WT or NDV-SARS-CoV-2 RBD were given an intraperitoneal injection of 150 μg of anti-mouse PD-L1 antibody (Figure 9A).

[0067] 4. Tumor growth curve recording: Observe tumor growth 5-6 days after tumor cell injection. Usually, the tumor grows to the size of soybeans around 6 days. Then, use a vernier caliper to measure and record the tumor size every three days (the formula for calculating tumor volume is: tumor volume = 1 / 2 length × width 2 The results show that compared with PBS or NDV-WT alone, the NDV-SARS-CoV-2 RBD alone significantly inhibited the growth of breast cancer tumors in mice immunized with the COVID-19 vaccine. In addition, the NDV-SARS-CoV-2 RBD and PD-L1 antibodies synergistically inhibited the growth of breast cancer tumors (Figure 9B).

[0068] NDV-SARS-CoV-2 RBD genome sequence (SEQ ID NO.7):

[0069] NDV-SARS-CoV-2 RBD genome sequence (continued) (SEQ ID NO.8):

[0070] NDV-H1N1-NP genome sequence (SEQ ID NO.9):

[0071] NDV-H1N1-NP genome sequence (continued) (SEQ ID NO.10):

Claims

1. Targeting intratumoral bystander T cells (Tbys cells, including CD8 + Tbys cells and / or CD4 + activators of T cells) for the treatment of tumors.

2. The activator according to claim 1, wherein the activator comprises a recombinant oncolytic virus, wherein the recombinant oncolytic virus comprises a nucleic acid molecule encoding an antigen of the target virus recognized by the Tbys cell.

3. The activator according to claim 2, wherein the recombinant oncolytic virus comprises any one of the following viruses: (1) Single-stranded DNA virus, double-stranded DNA virus, single-stranded positive-sense RNA virus, single-stranded negative-sense RNA virus, double-stranded RNA virus; (2) Parvovirus, adenovirus, vaccinia virus, herpes simplex virus, poliovirus, measles virus, Newcastle disease virus, respiratory enterovirus.

4. The activator according to claim 2 or 3, wherein the target virus includes an epidemic virus, such as a coronavirus, such as a SARS-CoV-2 virus, an influenza virus such as influenza A, B, C or D virus, Epstein-Barr virus and cytomegalovirus, optionally, wherein the recombinant oncolytic virus includes, for example, a Newcastle disease virus NDV-SARS-CoV-2 RBD recombinant strain and a Newcastle disease virus NDV-H1N1 NP recombinant strain.

5. The activator according to any one of claims 2 to 4, wherein the antigen of the target virus is inserted into the genome of the recombinant oncolytic virus, and optionally, the recombinant oncolytic virus may further include a nucleic acid molecule encoding one or more payload genes, such as a nucleic acid molecule encoding an immune checkpoint inhibitor, a cytokine, a T cell stimulating molecule, a tumor-associated antigen and / or a microRNA.

6. The activator according to any one of claims 1 to 5, wherein the tumor comprises melanoma, lung cancer, bladder cancer, ovarian cancer, brain cancer, pancreatic cancer, prostate cancer, sarcoma, breast cancer, cervical cancer, bone cancer, liver cancer, head and neck cancer, stomach cancer, kidney cancer, lymphoma, leukemia, thyroid cancer, colon cancer and rectal cancer.

7. The activator according to any one of claims 1 to 4, wherein the drug comprises a systemic, intraperitoneal or intratumoral injection drug, preferably an intratumoral injection drug, preferably, the drug is used in combination with another anti-cancer therapy, wherein the other anti-cancer therapy includes, for example, an immune checkpoint blockade therapy, preferably a PD-1 / PD-L1 immune checkpoint blockade therapy.

8. The recombinant oncolytic virus as defined in any one of claims 2 to 7.

9. A method for preparing the recombinant oncolytic virus of claim 8, wherein the method comprises introducing a nucleic acid molecule encoding an antigen of the target virus into the genome of the recombinant oncolytic virus.

10. A pharmaceutical composition comprising the recombinant oncolytic virus according to claim 8 or the recombinant oncolytic virus prepared by the method according to claim 9. Preferably, the pharmaceutical composition further comprises a suitable excipient, a pharmaceutical carrier or a buffer.

11. A method for treating tumors, comprising administering an effective amount of the activator defined in any one of claims 1 to 7 or the pharmaceutical composition according to claim 10 to an individual in need thereof.

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