Oncolytic virus vector and its application

Abstract

The embodiments of the present disclosure may provide an oncolytic virus vector and its application. The oncolytic virus vector may comprise a recombinant nucleic acid. The recombinant nucleic acid may comprise (i) a first nucleic acid fragment encoding a soluble PD-1 molecule, (ii) a second nucleic acid fragment encoding a CD86 molecule, and (iii) a third nucleic acid fragment encoding an antibody against a CD3 molecule.

Description

Technical Field

[0001] Sequence Listing This application is electronically filed in XML format and includes a sequence listing that is hereby incorporated by reference in its entirety. The XML copy created on May 31, 2021, is named "Sequence Listing-20699-0007WO00" and is 18,925 bytes in size.

[0002] The present disclosure relates to the field of biotechnology, and more particularly to oncolytic virus vectors containing recombinant nucleic acids and their applications.

Background Art

[0003] Currently, virus drugs represented by oncolytic viruses are playing an increasingly important role in the treatment of tumors. Oncolytic viruses refer to a class of viruses that can effectively infect cancer cells and destroy them. Oncolytic viruses replicate and proliferate within cancer cells, release new infectious virus particles, infect other cancer cells, and destroy other cancer cells. Oncolytic viruses also act on cancer cells to affect the tumor microenvironment, stimulate the host to produce anti-tumor immune responses or produce proteins that directly lyse tumors. Due to the characteristics of oncolytic viruses, such treatments are usually administered systemically or locally to treat primary tumors and metastatic tumors. When cancer cells rupture and die under the infection of oncolytic viruses, newly generated virus particles are released and further infect surrounding cancer cells. Oncolytic viruses not only directly kill tumor cells but also stimulate the body's immune response and enhance the anti-tumor effect. In addition to being used alone, oncolytic viruses are administered in combination with other anti-cancer agents. Furthermore, oncolytic viruses can also be recombined with foreign genes beneficial for cancer treatment. Thus, on the one hand, such viruses can exert an oncolytic effect through oncolytic proteins, and on the other hand, the anti-cancer effects of other drugs can also be obtained. Therefore, oncolytic viruses containing recombinant nucleic acids are still needed to achieve better treatment results for cancer.

Summary of the Invention

Means for Solving the Problems

[0004] One aspect of the present disclosure may provide an oncolytic virus containing a recombinant nucleic acid. The recombinant nucleic acid may include (i) a first nucleic acid fragment encoding a soluble PD-1 molecule, (ii) a second nucleic acid fragment encoding a CD86 molecule, and (iii) a third nucleic acid fragment encoding an antibody against a CD3 molecule.

[0005] In some embodiments, the similarity between the first nucleic acid fragment and the sequence shown in SEQ ID NO: 1 may be 90% or more. ; The similarity between the second nucleic acid fragment and the sequence shown in SEQ ID NO: 2 is 90% or more; the similarity between the third nucleic acid fragment and the sequence shown in SEQ ID NO: 3 is 90% or more.

[0006] In some embodiments, the recombinant nucleic acid may further comprise a fourth nucleic acid fragment encoding a US11 protein , The similarity between the fourth nucleic acid fragment and the sequence shown in SEQ ID NO: 4 may be 90% or more.

[0007] In some embodiments, the oncolytic virus may belong to the genus Simplexvirus

[0008] In some embodiments, the oncolytic virus may be an HSV-1 virus, and the similarity between the recombinant nucleic acid and the sequence shown in SEQ ID NO: 5 may be 80% or more ; The oncolytic virus is an HSV-2 virus, and the fourth nucleic acid fragment contains an exogenous nucleic acid fragment inserted into the recombinant nucleic acid.

[0009] In some embodiments, the recombinant Nucleic acid may comprise at least one of a nucleic acid fragment encoding a cytokine, a nucleic acid fragment encoding a molecule that enables an oncolytic virus to target and infect cancer cells, a nucleic acid fragment encoding an anti-angiogenic factor, and a nucleic acid fragment encoding a matrix metalloproteinase

[0010] In some embodiments, when the oncolytic virus acts at a multiplicity of infection of 2 on human non-small cell lung cancer cells, human liver cancer cells, human breast cancer cells or human pancreatic cancer cells in a culture environment 1 or it may kill at least 70%, 90%, 60% or 40% of the cancer cells within 48 hours respectively

[0011] In some embodiments, when the oncolytic virus is injected into a non-small cell lung cancer tumor by a single injection at a dose of 8×10 6 pfu, at least 60% of the tumor volume may decrease within 100 days, or ; Tumor when the oncolytic virus is injected into a non-small cell lung cancer tumor by three injections at a dose of 8×10 6 pfu, at least 80% of the tumor volume may decrease within 100 days, or ; Or when the oncolytic virus is injected into a subject suffering from colorectal cancer at a dose of at least 3×10 6 pfu, the survival period of the subject is extended.

[0012] One aspect of the present disclosure is a composition for treating cancerAnd application can be provided. The composition may include any one of the above-described oncolytic viruses and a pharmaceutically acceptable vector or excipient.

[0013] In some embodiments, cancers may include melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, rectal cancer, liver cancer, cervical cancer , Colorectal cancer or osteosarcoma.

[0014] One aspect of the present disclosure may provide a composition for treating non-small cell lung cancer or colorectal cancer. And application can be provided. The composition may include any one of the above-described oncolytic viruses and a pharmaceutically acceptable vector or excipient.

[0015] One aspect of the present disclosure may provide a method for treating cancer. The method may include the step of administering an effective dose of the above-described composition to a subject suffering from cancer. , The subject is a mammal to be.

[0016] In some embodiments, the ratio of the amount of oncolytic virus in the effective dose of the composition to the body weight of the subject may be in the range of 1×10 6 pfu / kg to 2×10 6 pfu / kg.

[0017] In some embodiments, the ratio of the amount of oncolytic virus in the effective dose of the composition to the body weight of the subject may be in the range of 1.30×10 6 pfu / kg to 1.70×10 6 pfu / kg.

[0018] In some embodiments, the step of administering the effective dose of the composition to a subject suffering from cancer may include the step of administering the composition to the subject by injection. At a site within or near the tumor The step of administering the composition to the subject may be included.

[0019] The present disclosure will be further described with respect to exemplary embodiments. These exemplary embodiments will be described in detail with reference to the drawings. These embodiments are non-limiting exemplary embodiments.

Brief Description of the Drawings

[0020]

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Modes for Carrying Out the Invention

[0021] The technical solutions of the embodiments of the present disclosure are further clearly described below, and the accompanying drawings that need to be formed in the description of the embodiments are briefly described below. Obviously, the drawings described below are only some examples or embodiments of the present disclosure. A person skilled in the art can apply the present disclosure to other similar situations according to these drawings without further creative efforts. Unless clearly obtained from the context or the context indicates otherwise, the same reference numerals in the drawings refer to the same structure or operation.

[0022] As shown in the present disclosure and the claims, unless the context clearly indicates an exception, "a", "one" and / or "the" are not particularly singular, and the plural form may be included. The terms "comprise", "comprises" and / or "comprising", "include", "includes" and / or "including", when used in the present disclosure, merely imply the inclusion of the specifically identified steps and elements, and it is further understood that these steps and elements do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0023] As shown in the present disclosure and the claims, unless the context clearly indicates an exception, the terms "comprise", "comprises" and / or "comprising", "include", "includes" and / or "including", when used in the present disclosure, can be understood to mean including the described elements, but do not exclude the presence or addition of one or more other steps and their elements.

[0024] The following are the definitions of some terms used in the present disclosure.

[0025] As used herein, a "subject" (also referred to as an "individual" or "patient") can be an individual to be treated by the oncolytic viruses or compositions of the present disclosure. In some embodiments, the subject can be a vertebrate. Vertebrates can include fish (e.g., sharks), amphibians (e.g., frogs, Xenopus laevis, giant salamanders), reptiles (e.g., turtles, snakes, lizards), birds (e.g., ostriches), mammals, and the like. In some embodiments, the vertebrate can be a mammal. Mammals can include, but are not limited to, primates (including humans and non-human primates) and rodents (e.g., mice and rats). In some embodiments, the mammal can be a human. In some embodiments, the subject can have cancer and may have received other treatments (e.g., chemotherapy) or may not have been treated at all.

[0026] The term "treating" (or "treatment") can refer to ameliorating or curing a subject's disease (e.g., cancer). In some embodiments, treatment can include alleviating, delaying, or mitigating the severity of cancer symptoms (e.g., reducing tumor volume), reducing the frequency of cancer symptoms (e.g., pain), extending survival time, increasing survival rate, decreasing cancer cell survival rate, killing cancer cells, and the like.

[0027] The term "effective dose" can refer to an amount of a composition (e.g., a dose sufficient to treat a disease) that is sufficient to bring about a useful event or reduce an adverse undesirable event. In the present disclosure, the composition can include an oncolytic virus. The amount of oncolytic virus in the effective dose of the composition can depend on various factors including, but not limited to, the purpose of treatment, the weight, sex, age, and general health of the subject, the route of administration, the time of administration, and the nature of the disease being treated.

[0028] The term "herpes simplex virus (HSV)" can be an enveloped virus, a neurotropic virus, and a double-stranded DNA virus. For example, the virus can be classified into herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2).

[0029] The term "immune checkpoint inhibitor" can refer to an antibody that inhibits or blocks an inhibitory immune checkpoint molecule. Immune checkpoints can be regulators and modifiers of the immune system, and their role is to prevent the immune system from attacking cells indiscriminately, which can be essential for self-tolerance.

[0030] The present disclosure can provide an oncolytic virus containing a recombinant nucleic acid. The oncolytic virus can be a herpes simplex virus, for example, an HSV-1 virus or an HSV-2 virus. The recombinant nucleic acid can include a first nucleic acid fragment encoding a soluble programmed death-1 (sPD-1) molecule, a second nucleic acid fragment encoding a CD86 molecule, a third nucleic acid fragment encoding an antibody against a surface antigen classification 3 (CD3) molecule, and a fourth nucleic acid fragment encoding a US11 protein. The recombinant nucleic acid can also include a nucleic acid fragment encoding a cytokine, a nucleic acid fragment encoding a molecule that facilitates the oncolytic virus targeting and infecting cancer cells, a nucleic acid fragment encoding an anti-angiogenic factor, a nucleic acid fragment encoding a matrix metalloproteinase, etc., or any combination thereof.

[0031] The present disclosure can also provide a composition for treating cancer. The composition can include any one of the above-described oncolytic viruses and a pharmacologically acceptable vector or excipient. Cancers that can be treated by the composition can include, but are not limited to, melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, rectal cancer, liver cancer, cervical cancer, osteosarcoma.

[0032] The present disclosure may also provide the application of the above-described oncolytic virus in the preparation of a drug for treating cancer. Specifically, the present disclosure may also provide the application of the above-described oncolytic virus in the preparation of a drug for treating lung cancer.

[0033] The present disclosure may further provide a method for treating cancer. The method may include the step of administering an effective dose of the above-described composition to a subject suffering from cancer. The composition may be injected into the subject by means such as injection. For example, the composition may be injected into the subject's tumor or a site near the tumor. In some embodiments, the composition may be injected into the subject in combination with other drugs (such as anti-cancer agents).

[0034] The oncolytic virus and its composition disclosed in the present disclosure may express proteins / antibodies that can activate T cells, may express proteins / antibodies that can reduce T cell inhibition, thereby improving the body's immune ability, enhancing the ability of the virus to escape from the host's immune clearance, extending the survival time in the body, thereby inhibiting and killing cancer cells, effectively reducing the survival rate of cancer cells in the administered subject, effectively improving or delaying the symptoms of cancer (such as reducing the tumor volume), and improving the survival ability of the subject.

[0035] One aspect of the present disclosure may provide an oncolytic virus containing a recombinant nucleic acid (also referred to as a "recombinant oncolytic virus").

[0036] According to various genetic materials of the virus, oncolytic viruses can be classified into DNA-type oncolytic viruses and RNA-type oncolytic viruses. Exemplary DNA-type oncolytic viruses may include, but are not limited to, adenovirus, vaccinia virus, parvovirus, herpes simplex virus, etc. Exemplary RNA-like oncolytic viruses may include, but are not limited to, reovirus, poliovirus, Semliki Forest virus, etc.

[0037] In some embodiments of the present disclosure, the oncolytic virus can be an HSV belonging to the Herpesviridae family and the genus Simplexvirus. HSV can include HSV-1 and HSV-2. In some embodiments, the oncolytic virus can be an artificially engineered oncolytic virus. For example, by removing from the wild-type HSV-1 virus an oncolytic virus encoding the neurotropic ICP34.5 (or γ-34.5) gene, it can be made even less neurotoxic, that is, the resulting oncolytic virus can be non-pathogenic / non-neurotoxic and oncolytic.

[0038] In some embodiments, HSV-1 can be used as an oncolytic virus that selectively attacks cancer cells because it is easy to handle and relatively harmless in its natural state. By modifying the gene of an oncolytic virus (e.g., HSV-1), such as inserting other gene fragments that can inhibit and kill cancer cells, the ability of the oncolytic virus to target and infect cancer cells, and / or the ability of the oncolytic virus to kill cancer cells can be improved. As a result, the oncolytic virus can have a better anti-tumor therapeutic effect.

[0039] The recombinant nucleic acid can include one or more exogenous nucleic acid fragments. The exogenous nucleic acid fragments can include, but are not limited to, nucleic acid fragments encoding immune checkpoint inhibitors, nucleic acid fragments encoding co-stimulatory molecules, nucleic acid fragments encoding antibodies against surface antigens of effector cells, nucleic acid fragments encoding molecules that facilitate the oncolytic virus to evade or resist the host immune response, nucleic acid fragments encoding molecules that facilitate the oncolytic virus to target and infect cancer cells, nucleic acid fragments encoding cytokines, nucleic acid fragments encoding anti-angiogenic factors, nucleic acid fragments encoding matrix metalloproteinases, antisense RNAs or small RNAs that block or down-regulate tumor overexpressed oncogenes and metabolic genes, prodrug converting enzymes, etc., or any combination thereof.

[0040] In some embodiments, the nucleic acid fragment encoding an immune checkpoint inhibitor may include, but is not limited to, a nucleic acid fragment encoding a soluble PD-1 molecule (i.e., the first nucleic acid fragment), a nucleic acid fragment encoding a PD-1 inhibitor, a nucleic acid fragment encoding a PD-L2 (or B7-DC, CD273) inhibitor, a nucleic acid fragment encoding a CTLA-4 inhibitor, a nucleic acid fragment encoding a LAG-3 inhibitor, a nucleic acid fragment encoding a TIM-3 inhibitor, a nucleic acid fragment encoding a neuropilin inhibitor, a nucleic acid fragment encoding a CCR4 inhibitor, a nucleic acid fragment encoding a TIGIT (or Vsig9, Vstm3, WUCAM) inhibitor, a nucleic acid fragment encoding a VISTA (or Dies1) inhibitor, etc., or a combination thereof.

[0041] In some embodiments, a co-stimulatory molecule can refer to a molecule whose encoding can stimulate the proliferation of T cells or help activate the function of T cells. Nucleic acid fragments encoding co-stimulatory molecules can include, but are not limited to, nucleic acid fragments encoding the B7 family, nucleic acid fragments encoding CD27, nucleic acid fragments encoding CD28, nucleic acid fragments encoding CD70, nucleic acid fragments encoding CD83, nucleic acid fragments encoding CD134 (or OX-40), nucleic acid fragments encoding CD134L (or OK-40L), nucleic acid fragments encoding CD137 (or 41BB), nucleic acid fragments encoding CD137L (or 41BBL), nucleic acid fragments encoding CD224, nucleic acid fragments encoding GITR, nucleic acid fragments encoding ICOS, etc., or any combination thereof. In some embodiments, nucleic acid fragments encoding co-stimulatory molecules can include, but are not limited to, nucleic acid fragments encoding B7-1 (or CD80), nucleic acid fragments encoding B7-2 (or CD86), nucleic acid fragments encoding B7-H1 (or PD-L1, CD274), nucleic acid fragments encoding ICOS-L (or CD275, B7-H2), nucleic acid fragments encoding B7-H3 (or CD276), nucleic acid fragments encoding B7-H4, nucleic acid fragments encoding B7-DC (or PD-L2, CD273), or nucleic acid fragments encoding BT3.1 (or CD277), i.e., nucleic acid fragments encoding the B7 family.

[0042] In some embodiments, nucleic acid fragments encoding antibodies against surface antigens of effector cells can include nucleic acid fragments encoding antibodies against surface antigens of T cells and nucleic acid fragments encoding antibodies against surface antigens of B cells. For example, nucleic acid fragments encoding antibodies against CD3 molecules (i.e., the third nucleic acid fragment), nucleic acid fragments encoding antibodies against CD4 molecules, nucleic acid fragments encoding antibodies against CD5 molecules, nucleic acid fragments encoding antibodies against CD8 molecules, nucleic acid fragments encoding antibodies against CD45RO molecules, nucleic acid fragments encoding antibodies against CD20 molecules, nucleic acid fragments encoding antibodies against CD21 molecules, nucleic acid fragments encoding antibodies against CD45RA molecules, etc., or any combination thereof.

[0043] Nucleic acid fragments encoding molecules that enable oncolytic viruses to avoid or facilitate resistance to the host immune response may include, but are not limited to, nucleic acid fragments encoding US11 (i.e., the fourth nucleic acid fragment), nucleic acid fragments encoding UL82, etc., or any combination thereof.

[0044] Nucleic acid fragments encoding molecules that enable oncolytic viruses to target and infect cancer cells may include, but are not limited to, nucleic acid fragments encoding CD86 molecules, etc.

[0045] Nucleic acid fragments encoding cytokines may include, but are not limited to, nucleic acid fragments encoding GM-CSF, nucleic acid fragments encoding G-CSF, nucleic acid fragments encoding M-CSF, nucleic acid fragments encoding IL-1, nucleic acid fragments encoding IL-2, nucleic acid fragments encoding IL-3, nucleic acid fragments encoding IL-4, nucleic acid fragments encoding IL-5, nucleic acid fragments encoding IL-6, nucleic acid fragments encoding IL7, nucleic acid fragments encoding IL-8, nucleic acid fragments encoding IL-10, nucleic acid fragments encoding IL-12, nucleic acid fragments encoding IL-13, nucleic acid fragments encoding IL-15, nucleic acid fragments encoding IL-18, nucleic acid fragments encoding IL-21, nucleic acid fragments encoding IL-23, nucleic acid fragments encoding IFN-α, nucleic acid fragments encoding IFN-γ, nucleic acid fragments encoding TGF-β, nucleic acid fragments encoding TNF-α, etc., or any combination thereof.

[0046] The nucleic acid fragments encoding anti-angiogenic factors include, but are not limited to, nucleic acid fragments encoding one or more interacting polypeptides that disrupt cell types (e.g., endothelial cells (EC), and circulating endothelial progenitor cells, pericytes, vascular smooth muscle cells, and mesenchymal cells including stem cells and parenchymal cells), nucleic acid fragments encoding one or more interacting polypeptides that disrupt secreted factors (e.g., vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) or angiogenin), etc., or combinations thereof.

[0047] The nucleic acid fragments encoding matrix metalloproteinases include, but are not limited to, matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 7 (MMP7), matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 12 (MMP12).

[0048] In some embodiments, the recombinant nucleic acid of the oncolytic virus may include a nucleic acid fragment encoding an immune checkpoint inhibitor, a nucleic acid fragment encoding a costimulatory molecule, a nucleic acid fragment encoding an antibody against a surface antigen of effector cells, and a nucleic acid fragment encoding a molecule that facilitates the oncolytic virus to avoid or resist the host immune response.

[0049] In some embodiments, the recombinant nucleic acid may include a first nucleic acid fragment encoding a soluble PD-1 molecule, a second nucleic acid fragment encoding a CD86 molecule, and a third nucleic acid fragment encoding an antibody against a CD3 molecule. Additionally or alternatively, the recombinant nucleic acid may include a fourth nucleic acid fragment encoding a US11 protein.

[0050] The soluble PD-1 molecule can be the extracellular region of a PD-1 immunosuppressive molecule that competitively binds to the ligand PD-L1 expressed by cancer cells, thereby releasing the inhibitory effects of PD-1 and PD-L1 on T cells. Tumor-lytic viruses recombined with the soluble PD-1 molecule can effectively promote the immune response.

[0051] The CD3 molecule can be an important marker on the surface of T cells and consists of five polypeptide chains, γ, δ, ε, ζ, and η. Antibodies against the CD3 molecule can specifically mobilize T cells expressing the CD3 molecule. Therefore, by inserting a gene fragment capable of expressing an antibody against the CD3 molecule into the nucleic acid of a tumor-lytic virus, the activation and / or proliferation of T cells can be promoted, and the antitumor effect of the tumor-lytic virus can be enhanced. Exemplary nucleic acid fragments encoding antibodies against the CD3 molecule can include nucleic acid fragments encoding OKT3, nucleic acid fragments encoding L2K, nucleic acid fragments encoding UCHT1, etc., or any combination thereof. In some embodiments, the third nucleic acid fragment can be a nucleic acid fragment of OKT3.

[0052] Generally, the activation of T cells can depend on two signals. One can be an antigenic peptide-MHC complex that can be recognized by the TCR. The second signal can be a co-stimulatory signal that can affect the activation, proliferation, and cytokine secretion of T cells. The B7 family can be the most important co-stimulatory molecules, among which CD80 and CD86 are homologous and can be important members of the B7 family. Nucleic acid fragments expressing OKT3 within tumor-lytic viruses can be used for the specific mobilization of T cells. Nucleic acid fragments expressing CD86 can further activate T cells and enable T cells to proliferate and differentiate. In some embodiments, the second nucleic acid fragment can also be a nucleic acid fragment of CD80.

[0053] The US11 protein can interact with the endogenous pattern recognition receptors RIG-I and MDA-5, interfere with the interaction between RIG-I and MDA-5 and the adapter protein MAVS, thereby inhibiting the activation of the RLR-mediated innate immune downstream signaling pathway IRF3 and preventing the production of beta interferon. Adding a nucleic acid fragment encoding the US11 protein (i.e., the fourth nucleic acid fragment) can be used to enhance the ability of oncolytic viruses to evade the host's natural immune defense and extend the retention time of oncolytic viruses in the body. In some embodiments, the fourth nucleic acid fragment may contain an exogenous nucleic acid fragment inserted into the recombinant nucleic acid. For example, the exogenous nucleic acid fragment can be a nucleic acid fragment encoding an exogenous US11 protein (e.g., human or animal). In some embodiments, the fourth nucleic acid fragment may contain a non-exogenous nucleic acid fragment inserted into the recombinant nucleic acid, such as a nucleic acid fragment encoding the US11 protein of an oncolytic virus.

[0054] In some embodiments, the similarity between the first nucleic acid fragment and the sequence shown in SEQ ID NO: 1 can be 95%, 90%, 85%, 80% or more. In some embodiments, the similarity between the second nucleic acid fragment and the sequence shown in SEQ ID NO: 2 can be 95%, 90%, 85%, 80% or more. In some embodiments, the similarity between the third nucleic acid fragment and the sequence shown in SEQ ID NO: 3 can be 95%, 90%, 85%, 80% or more. In some embodiments, the similarity between the fourth nucleic acid fragment and the sequence shown in SEQ ID NO: 4 can be 95%, 90%, 85%, 80% or more. In some embodiments, the similarity between the recombinant nucleic acid of the oncolytic virus and the sequence shown in SEQ ID NO: 5 can be 95%, 90%, 85%, 80% or more.

[0055] In some embodiments, the oncolytic virus may further comprise a fifth nucleic acid fragment, for example, enhanced yellow fluorescent protein (EYFP), whose coding is used to screen recombinant oncolytic viruses. In some embodiments, one or more of the first nucleic acid fragment, the second nucleic acid fragment, the third nucleic acid fragment, the fourth nucleic acid fragment, and the fifth nucleic acid fragment may be linked to one or more expression control sequences. The one or more expression control sequences may include a promoter, an enhancer, a polynucleotide (e.g., a terminator), or a combination thereof. Exemplary promoters may include the SV40 promoter, the CMV promoter, the MSV promoter, the EF1 promoter, the MMLV promoter, the U6 promoter, the H1 promoter, etc. Exemplary enhancers may include the SV40 enhancer, the CMV enhancer, etc. Terminators may include SV40 PolyA, TK PolyA, BGH PolyA, etc. For example, the first nucleic acid fragment may be operably linked to a promoter. As another example, the fifth nucleic acid fragment may be operably linked to a CMV promoter, a CMV enhancer, or a BGH PolyA.

[0056] In some embodiments, each exogenous nucleic acid fragment may be inserted into the nucleic acid of the oncolytic virus by one or more common methods in the art for obtaining the recombinant nucleic acids described above, which may not be limited in the present disclosure. For example, one or more exogenous nucleic acid fragments can be inserted into the vector using ligase, fusion polymerase chain reaction (PCR) technology, etc., or combinations thereof. For example, ligase can be continuously used to ligate the digested vector and the gene fragment to be inserted. As another example, fusion PCR can be used to continuously ligate various fragments and then fuse them with the vector. As just one example, a first and a second vector (such as a plasmid) may be constructed, and multiple gene fragments (e.g., three gene fragments) may be continuously connected to the first vector using ligase, and other gene fragments may be continuously connected to the second vector using ligase, and PCR can be used to obtain the remaining gene fragments connected together so as to be connected to the first vector. In some embodiments, the vector can be the nucleic acid of a wild-type oncolytic virus. For example, the vector can be the nucleic acid of a wild-type HSV-1 virus. In some embodiments, the vector can be the nucleic acid of an oncolytic virus in which one or more coding genes (e.g., ICP34.5) are deleted.

[0057] It should be noted that the present disclosure does not limit the sequences of each nucleic acid fragment within the recombinant nucleic acid. In some embodiments, the first, second, third, and fourth nucleic acid fragments can be inserted into different sites of the nucleic acid of the oncolytic virus, respectively. In some embodiments, one or more of the first, second, third, and fourth nucleic acid fragments can be inserted into the same site of the nucleic acid of the oncolytic virus. In some embodiments, the insertion site of the nucleic acid fragment described above can be any suitable site within the coding region of the nucleic acid of the oncolytic virus. For example, the insertion site of the nucleic acid fragment described above can be a position where one or more coding genes (e.g., ICP34.5) are deleted in the HSV-1 virus. In some embodiments, the exogenous nucleic acid fragment can be inserted continuously into the same or different sites within the nucleic acid of the oncolytic virus. The sequence of the exogenous nucleic acid fragment within the recombinant nucleic acid can be arbitrary. In some embodiments, the sequences of the nucleic acid fragments (from the 5'-end to the 3'-end) within the recombinant nucleic acid of the oncolytic virus can be the fourth nucleic acid fragment, the fifth nucleic acid fragment, the first nucleic acid fragment, the second nucleic acid fragment, and the third nucleic acid fragment.

[0058] One aspect of the present disclosure can provide a composition for treating cancer. The composition can include any one of the oncolytic viruses described above and a pharmaceutically acceptable vector or excipient.

[0059] Pharmacologically acceptable vectors can include coating layers, capsules, microcapsules, nanocapsules, etc., or any combination thereof. It should be noted that the vector needs to be non-toxic and should not significantly affect the activity of important components in the composition (for example, the above-mentioned oncolytic virus, molecules that promote the anti-cancer effect expressed by the oncolytic virus, such as soluble PD-1 molecules). In some embodiments, the vector can protect important components in the composition and reduce or avoid the inactivation or degradation of important components under negative conditions (such as denaturation caused by oxidation, strong acids or strong alkalis, etc.). For example, enzymes, or the relatively low pH value in gastric juice, may decompose or inactivate important components. The vector can help maintain or enhance the effectiveness of the pharmaceutical composition by protecting important components in the composition.

[0060] In some embodiments, the vector can be used to control the release of important components (such as oncolytic viruses). Release can include, but is not limited to, sustained release, controlled release, targeted release, etc. For example, the vector can include hydrogel capsules, microcapsules or nanocapsules made from collagen, gelatin, chitosan, alginate, polyvinyl alcohol, polyethylene oxide, starch, cross-linked starch, etc., or any combination thereof.

[0061] In some embodiments, pharmaceutically acceptable vectors can include dispersion media (such as solvents), coatings, buffers, stabilizing agents, isotonic agents, absorption retardants, etc. Exemplary pharmacologically acceptable vectors can include phosphate buffered saline, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, gels, bioabsorbable matrix materials, other suitable materials, etc., or any combination thereof.

[0062] In some embodiments, excipients may include, but are not limited to, water, physiological saline, polyethylene glycol, hyaluronic acid, ethanol, and pharmaceutically acceptable salts, such as salts of inorganic acids (e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.) and salts of organic acids (e.g., acetate, propionate, benzoate, etc.).

[0063] In some embodiments, cancers that the composition can treat may include, but are not limited to, glioblastoma, melanoma, liver cancer, lung cancer, colorectal cancer, rectal cancer, head and neck tumors, breast cancer, renal cell carcinoma, ovarian cancer, prostate cancer, gastric cancer, lymphoma, pancreatic cancer, bladder cancer, endometrial cancer, cervical cancer, sarcoma (such as soft tissue sarcoma and osteosarcoma), etc.

[0064] The composition can be injected into a subject suffering from cancer, such as a human or an animal. In some embodiments, the composition can be injected into the subject by one or more modes of administration. The one or more modes of administration may include, but are not limited to, oral administration, injection or topical administration. Forms of the composition suitable for oral administration may include, but are not limited to, tablets, liposome formulations, sustained release capsules, microparticles, microspheres or any other suitable form. Forms of the composition suitable for injection may include, but are not limited to, sterile aqueous preparations, oily preparations, etc. Forms of the composition suitable for topical administration may include, but are not limited to, sterile aqueous or non-aqueous solutions, suspensions and emulsions. Taking nasal administration as an example, forms of the composition may include aerosols, mists, powders, solutions, suspensions, gels, etc.

[0065] In some embodiments, the composition can be stored at a suitable temperature, which can include room temperature (about 20°C), 4°C, -20°C, -80°C, etc. The composition can also be prepared in various forms convenient for storage and transportation, such as powder. The powder can be a sterile powder, and a solvent can be added to the sterile powder and uniformly mixed before use to prepare a solution for oral administration, injection, or topical administration. In some embodiments, the composition can also contain components that have an antibacterial effect but do not significantly adversely affect the survival of oncolytic viruses and stabilize the composition under specific storage conditions (such as refrigeration and freezing), preventing contamination by microorganisms (such as bacteria and fungi).

[0066] In some embodiments, the multiplicity of infection of the oncolytic virus acting on cancer cells in the culture environment can be 0.1, 0.2, 0.5, 0.8, 1.0, 1.5, 2.0, 2.5, etc.

[0067] In some embodiments, when the oncolytic virus acts on human non-small cell lung cancer cells in the culture environment at a multiplicity of infection of 2, at least 30% of the cancer cells can be killed within 48 hours accordingly. In some embodiments, when the oncolytic virus acts on human non-small cell lung cancer cells in the culture environment at a multiplicity of infection of 2, at least 50% of the cancer cells can be killed within 48 hours. In some embodiments, when the oncolytic virus acts on human non-small cell lung cancer cells in the culture environment at a multiplicity of infection of 2, at least 70% of the cancer cells can be killed within 48 hours accordingly.

[0068] In some embodiments, when the oncolytic virus acts on human liver cancer cells at a multiplicity of infection of 2 in a culture environment, correspondingly, at least 60% of the cancer cells can be killed within 48 hours. In some embodiments, when the oncolytic virus acts on human liver cancer cells at a multiplicity of infection of 2 in a culture environment, at least 70% of the cancer cells can be killed within 48 hours. In some embodiments, when the oncolytic virus acts on human liver cancer cells at a multiplicity of infection of 2 in a culture environment, at least 80% of the cancer cells can be killed within 48 hours. In some embodiments, when the oncolytic virus acts on human liver cancer cells at a multiplicity of infection of 2 in a culture environment, at least 90% of the cancer cells can be killed within 48 hours.

[0069] In some embodiments, when the oncolytic virus acts on human breast cancer cells at a multiplicity of infection of 2 in a culture environment, correspondingly, at least 30% of the cancer cells can be killed within 48 hours. In some embodiments, when the oncolytic virus acts on human breast cancer cells at a multiplicity of infection of 2 in a culture environment, at least 40% of the cancer cells can be killed within 48 hours. In some embodiments, when the oncolytic virus acts on human breast cancer cells at a multiplicity of infection of 2 in a culture environment, correspondingly, at least 60% of the cancer cells can be killed within 48 hours.

[0070] In some embodiments, when the oncolytic virus acts on human pancreatic cancer cells at a multiplicity of infection of 2 in a culture environment, correspondingly, at least 20% of the cancer cells can be killed within 48 hours. In some embodiments, when the oncolytic virus acts on human pancreatic cancer cells at a multiplicity of infection of 2 in a culture environment, correspondingly, at least 30% of the cancer cells can be killed within 48 hours. In some embodiments, when the oncolytic virus acts on human pancreatic cancer cells at a multiplicity of infection of 2 in a culture environment, at least 40% of the cancer cells can be killed within 48 hours.

[0071] In some embodiments, when the oncolytic virus acts on human non-small cell lung cancer cells, human liver cancer cells, human breast cancer cells or human pancreatic cancer cells in a culture environment at a multiplicity of infection of 0.1, correspondingly, about 50%, 40%, 60% or 40% of the cancer cells can be killed within 48 hours.

[0072] In some embodiments, when the oncolytic virus acts on human non-small cell lung cancer cells, human liver cancer cells, human breast cancer cells or human pancreatic cancer cells in a culture environment at a multiplicity of infection of 1, correspondingly, at least 70%, 80%, 60% or 40% of the cancer cells can be killed within 48 hours.

[0073] In some embodiments, when the oncolytic virus is injected into a subject by injection, the tumor volume can be reduced or, further, the tumor can be eliminated. For example, when the oncolytic virus is injected into a human non-small cell lung cancer tumor by a single injection at a dose of 8×10 6 pfu or three injections at a dose of 8×10 6 pfu, it can cause a reduction of at least 60% or 80% in tumor volume within 100 days, respectively.

[0074] In some embodiments, when the oncolytic virus is injected into a subject by injection, the survival period of the subject can be extended and / or the survival rate of the subject can be improved. When the oncolytic virus is injected into a subject suffering from colorectal cancer by three injections at a dose of at least 1×10 6 pfu, the survival period of the subject can be extended. For example, the survival rate of the subject can reach 90% within 111 days of the injection of the oncolytic virus. In some embodiments, when the oncolytic virus is injected into a subject suffering from colorectal cancer by three injections at a dose of at least 1×10 7 pfu, the survival period of the subject can be extended. For example, the survival rate of the subject can reach 40% within 111 days of the injection of the oncolytic virus.

[0075] One aspect of the present disclosure may provide the application of oncolytic viruses in the preparation of drugs for treating cancer. Oncolytic viruses can be used to treat subjects suffering from cancer, such as mammals.

[0076] Exemplary cancers may include melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, colorectal cancer, rectal cancer, liver cancer, cervical cancer, or osteosarcoma. In some embodiments, the cancer may include lung cancer, liver cancer, breast cancer, or pancreatic cancer.

[0077] One aspect of the present disclosure may provide a method for treating cancer. The method may include the step of administering an effective dose of the above-described composition to a subject suffering from cancer. The subject may be a mammal, such as a human.

[0078] In some embodiments, the effective dose of the composition may be injected into a subject having cancer. For example, the effective dose may be determined based on the characteristics of the subject to be treated, the route of administration, and / or the characteristics of the cancer (e.g., type of cancer, progression of cancer, etc.). Specifically, the characteristics of the subject may include, but are not limited to, age, gender, height, weight, health status, etc. Therefore, the effective doses described in the embodiments of the present disclosure are exemplary and may be modified by those skilled in the art according to specific situations. For example, the ratio of the amount of oncolytic virus in the composition to the body weight of the subject may be in the range of 0.5×10 6 pfu / kg to 2.5×10 6 pfu / kg, 0.70×10 6 pfu / kg to 2.3×10 6 pfu / kg, 1×10 6 pfu / kg to 2×10 6 pfu / kg, 1.30×10 6 pfu / kg to 1.70×10 6 pfu / kg, 1.36×10 6 pfu / kg to 1.67×10 6 pfu / kg, 1.40×10 6 pfu / kg to 1.60×10 6 pfu / kg, etc.

[0079] In some embodiments, the composition can be injected into a subject by various modes of administration. The modes of administration can include, but are not limited to, oral administration, injection, or topical administration. In some embodiments, the composition can be injected into a subject by injection. Exemplary injection modes can include, but are not limited to, intraperitoneal injection, subcutaneous injection, intramuscular injection, intravenous injection, etc. In some embodiments, the composition can be injected into a site within or near the tumor of the subject. In some embodiments, the composition can be injected into a tissue or organ of the subject, such as the kidney, liver, heart, thyroid, or joint. In some embodiments, topical administration can include, for example, administering the composition to the skin to relieve cancers such as skin cancer, lymphoma, etc. In some embodiments, topical administration can include intravaginal administration, rectal administration, nasal administration, auricular administration, intramedullary administration, intra-articular administration, intrapleural administration, etc., or any combination thereof. In some embodiments, the composition can be injected into a subject by a combination of different modes of administration. In some embodiments, the method can include administering to the subject three times a day, twice a day, once a day, every other day, etc.

[0080] The compositions of the present disclosure can be used before or after the administration of other pharmaceutical compositions used for the treatment of cancer. Optionally, the compositions disclosed in the present disclosure may be combined with other treatment modalities to treat the cancer of the subject. For example, other treatment modalities can include, but are not limited to, administration of other pharmaceutical compositions capable of treating the cancer of the subject, resection of the tumor of the subject by surgery, radiotherapy, etc. Specifically, pharmaceutical compositions that can be used for treating cancer can include, but are not limited to, cytotoxic anti-cancer agents and non-cytotoxic anti-cancer agents. Non-cytotoxic anti-cancer agents can include hormonal drugs (e.g., tamoxifen, exemestane), targeted drugs (e.g., bevacizumab), and immunotherapy drugs (e.g., monoclonal antibodies, tumor vaccines), etc.

[0081] The experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments were purchased from conventional biochemical reagent companies unless otherwise specified.

[0082] Embodiment 1: Construction of a vector of a recombinant oncolytic virus (i.e., an oncolytic virus containing a recombinant nucleic acid) strain. 1.1 A recombinant viral vector R1 is obtained by inserting a gene fragment encoding the US11 protein, a BGH Poly A fragment, an EYFP gene fragment, a CMV promoter, a CMV enhancer, and a soluble PD-1 gene fragment into an HSV-1 viral vector.

[0083] 1.2 A linearized viral vector R1 is obtained by digesting the constructed viral vector R1 with Hind III and Kpn I.

[0084] 1.3 Using agarose gel electrophoresis, the length of the digested viral vector R1 is verified, and the linearized viral vector R1 is recovered using a recovery kit.

[0085] 1.4 A recombinant viral vector R2 is obtained by inserting gene fragments encoding the CD86 molecule and the OKT3 molecule (i.e., an antibody against the CD3 molecule) into another HSV-1 viral vector.

[0086] 1.5 By digesting the constructed viral vector R2 with Hind III and Kpn I, gene fragments encoding the CD86 molecule and the OKT3 molecule are obtained.

[0087] 1.6 Using agarose gel electrophoresis, the lengths of the gene fragments encoding the CD86 molecule and the OKT3 molecule after digestion are verified, and the gene fragments encoding the CD86 molecule and the OKT3 molecule are recovered using a recovery kit.

[0088] 1.7 A recombinant viral vector R130 is obtained by connecting the purified linearized viral vector R1 with the gene fragments encoding the CD86 molecule and the OKT3 molecule using ligase.

[0089] 1.8 Digest the viral vector R130 using Hind III and Kpn I, verify whether the gene fragments encoding the CD86 molecule and the OKT3 molecule are connected to the linearized viral vector R1, and perform sequencing verification on the viral vector R130.

[0090] Figure 1 is a schematic diagram showing the vector plasmid structure of a recombinant oncolytic virus according to some embodiments of the present disclosure. The connection sequences and positions of each fragment are shown in Figure 1.

[0091] Simultaneously transfect the wild-type HSV-1 virus and the R130 plasmid into green monkey kidney cells (Vero cells), and remove cell debris after collection. The virus suspension obtained after high-speed centrifugation and purification was used as a mixed virus solution and stored at -80°C for subsequent use. Vero cells were purchased from the American Type Culture Collection (ATCC).

[0092] Embodiment 2: Screening of recombinant oncolytic virus strains: 2.1 Transplant about 4×10 5 individual green monkey kidney cells (Vero cells) into the wells of a 6-well plate.

[0093] 2.2 After 24 hours, aspirate and remove the culture medium of Vero cells, rinse once with 1 ml of preheated serum-free DMEM, and then add 680 μl of preheated serum-free DMEM medium.

[0094] 2.3 Take out the mixed virus solution from -80°C and dissolve it in a 4°C refrigerator.

[0095] 2.4 Divide the mixed virus solution into three gradients of 5 μl, 10 μl, and 20 μl, and repeat each gradient twice.

[0096] 2.5 Inoculate a 6-well plate with a mixed virus solution of three gradients, culture Vero cells, mix the wells, then shake back and forth several times, place the 6-well plate in a 37°C, 5% CO2 cell incubator for 1.5 hours of virus adsorption, and shake every 15 minutes.

[0097] 2.6 Preheat water baths at three temperatures of 72°C, 42°C, and 37°C in advance. Dissolve 2% agarose gel in the 72°C water bath (autoclave), preheat DMEM medium containing 4% FBS in the 37°C water bath, and preheat DMEM medium containing 1% low melting point agarose and 2% FBS prepared according to the number of cultured cells in the 42°C water bath. Each 6-well plate requires 2 ml of DMEM medium containing 1% agarose gel and 2% FBS.

[0098] 2.7 Aspirate and remove the virus-containing serum-free medium in the 6-well plate, and gently add 2 ml of DMEM medium containing 1% agarose gel and 2% FBS to the wells.

[0099] 2.8 Seal the 6-well plate with parafilm, place it in a 4°C refrigerator to solidify the low melting point agarose, and transfer it to a cell culture incubator for normal culture after 10 minutes.

[0100] 2.9 Check the appearance of plaques with green fluorescence every day. When the plaques with green fluorescence are large enough, use a 200 μl sterile pipette tip to pick them out, transfer them to a 0.6 ml sterile centrifuge tube containing 200 μl of serum-free DMEM medium, count the selected recombinant oncolytic virus strains, and store the recombinant oncolytic virus strains at -80°C for at least 15 minutes.

[0101] 2.10 Use a pipette tip to mix the stored recombinant oncolytic virus strains, re-inoculate them into Vero cells in a 6-well plate according to a specific ratio, and if all the formed plaques contain green fluorescence following the above steps for the next selection round, select good plaques for amplification and storage to obtain the selected recombinant oncolytic virus strains.

[0102] Embodiment 3, Identification of Recombinant Oncolytic Virus Strains: 3.1 Culture the collected medium and the cells collected after amplification on a 10 cm Vero cell culture dish, repeat freezing and thawing 2 - 3 times at -80°C, centrifuge at 4°C, 3500 rpm for 15 minutes, and collect 200 μl of the supernatant.

[0103] 3.2 Transfer 200 μl of the supernatant to a 1.5 ml centrifuge tube, add 400 μl of the lysate, and immediately vortex to mix thoroughly.

[0104] 3.3 Let it stand at room temperature for 10 minutes, and shake and mix every 5 minutes.

[0105] 3.4 Add 450 μl of absolute ethanol, and immediately vortex to mix thoroughly.

[0106] 3.5 Add the above mixture to the adsorption column, place the adsorption column in the collection tube, centrifuge at 13000 rpm for 30 - 60 seconds, and discard the waste liquid in the collection tube.

[0107] 3.6 Add 500 μl of the protein removal solution, centrifuge at 12000 rpm for 30 seconds, and discard the waste liquid.

[0108] 3.7 Add 500 μl of the rinsing solution, centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, add 500 μl of the rinsing solution, and repeat once.

[0109] 3.8 Return the adsorption column to an empty collection tube, centrifuge at 13,000 rpm for 2 minutes, and remove the rinse solution as much as possible so that the residual ethanol in the rinse solution does not inhibit the downstream reaction.

[0110] 3.9 Take out the adsorption column, place it in an RNase-free centrifuge tube, add 30 - 50 μl of RNase-free water to the middle part of the adsorption membrane, leave it at room temperature for 1 minute, and centrifuge at 12,000 rpm for 1 minute to obtain the genome of the recombinant oncolytic virus strain.

[0111] 3.10 Design 4 pairs of PCR primers for those inserted to amplify the exogenous gene fragments, and verify the 4 amplified exogenous gene fragments by agarose gel electrophoresis. The agarose gel electrophoresis results of the PCR-amplified DNA fragments are shown in Figure 2. As shown in Figure 2, a single DNA fragment was amplified, the fragment size was accurate, and it was shown that each exogenous gene fragment was inserted into the oncolytic virus strain.

[0112] 3.11 Perform next-generation sequencing (NGS) verification on the amplified fragments and conduct sequence comparison using snapgene biological software. The comparison result was 100%. Thereby, it was verified that the PCR-amplified DNA fragment was a target gene sequence without mutation. Therefore, the recombinant HSV-1 virus containing the recombinant nucleic acid is called HSV1-R130 and has been deposited with the General Microbiology Center of the China Microbiological Culture Collection Management Committee (CGMCC). The deposit date was May 8, 2021. The registration number of the deposit center is CGMCC No.20319, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Courtyard, Beichen West Road, Chaoyang District, Beijing.

[0113] Embodiment 4. The recombinant oncolytic virus has a significant killing effect on cancer cells: 4.1 Inoculate human non-small cell lung cancer cells A549, human hepatocellular carcinoma cells HepG2, human breast cancer cells MCF-7, and human pancreatic cancer cells SW1990 into a 12-well plate over 24 hours according to an appropriate inoculum amount. The cells basically covered a monolayer.

[0114] 4.2 After 24 hours, aspirate and remove the original culture medium in the 12-well plate, wash twice with DPBS or serum-free DMEMP medium, and then add 300 μl of serum-free DMEM medium.

[0115] 4.3 Dilute the stock solution of the recombinant oncolytic virus 10-fold and 100-fold.

[0116] 4.4 Calculate the amount of virus added per well according to the initial cell inoculum amount and virus titer, and classify them into the following four groups: the multiplicity of infection (MOI) of the control group is 0, the MOI of the low-dose group is 0.1, the MOI of the medium-dose group is 1.0, and the MOI of the high-dose group is 2.0.

[0117] 4.5 After 1 hour of virus adsorption, replace it with DMEM maintenance medium containing 2% FBS and continue the culture.

[0118] 4.6 Treat the cells at 24 hours and 48 hours respectively, stain the cells with trypan blue, and measure the viability of live cells.

[0119] Figure 3 is a schematic diagram of human non-small cell lung cancer cell A549 transfected with recombinant oncolytic virus after staining according to some embodiments of the present disclosure. Figure 4 is a schematic diagram of human hepatocellular carcinoma cell HepG2 transfected with recombinant oncolytic virus after staining according to some embodiments of the present disclosure. Figure 5 is a schematic diagram of human breast cancer cell MCF-7 transfected with recombinant oncolytic virus after staining according to some embodiments of the present disclosure. Figure 6 is a schematic diagram of human pancreatic cancer cell SW1990 transfected with recombinant oncolytic virus after staining according to some embodiments of the present disclosure. The survival rates (or cell survival rates) of the four stained cancer cells shown in Figures 3-6 were calculated. The results are shown in Figure 7. Figure 7 is a schematic diagram of the cell survival rates of human non-small cell lung cancer cell A549, human hepatocellular carcinoma cell HepG2, human breast cancer cell MCF-7, and human pancreatic cancer cell SW1990 transfected with recombinant oncolytic virus according to some embodiments of the present disclosure.

[0120] As shown in Figure 7, in the control group without adding virus, four groups of cancer cells A549, HepG2, MCF-7, and SW1990 were cultured for 24 hours and 48 hours, and the cell survival rates were all about 100%. In the experimental group with virus added, low dose (0.1 MOI), medium dose (1 MOI), and high dose (2 MOI) could effectively kill the four cancer cells. The killing effect at a virus action time of 48 hours was significantly higher than that at an action time of 24 hours.

[0121] After treating cancer cells A549 and MCF-7 with recombinant oncolytic virus for 24 hours, the cell survival rates of the low dose group, medium dose group, and high dose group were about 80%, 50%, and 50%, respectively. When the treatment time was extended to 48 hours, the cell survival rates of the low dose group, medium dose group, and high dose group were about 50%, 38%, and 38%, respectively. Extending the action time of the recombinant oncolytic virus or increasing the dose of the recombinant oncolytic virus significantly increased the mortality rates of cancer cells A549 and MCF-7 and decreased the viability of cancer cells A549 and MCF-7.

[0122] After treating cancer cells HepG2 with recombinant oncolytic virus for 24 hours, the cell survival rates of the medium-dose group and the high-dose group were approximately 80% and 60%, respectively. When the treatment time was extended to 48 hours, the cell survival rate of the low-dose group was 70%, and the cell survival rates of the medium-dose group and the high-dose group were only 20% and 10%, respectively. This indicates that extending the treatment time of the recombinant oncolytic virus or increasing the dose of the recombinant oncolytic virus can significantly increase the mortality rate of human liver cancer cells HepG2 and reduce the viability of human liver cancer cells HepG2.

[0123] After treating cancer cells SW1900 with recombinant oncolytic virus for 24 hours, the cell survival rates of the low-, medium- and high-dose groups were slightly lower than the cell survival rate of the control group. After 48 hours of virus action, the effect was more significant than that after 24 hours, that is, the cell survival rate was low. The low-dose group, the medium-dose group and the high-dose group can effectively kill SW1900 cancer cells within 48 hours. The cell survival rate of the low-dose group was less than 80%. There was little difference between the medium-dose group and the high-dose group, and the cell survival rate was less than 60%. The above results indicate that the recombinant oncolytic virus can rapidly kill cancer cells SW1900 in a short period (for example, 48 hours), and the higher the dose of the recombinant oncolytic virus, the lower the cell survival rate of cancer cells.

[0124] Therefore, the recombinant oncolytic virus can rapidly and effectively kill four cancer cells, A549, HepG2, MCF-7 and SW1990, and has an obvious inhibitory effect on lung cancer, liver cancer, breast cancer and pancreatic cancer. The inhibitory effect on liver cancer was the most effective. Extending the virus action time or increasing the virus dose significantly reduced the cell survival rate of cancer cells.

[0125] Embodiment 5. The recombinant oncolytic virus has an obvious curative effect on immunodeficient nude mice suffering from non-small cell lung cancer: To test the effectiveness of R130, non-small cell lung cancer cells A549 were intraperitoneally injected into immunocompetent mice to construct a non-small cell lung cancer mouse model. An R130 virus solution was injected into the peritoneal cavity of the mice suffering from non-small cell lung cancer. Control group CK directly injected with 100 μL of PBS (pH 7.4) buffer solution, high-dose group injected with a dose of 8×10 6 pfu every three days for three consecutive injections, and the experiment was classified into three groups: low-dose group injected with a dose of 8×10 6 pfu once. The physical condition of the mice was observed daily, and the body weight of the mice was monitored every two days. The tumor diameter was monitored using calipers, the tumor volume was calculated, and the mice were euthanized if they survived the treatment for 111 days.

[0126] Figure 8 is a schematic diagram of a lung cancer mouse model constructed by treating immunodeficient nude mice with recombinant oncolytic virus according to some embodiments of the present disclosure. As shown in Figure 8, when the oncolytic virus was injected into non-small cell lung cancer tumors by a single injection of a dose of 8×10 6 pfu, or three injections of a dose of 8×10 6 pfu, it caused at least a 60% or 80% reduction in tumor volume.

[0127] Embodiment 6. The recombinant oncolytic virus has a significant curative effect on immune intact mice suffering from colorectal cancer: To test the effectiveness of R130, a CT26 colorectal cancer mouse model was established by intraperitoneally injecting colorectal cancer cells CT26 into immune intact mice. An R130 virus solution was injected into the peritoneal cavity of the immune intact mice suffering from colorectal cancer. Control group CK directly injected with 100 μL of PBS (pH 7.4) buffer solution, high-dose group injected with a dose of 1×10 7 pfu every three days for three consecutive injections, and the test was classified into a low-dose group injected with a dose of 1×10 6 pfu every three days for three consecutive injections.

[0128] Figure 9 is a schematic diagram of the time and mouse survival rate of a CT26 peritoneal colon cancer mouse model treated with different doses of recombinant oncolytic virus according to some embodiments of the present disclosure. As shown in Figure 9, in the control group injected with PBS, after 33 days of injection, the survival rate of the mice was 10%, and 90% of the mice died. However, in the experimental groups injected with virus amounts of 3×10 7 pfu and 3×10 6 pfu, the survival rates of the mice were 100% and 80% respectively. At the end of the 111-day experiment, the mice in the experimental groups with virus amounts of 3×10 7 pfu and 3×10 6 pfu still had relatively high survival rates, namely 90% and 40% respectively.

[0129] Therefore, the recombinant oncolytic virus significantly improved the survival rate of tumor-bearing mice. After injecting the virus three times at a dose of 10 7 pfu, 90% of the mice with abdominal tumors were able to survive for a long time.

[0130] The recombinant oncolytic virus and its application disclosed in the present disclosure may bring beneficial effects including, but not limited to, the following: (1) The nucleic acid fragment encoding the soluble PD-1 molecule in the recombinant oncolytic virus can reduce or mitigate the inhibitory effect of T cells on the oncolytic virus, thereby enhancing the survival and proliferation of the oncolytic virus in the host cells; (2) The nucleic acid fragment encoding the antibody of the CD3 molecule in the recombinant oncolytic virus can activate the proliferation and activation of T cells and enhance the anti-tumor effect of the immune system; (3) The nucleic acid fragment encoding the CD86 molecule in the recombinant oncolytic virus can further activate T cells and enable T cells to proliferate and differentiate; and (4) The nucleic acid fragment encoding the US11 protein in the recombinant oncolytic virus can enhance the ability of the oncolytic virus to avoid the host's innate immune defense, extend the residence time of the oncolytic virus in the body, thereby enhancing the target infection and killing effect on cancer cells.

[0131] It should be noted that different embodiments may have different beneficial effects. In different embodiments, the possible beneficial effects may be any one or combination of the above, or any other possible beneficial effects.

[0132] Those skilled in the art should understand that the above embodiments are merely illustrative of the present disclosure and do not limit the present disclosure. Modifications, equivalent substitutions, and changes made within the scope of the spirit and principle of the present disclosure shall be included within the protection scope of the present disclosure.

Claims

1. A oncolytic virus comprising a recombinant nucleic acid, wherein the recombinant nucleic acid comprises (i) a first nucleic acid fragment encoding a soluble PD-1 molecule, (ii) a second nucleic acid fragment encoding a CD86 molecule, (iii) a third nucleic acid fragment encoding an antibody against a CD3 molecule, (iv) a fourth nucleic acid fragment encoding a US11 protein, and the oncolytic virus is an HSV-1 virus, the recombinant nucleic acid has the sequence of SEQ ID NO: 5, the oncolytic virus.

2. The recombinant nucleic acid comprises at least one of a nucleic acid fragment encoding a cytokine, a nucleic acid fragment encoding a molecule that targets cancer cells and facilitates infection of cancer cells, a nucleic acid fragment encoding an anti-angiogenic factor, and a nucleic acid fragment encoding a matrix metalloproteinase, the oncolytic virus according to claim 1.

3. When the oncolytic virus acts on human non-small cell lung cancer cells, human liver cancer cells, human breast cancer cells or human pancreatic cancer cells at a multiplicity of infection of 1 or 2 in a culture environment, at least 70%, 90%, 60% or 40% of the cancer cells are killed within 48 hours respectively, the oncolytic virus according to claim 1.

4. When the oncolytic virus is injected into a non-small cell lung cancer tumor by a single injection at a dose of 8×10 6 pfu, at least 60% of the tumor volume decreases within 100 days; when the oncolytic virus is injected into the non-small cell lung cancer tumor by three injections at the dose of 8×10 6 pfu, at least 80% of the tumor volume decreases within 100 days; or when the oncolytic virus is injected into a subject suffering from colorectal cancer at a dose of at least 3×10 6 pfu, the survival period of the subject is prolonged, the oncolytic virus according to claim 1.

5. A composition for treating cancer, comprising the oncolytic virus according to any one of claims 1 to 4 and a pharmaceutically acceptable vector or excipient.

6. The composition according to claim 5, wherein the cancer includes melanoma, lung cancer, leukemia, gastric cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, rectal cancer, liver cancer, cervical cancer, colorectal cancer or osteosarcoma.

7. A composition for treating non-small cell lung cancer or colorectal cancer, comprising the oncolytic virus according to any one of claims 1 to 4 and a pharmaceutically acceptable vector or excipient.

8. The ratio of the amount of the oncolytic virus in the composition at an effective dose to the body weight of the subject is in the range of 1×10 6 pfu / kg to 2×10 6 pfu / kg. The composition according to claim 5.

9. The ratio of the amount of the oncolytic virus in the composition at an effective dose to the body weight of the subject is in the range of 1.30×10 6 pfu / kg to 1.70×10 6 pfu / kg. The composition according to claim 5.

10. The composition according to claim 9, wherein the composition is administered to the subject by injection at a site within or near the tumor.

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