TWI894357B - Oncolytic virus and a modified immune cell for treating tumors - Google Patents

Abstract

The present application provides a combination of an oncolytic virus and a modified immune effector cell for treating tumors. The present application provides a composition, a pharmaceutical composition, and a reagent kit comprising the oncolytic virus and the modified immune effector cell. The present application further provides a preparation method and a use for preparation of a drug for treating tumors of the composition, the pharmaceutical composition, and the reagent kit.

Description

Combining oncolytic viruses with engineered immune cells for tumor treatment

This application involves the field of biomedicine, specifically the combined use of oncolytic viruses and engineered immune cells for the treatment of tumors.

Immune effector cells are cells in the human body that participate in immune responses. Immune checkpoints are inhibitory molecules present in the immune system. They regulate the intensity and breadth of immune responses through ligand-receptor interactions, preventing damage and destruction caused by excessive responses. They are one of the normal body's negative feedback regulatory mechanisms. However, during the development and progression of tumors, immune checkpoints become a major factor in inducing tumor immune tolerance, resulting in an inefficient anti-tumor immune response.

Viral cancer treatments have developed rapidly over the past two decades. One of the greatest advances in viral therapy is the ability to exploit the differences between tumor cells and normal cells to modify the structure of certain viruses, enabling them to selectively replicate in tumor cells and ultimately kill them. These modified viruses are called oncolytic viruses and are derived from viruses such as herpes viruses and pox viruses. It has been discovered that some wild-type viruses also have the ability to selectively replicate in cells, thereby dissolving tumors. However, the efficacy of oncolytic viruses alone in treating tumors is sometimes suboptimal. Combining oncolytic viruses with chemotherapy can cause severe side effects, as the chemotherapy drugs also damage normal cells.

Therefore, more effective treatment options and drugs are still needed in the immunotherapy of tumors and/or cancers.

The present application provides a composition comprising: (a) an oncolytic virus; and (b) a modified immune effector cell, wherein the expression amount and/or activity of the immune checkpoint of the modified immune effector cell is adjusted compared to the immune effector cell without the modification. The present application also provides a pharmaceutical composition comprising: (a) at least one oncolytic virus and/or a pharmaceutically acceptable carrier; and (b) at least one immune effector cell and/or a pharmaceutically acceptable carrier. The present application also provides a reagent kit comprising the oncolytic virus, a tool for preparing the oncolytic virus, the modified immune effector cell, a tool for preparing the immune effector cell and/or a pharmaceutically acceptable carrier. On the other hand, the present application also provides a method for preparing the composition, the pharmaceutical composition, and the reagent kit. This application also provides the use of the oncolytic virus in combination with the oncolytic virus and the modified immune effector cells, and the use of the modified immune effector cells in combination with the oncolytic virus and the modified immune cells. This application also provides a method for treating tumors, comprising administering the composition, the pharmaceutical composition, and/or the kit to a subject in need thereof. This application also provides the use of the composition, the pharmaceutical composition, and the kit in the preparation of a medicament for treating tumors. The combination of oncolytic virus and modified immune effector cells effectively exerts a synergistic effect, enhancing the killing power of tumors and improving the therapeutic efficacy of tumors.

In one aspect, the present application provides a composition comprising: (a) an oncolytic virus; and (b) a modified immune effector cell, wherein the expression level and/or activity of an immune checkpoint in the modified immune effector cell is modulated compared to an unmodified immune effector cell.

In certain embodiments, the oncolytic virus comprises a natural oncolytic virus and a modified oncolytic virus.

In certain embodiments, the oncolytic virus is selected from the group consisting of vesicular stomatitis virus (VSV), poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, poliovirus, reovirus, Seneca virus, echovirus, coxsackievirus, Newcastle disease virus, and Malaba virus.

In certain embodiments, the bronchiolitis virus comprises matrix protein M, nucleocapsid protein N, phosphoprotein P, large polymerase protein L, and/or glycoprotein G.

In certain embodiments, the matrix protein M of the bronchiolitis virus is modified.

In certain embodiments, the modification of the bronchiolitis virus matrix protein M comprises mutations at more than one site.

In certain embodiments, the mutation site of the bronchiolitis virus matrix protein M is selected from one or more of the following groups: M51, L111, V221, and S226.

In certain embodiments, the mutation site of the bronchiolitis virus matrix protein M is selected from the following group of single site mutations:

a)M51R;

b)△L111;

c) V221F; and

d) S226R.

In certain embodiments, the combination of mutation sites of the bronchiolitis virus matrix protein M is selected from any one of the following groups:

a) M51R and △L111;

b) M51R and V221F;

c) M51R and S226R;

d) △L111 and V221F;

e) △L111 and S226R;

f) V221F and S226R;

g) M51R, △L111 and V221F;

h) M51R, △L111 and S226R;

i) M51R, V221F, and S226R;

j) △L111, V221F and S226R; and,

k)M51R, △L111, V221F and S226R.

In certain embodiments, the combination of mutation sites selected from M51R, V221F, and S226R in the basal protein M of stomatitis virus comprises the amino acid sequence shown in SEQ ID NO: 6.

In certain embodiments, the combination of mutation sites selected from M51R, ΔL111, V221F, and S226R in the basal protein M of stomatitis virus comprises the amino acid sequence shown in SEQ ID NO: 8.

In certain embodiments, the immune effector cells are selected from the group consisting of T cells, B cells, NK cells, and macrophages.

In certain embodiments, the immune effector cells comprise autologous cells and allogeneic cells.

In certain embodiments, the number of the immune checkpoints is more than one.

In certain embodiments, the immune detection point is selected from one or more of the following groups: PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, BTLA, VISTA, TIGIT, B7-H2, B7-H3, B7-H4, and B7-H6.

In certain embodiments, the immune checkpoint is PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, BTLA, VISTA, TIGIT, B7-H2, B7-H3, B7-H4, or B7-H6.

In certain embodiments, the immune checkpoint is a combination selected from the following groups:

a) PD-1 and CTLA-4;

b) PD-1 and PD-L1;

c) PD-L1 and CTLA4;

d) LAG-3 and CTLA-4;

e) TIM-3 and CTLA-4;

f)VISTA and CTLA-4; and

g) PD1, CTLA-4, and PD-L1.

In certain embodiments, the modulation of the expression of the immune checkpoint comprises upregulation, downregulation, and/or deletion of the expression of the immune checkpoint compared to unmodified immune effector cells.

In certain embodiments, modulation of the immune checkpoint activity comprises an increase, decrease, and/or absence of the immune checkpoint activity compared to unmodified immune cells.

In certain embodiments, the modulation of the immune checkpoint comprises regulation of immune checkpoint gene expression levels, transcription levels, and/or translation levels.

In certain embodiments, the regulation of gene expression levels comprises gene editing, overexpression, point mutation, and/or homologous recombination.

In certain embodiments, the gene editing method is selected from one or more of the following groups: CRISPR/Cas9, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN) and monobase editing (BE).

In certain embodiments, the gene editing comprises designing a sequence of sgRNA.

In certain embodiments, the immune effector cell knocks out a target sequence of the PD-1 gene selected from SEQ ID NO: 1.

In certain embodiments, the immune effector cell knocks out a target sequence of the CTLA-4 gene selected from SEQ ID NO: 2.

On the other hand, the present application also provides a pharmaceutical composition comprising: (a) the at least one oncolytic virus and/or a pharmaceutically acceptable carrier; and (b) the at least one modified immune effector cell and/or a pharmaceutically acceptable carrier.

On the other hand, the present application also provides a test kit comprising one or more selected from the following group:

a) the at least one oncolytic virus and means for preparing the at least one modified immune effector cell, and/or a pharmaceutically acceptable carrier;

b) the at least one modified immune effector cell and means for preparing the at least one oncolytic virus, and/or a pharmaceutically acceptable carrier; and,

c) means for preparing the at least one oncolytic virus and the at least one modified immune effector cell, and/or a pharmaceutically acceptable carrier;

On the other hand, this application also provides methods for preparing the composition, the pharmaceutical composition, and the reagent kit.

On the other hand, the present application also provides the use of the oncolytic virus in combination with the modified immune effector cells.

On the other hand, this application also provides the use of the modified immune effector cells in combination with the oncolytic virus and the modified immune cells.

On the other hand, the present application also provides a method for treating tumors, comprising administering the composition, the pharmaceutical composition, and the preparation method of the kit to a subject in need thereof.

In certain embodiments, the oncolytic virus is administered to a subject in need thereof via an intravenous and/or intratumoral route, wherein the immune effector cells are administered to a subject in need thereof via an intravenous and/or local administration route.

In certain embodiments, the oncolytic virus and the modified immune cell are administered simultaneously in therapeutically effective amounts.

In certain embodiments, the oncolytic virus and the modified immune cell are administered separately, and the administered dosage is a therapeutically effective amount.

In certain embodiments, the oncolytic virus and the modified immune cell are administered once or multiple times.

On the other hand, this application also provides the use of the composition, the pharmaceutical composition, and the reagent kit in the preparation of drugs for treating tumors.

In certain embodiments, the tumor described in this application comprises a solid tumor and/or a hematological tumor.

In certain embodiments, the tumor is selected from one or more of the following groups: head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.

Those skilled in the art will readily appreciate other aspects and advantages of the present application from the detailed description that follows. The detailed description that follows merely illustrates and describes exemplary embodiments of the present application. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to modify the specific embodiments disclosed without departing from the spirit and scope of the inventions to which the present application relates. Accordingly, the drawings and descriptions in the specification are intended to be illustrative only and not restrictive.

The specific features of the invention involved in this application are shown in the attached patent scope. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

Figure 1 shows a schematic diagram of the replication ability of each modified oncolytic virus in LLC cells and MEF cells in vitro;

Figure 2 shows a schematic diagram of the killing ability of each modified oncolytic virus on LLC cells, Hela cells, and MEF cells in vitro;

Figure 3 shows a schematic diagram of the effects of various modified oncolytic viruses on the expression levels of IFN-β in LLC cells and MEF cells in vitro;

Figure 4 shows a schematic diagram of the CRISPR/Cas9 fusion plasmid elements;

Figure 5 shows flow cytometry analysis of the expression of PD-1 molecules on the surface of modified human peripheral blood T cells.

Figure 6 shows the in vitro anti-tumor effect test of the modified oncolytic virus;

Figure 7 shows the T cell therapy strategy of combining a modified oncolytic virus with PD-1 molecule deletion;

Figure 8 shows the tumor-killing effect of a modified oncolytic virus combined with PD-1-deficient T cells.

The following describes the implementation of the present invention using specific embodiments. Those skilled in the art can readily understand the other advantages and effects of the present invention from the disclosure in this specification.

Definition of terms

In this application, the term "composition" includes at least two components. In this application, the components of the composition can be mixed or separated. In this application, the components of the composition can be administered simultaneously or separately. The separate administration can be sequential. For example, one component of the composition can be administered first, followed by another component. In a specific embodiment, the composition comprises the oncolytic virus and the modified immune effector cell.

In this application, the term "oncolytic virus" generally refers to a virus that can replicate in and kill tumor cells. In certain embodiments, the virus is engineered to increase tumor cell selectivity. Oncolytic viruses include, but are not limited to, stomatitis virus, poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, poliovirus, reovirus, Seneca virus, echovirus, coxsackievirus, Newcastle disease virus, and Malaba virus. In preferred embodiments, the oncolytic virus is stomatitis virus. A preferred stomatitis virus can be a stomatitis virus Mudd Summer subtype strain in which the matrix protein M gene has undergone site-directed mutation.

In this application, the term "modified" refers to the altered state or structure of a molecule or cell of the invention. Molecules can be modified in a variety of ways, including chemically, structurally, and functionally. Cells can be modified by the introduction of nucleic acids.

In this application, the term "immune effector cells" is used interchangeably with "immune cells" and generally refers to cells that play a role in immune responses. Immune effector cells include: lymphocytes, such as T cells and B cells, and natural killer cells; and bone marrow cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

As used herein, the term "T cell" refers to thymus-derived cells that participate in various cell-mediated immune responses, including thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. In certain embodiments, T cell populations include, but are not limited to, helper T cells (HTLs; CD4+ T cells), cytotoxic T cells (CTLs; CD8+ T cells), CD4+CD8+ T cells, CD4-CD8- T cells, or any other subset of T cells. In certain embodiments, the T cell population includes, but is not limited to, T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR, and can be further isolated by positive or negative selection techniques, if desired.

In this application, the term "immune checkpoint" generally refers to a group of molecules on the surface of immune cells that modulate immune responses by downregulating or inhibiting tumor immune responses. Immune checkpoints include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, PD-L2, TIM-3, TIM-4, LAG-3, BTLA, SIRPα, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, arginase, CD73, and A2aR (see, e.g., WO 2012/177624). The term also encompasses biologically active protein fragments, as well as nucleic acids encoding full-length immune checkpoint proteins and fragments of biologically active proteins thereof. In certain embodiments, the term also encompasses any and fragments of the homology descriptions provided herein.

In this application, the term "reduced expression" is used interchangeably with "decreased expression" or "downregulated expression," and generally refers to a decrease in the expression of a molecule in a modified cell compared to the expression of the molecule in a wild-type cell. The term "increased expression" is used interchangeably with "increased expression" or "upregulated expression," and generally refers to an increase in the expression of a molecule in a modified cell compared to the expression of the molecule in a wild-type cell. In certain embodiments, the decrease in expression can be a decrease in protein expression of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In certain embodiments, the decrease in molecule expression can be assessed by measuring a decrease in the level of RNA encoded by the target gene and/or a decrease in the level of molecule expression.

In this application, the term "reduced activity" is used interchangeably with "decreased activity" or "downregulated activity," and generally refers to a decrease in the amount of activity of a molecule in a modified cell compared to the amount of activity in a wild-type cell. The term "increased activity" is used interchangeably with "increased activity" or "upregulated activity," and generally refers to an increase in the amount of activity of a molecule in a modified cell compared to the amount of activity in a wild-type cell. A decreased activity level or decreased expression level can mean a decrease in the activity of the molecule by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In certain embodiments, the amount of molecule synthesized is not decreased, but the amino acid sequence is modified such that the activity of the molecule is decreased directly or indirectly.

In this application, the term "deletion" generally refers to the loss of expression and/or activity of a target molecule. Deletion can occur at the genetic level, transcriptional level, or translational level. In certain embodiments, deletion can mean that expression and activity of the target molecule are undetectable.

In this application, the term "modulation" is used interchangeably with "regulation" and generally refers to a change or alteration in the expression level and/or activity of a molecule of interest. Modulation can be an increase or decrease in the magnitude of a specific activity or function of the molecule of interest. In certain embodiments, activities and functions include, but are not limited to, binding characteristics, enzymatic activity, cell receptor activation, and signal transduction.

In this application, the term "native" generally refers to naturally occurring, unmodified. For example, a naturally occurring polypeptide or polynucleotide sequence present in an organism (including a virus) can be isolated from a natural source and has not been intentionally modified in the laboratory or otherwise.

In this application, the term "engineered" generally refers to a naturally occurring molecule that has been altered through artificial means. This alteration can occur through modification, mutation, synthesis, or other means. "Engineered" can be distinguished from naturally occurring. For example, a cell or organism is considered "engineered" if it has been manipulated to alter its genetic information (e.g., by introducing new genetic material that did not previously exist, such as through transformation, mating, somatic cell hybridization, transfection, transduction, or other mechanisms, or by altering or removing pre-existing genetic material, such as through substitution or deletion mutations).

In this application, the term "large polymerase protein L" is used interchangeably with "L protein" and generally refers to the VSV RNA polymerase protein. The VSV L gene encodes the RNA poly E protein, which is involved in initiation, elongation, methylation, capping, and poly (A) tail formation.

In this application, the term "nucleocapsid protein N" is used interchangeably with "N protein" and generally refers to the nucleoprotein of stomatitis virus. The N protein effectively protects viral RNA from digestion by various nucleases. The N protein is group-specific, shared by many types and subtypes, has high antigenicity, and can stimulate the body to produce non-neutralizing antibodies.

In this application, the term "phosphoprotein P," also known as "P protein," generally refers to the phosphoprotein of stomatitis virus. The P gene is highly and differentially phosphorylated. The P, L, and N protein-RNA complexes are essential for transcriptase activity.

In this application, the term "matrix protein M" is used interchangeably with "M protein" and generally refers to the matrix protein of stomatitis virus (VSV). Matrix protein M is an important virulence factor of VSV and is also a protein in stomatitis virus known to disrupt the innate immune response in mice.

In this application, the term "glycoprotein G" is used interchangeably with "G protein" and generally refers to the glycoprotein of stomatitis virus.

In this application, the term "M51R" refers to the mutation of methionine M at position 51 to arginine R; the term "△L111" refers to the deletion of leucine at position 111.

In this application, the term "mutation" generally refers to a change in the nucleotide or amino acid sequence of a wild-type molecule, for example, a change in the DNA and/or amino acid sequence of the wild-type CTLA4 extracellular domain. A mutation in the DNA can alter codons, thereby resulting in a change in the amino acid sequence. DNA changes can include substitutions, deletions, insertions, alternative splicing, or truncations. Amino acid changes can include substitutions, deletions, insertions, additions, truncations, or errors in protein processing or cleavage. Alternatively, a mutation in the nucleotide sequence can result in a silent mutation in the amino acid sequence, as is well understood in the art. In this regard, certain nucleotide codons encode the same amino acid. Examples include the nucleotide codons CGU, CGG, CGC, and CGA, which encode the amino acid arginine (R); or the codons GAU and GAC, which encode the amino acid aspartic acid (D). Thus, a protein can be encoded by one or more nucleic acid molecules that differ in their specific nucleotide sequence but still encode protein molecules with the same sequence.

In this application, "single-base editing (BE)" generally refers to gene editing technology involving single-base conversion. This technology is based on a complex consisting of the nuclease-inactive dCas9 or the single-strand DNA nickase-active Cas9n (Cas9 nickase), cytosine deaminase, uracil glycosylase inhibitor (UGI), and sgRNA. It achieves precise single-base editing from C to T to G to A within a specific activity window without inducing double-strand DNA breaks.

In this application, the term "point mutation" has both broad and narrow meanings. A broad point mutation can be an alkali substitution, an alkali insertion, or an alkali deletion. A narrow point mutation, also known as a single alkali change, can be a single alkali substitution. Alkali substitutions can be further divided into transitions and transpositions.

Homologous recombination In this application, the term "homologous recombination" generally refers to the exchange of nucleotide sequences at conserved regions shared by two genomic loci. Homologous recombination includes symmetric homologous recombination and asymmetric homologous recombination. Asymmetric homologous recombination is also called unequal recombination.

In this application, the term "autologous cells" generally refers to cells that originate from an individual and are subsequently reintroduced into the same individual. The individual can be human or animal.

In this application, the term "allogeneic cells" generally refers to cells that originate from one individual and are then introduced into another individual. The individual can be human or animal.

In this application, the term "in vitro expansion" generally refers to the provision of certain nutrients and stimulatory factors in an environment outside the body to promote cell proliferation.

In this application, the term "transcription" generally refers to the process of producing a nucleic acid copy of a starting nucleic acid. Transcription typically involves the production of an RNA copy of a DNA nucleic acid and may also involve the production of a DNA copy of an starting RNA nucleic acid (e.g., reverse transcription).

In this application, the term "translation" refers to the process at the ribosome where a chain of mRNA controls the assembly of an amino acid sequence to produce a protein or peptide.

In this application, the term "overexpression," also known as "hyperexpression" or "overexpressed," generally refers to gene expression at a level higher than the gene's expression level in normal cells.

In this application, the term "gene editing" generally refers to directing or achieving an alteration (e.g., deletion) of one or more nucleic acids at or near a targeted genomic DNA site.

In this application, the term "transcription factor-like nuclease," also known as "TALEN," is generally used in a broad sense to include single TALENs that can cleave double-stranded DNA without assistance from another TALEN. The term "TALEN" also refers to one or both members of a TALEN pair that are engineered to work together to cleave DNA at the same site. TALENs working together may be referred to as left-handed TALENs and right-handed TALENs, which refers to the chirality of the DNA or TALEN pair.

The code for TALs has been reported (PCT Publication WO 2011/072246), in which each DNA-binding repeat sequence is responsible for recognizing a single base pair within a DNA target sequence. These base pairs can be assembled to target a specific DNA sequence. Briefly, the target site for TALEN binding is identified, and a fusion molecule is created containing a nuclease and a series of target-recognizing RVDs (repair devices). Upon binding, the nuclease cleaves the DNA, allowing the cellular repair machinery to operate and modify the gene at the cleaved end. The term TALEN refers to proteins containing a transcription initiator-like (TAL) effector binding domain and a nuclease domain. This includes both monomeric TALENs that function on their own and others that require dimerization with another monomeric TALEN. When the two monomeric TALENs are identical, dimerization results in a homodimer TALEN, or when the two monomeric TALENs are different, dimerization results in a heterodimer TALEN. TALENs have been shown to induce gene modification in immortalized human cells using two major eukaryotic DNA repair pathways: non-homologous end joining (NHEJ) and homology-directed repair. TALENs are typically used in pairs, but monomeric TALENs are also known.

Genetic modifications performed using TALENs or other tools can, for example, be selected from the group consisting of insertions, deletions, insertion of exogenous nucleic acid fragments, and substitutions. Generally, a target DNA site is identified and a TALEN pair is created that specifically binds to that site. The TALENs are delivered into cells or embryos, for example, as proteins, mRNA, or via vectors encoding the TALENs. TALENs cleave DNA to form double-strand breaks and then repair the double-strand breaks, which typically result in the formation of indels or incorporation of sequences or polymorphisms contained in the accompanying exogenous nucleic acid, which is inserted into the chromosome or serves as a template for repairing the break using the modified sequence. This template-driven repair is a useful method for altering chromosomes, providing efficient changes to cellular chromosomes.

In this application, the term "zinc finger nuclease," also known as "ZFN," generally refers to artificial restriction enzymes created by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. The zinc finger domain can be engineered to target a desired DNA sequence, enabling zinc finger nucleases to target unique sequences within complex genomes. By exploiting the endogenous DNA repair machinery, these agents can be used to alter the genomes of higher organisms. In certain embodiments, ZFNs can be used in gene inactivation methods.

The zinc finger DNA-binding domain consists of approximately 30 amino acids and folds into a stable structure. Each zinc finger primarily binds to a triplet within the DNA substrate. Amino acid residues at key positions facilitate the most sequence-specific interactions with DNA sites. These amino acids can be altered while maintaining the necessary structure of the remaining amino acids. By tandemly linking several domains, it is possible to bind to longer DNA sequences. Additional functionalities, such as the nonspecific Fok I cleavage domain (N), the transcription activator domain (A), the transcription repressor domain (R), and the methylase (M), can be fused to the ZFP to form ZFN, zinc finger transcription activator (ZFA), zinc finger transcription repressor (ZFR), and zinc finger methylase (ZFM), respectively. Materials and methods for producing genetically modified animals using zinc finger nucleases are disclosed in, for example, patents US8106255; US2012/0192298; US2011/0023159 and US2011/0281306.

In this application, the term "knockout" generally refers to the elimination of gene expression of one or more genes.

In this application, the term "kit" generally refers to a collection of components, which may or may not be packaged together. The components of the kit may be contained in separate containers (i.e., a kit having separate parts), may be provided in a single container, or may not be placed in a container. In addition, the kit may include instructions for performing the method. The instructions may be provided in the form of a user manual in paper or electronic format. For example, the manual may include instructions for explaining the results obtained when performing the above-described method using the kit of this application.

In this application, the term "tools" generally refers to elements, components, reagents, molecules, etc. required to prepare target molecules.

In this application, the term "subject" generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, improvement, prevention and/or treatment of a disease, particularly a subject in need of the oncolytic virus expression vector. In some embodiments, the subject may include a cancer patient. In certain embodiments, the subject is a human or non-human mammal. Non-human mammals can include any mammalian species other than humans, such as livestock animals (e.g., cattle, pigs, sheep, chickens, rabbits, or horses), or rodents (e.g., rats and mice), or primates (e.g., gorillas and monkeys), or domestic animals (e.g., dogs and cats). "Subjects" can be male or female, and can be of different ages. Human subjects may be Caucasian, African, Asian, Semitic, or other races, or a mixture of races. Human subjects may be elderly, adults, adolescents, children, or infants.

In this application, the term "pharmaceutical composition" generally refers to a formulation that is in a form effective to permit the biological activity of the active ingredient and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. In certain embodiments, these formulations may comprise the active ingredient of the drug and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises a pharmaceutical composition for parenteral, transdermal, intracavitary, intraarterial, intrathecal, and/or intranasal administration or direct injection into a tissue. The pharmaceutical composition can be administered by various routes, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration.

In this application, the term "pharmaceutically acceptable" generally refers to an adjuvant that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, and has a reasonable benefit/risk ratio, within the scope of sound medical judgment. In some embodiments, pharmaceutically acceptable adjuvants may refer to those adjuvants approved by regulatory agencies (such as the U.S. Food and Drug Administration, the China Food and Drug Administration or the European Medicines Agency) or listed in generally recognized pharmacopeias (such as the U.S. Pharmacopoeia, the Chinese Pharmacopoeia or the European Pharmacopoeia) for use in animals (more particularly humans). In this application, pharmaceutically acceptable adjuvants may be aluminum-based adjuvants, mineral salt adjuvants, tonicity-active adjuvants, bacterial-derived adjuvants, emulsified adjuvants, liposomal adjuvants, cytokine adjuvants, carbohydrate adjuvants, and DNA and RNA oligomer adjuvants, among others.

In this application, the terms "effective amount" and "effective dose" are used interchangeably and generally refer to an amount sufficient to achieve or at least partially achieve the desired therapeutic effect. The term "therapeutically effective amount" generally refers to an amount sufficient to cure or at least partially arrest the disease and its complications in a subject already suffering from the disease. The effective amount for this use will depend on the severity of the infection and the overall state of the patient's own immune system.

In this application, the term "combination," also referred to as "co-administration," generally refers to administration of one drug component before, after, or simultaneously with another drug component. The two or more drug components in the combination can be administered by the same route or by different routes, and can be administered simultaneously or sequentially. For example, one therapeutic agent in the combination can be the oncolytic virus, and the second therapeutic agent can be the modified immune effector cell. The combination can also include a third or even more therapeutic agents.

In this application, the term "treating" generally refers to eliminating or ameliorating a disease, or one or more symptoms associated with a disease. In certain embodiments, treatment generally refers to administering one or more drugs to a patient suffering from a disease to eliminate or alleviate the disease. In certain embodiments, "treatment" may be administered after the onset of symptoms of a particular disease, with or without other drugs.

In the present application, the term "carrier" generally refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject. In certain embodiments, the pharmaceutically acceptable carrier in the composition, pharmaceutical composition or kit of the present application may include, but is not limited to, for example, a pharmaceutically acceptable liquid, gel or solid carrier, an aqueous medium (e.g., sodium chloride injection, Ringer's solution injection, isotonic dextrose injection, sterile water injection, dextrose or lactated Ringer's injection), a non-aqueous medium (e.g., non-volatile oil of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil), an antimicrobial substance, an isotonic substance (e.g., sodium chloride or dextrose), a buffer ( For example, phosphate or citrate buffer), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending/dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropylmethylcellulose or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol bis(2-aminoethyl ether)tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, excipients, non-toxic auxiliary substances, other components well known in the art, or combinations thereof. Suitable ingredients may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, coloring agents, or emulsifiers.

In this application, the term "tumor" generally refers to any new, pathological tissue growth. Tumors may be benign, such as hemangiomas, gliomas, and teratomas, or malignant, such as adenocarcinomas, sarcomas, glioblastomas, astrocytomas, neuroblastomas, and retinoblastomas. For research purposes, these tissues may be isolated from readily available sources using methods known to those skilled in the art.

In this application, the term "Cas9" or "Cas9 molecule" refers to the enzyme from the bacterial type II CRISPR/Cas system that is responsible for DNA cleavage. Cas9 also includes the wild-type protein and its functional and non-functional mutants. In certain embodiments, Cas9 can be Cas9 from Streptococcus pyogenes.

In this application, the term "CTLA-4," also known as cytotoxic T-lymphocyte antigen-4, generally refers to an immunosuppressive receptor belonging to the CD28 family. CTLA-4 is specifically expressed on T cells in vivo and binds to two ligands, CD80 and CD86 (also known as B7-1 and B7-2, respectively). The complete hCTLA-4 sequence can be found under GenBank accession number AAB59385. For example, the amino acid sequence of human CTLA-4 can be shown as SEQ ID NO: 12.

The term "LAG-3," also known as "CD223," generally refers to lymphocyte activation gene 3. The term "human LAG-3" refers to the human sequence of LAG-3, such as the complete amino acid sequence of human LAG-3 with Genbank accession number NP_002277.

In this application, the term "PD-1" generally refers to programmed cell death 1, also known as "programmed death 1," "CD279," "cluster of differentiation 279," "PD1," and "PDCD1." PD-1 is typically expressed on T cells, B cells, natural killer T cells, activated monocytes, and dendritic cells (DCs) and is involved in apoptosis. PD-1 typically comprises an extracellular IgV domain, a transmembrane region, and an intracellular domain. PD-1 can bind to two ligands, PD-L1 and PD-L2. The amino acid sequence of human PD-1 can be found in GenBank under accession number NP_005009.2. For example, the amino acid sequence of human PD-1 can be set forth as SEQ ID NO: 11.

In this application, the term "PD-L1" generally refers to programmed death ligand 1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). PD-L1 is one of two cell surface glycoprotein ligands for PD-1. The PD-1 receptor/PD-L1 ligand complex can downregulate T cell activation and cytokine secretion. The complete hPD-L1 sequence can be found in GenBank under accession number Q9NZQ7.

In this application, the term "TIGIT" generally refers to a T cell immunoreceptor with Ig and ITIM domains. It can be derived from any native TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-cluster and immunoglobulin domain-containing protein 9, V-cluster and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM. The amino acid sequence of a human TIGIT can be found in UniProt accession number Q495A1.

In this application, the term "TIM-3" generally refers to T cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM-3 is galectin-9 (Gal9).

In this application, the term "BTLA" generally refers to a B and T lymphocyte depleting agent. In a specific embodiment, BTLA can be human BTLA.

In this application, the term "VISTA" generally refers to T cell-activating V-domain Ig inhibitory protein. In certain embodiments, VISTA may be human VISTA, which generally refers to a VISTA protein having a human amino acid sequence. Its amino acid sequence can be found under GenBank accession number NP_071436.

Description of the invention

Oncolytic viruses in the composition

In this application, the oncolytic virus can be derived from any member of the currently identified virus family. The oncolytic virus exhibits higher replication and cytocidal tropism in dividing cells compared to non-dividing cells. The oncolytic viruses described in this application include both engineered and wild-type oncolytic viruses, including but not limited to: bronchiolitis stomatitis virus, poxvirus, retrovirus, herpes simplex virus, measles virus, Semliki Forest virus, poliovirus, reovirus, Seneca virus, echovirus, coxsackievirus, Newcastle disease virus, Malaba virus, alphavirus, lentivirus, influenza virus, Sinbis virus, myxoma virus, bullet virus, picornavirus, and parvovirus.

In certain embodiments, the oncolytic virus can also be derived from, for example, herpes simplex virus, vaccinia virus, stomatitis virus, parvovirus, myxoma virus, Newcastle disease virus, reovirus, Seneca virus, measles virus, retrovirus, influenza virus, Sindbis virus, or poxvirus. An oncolytic virus "derived from" a reference virus comprises a nucleic acid sequence or amino acid sequence possessed by the reference virus. In certain embodiments, an oncolytic virus "derived from" a reference virus comprises one or more genes possessed by the reference virus. In certain embodiments, an oncolytic virus "derived from" a reference virus encodes one or more proteins encoded by the reference virus.

In certain embodiments, an oncolytic virus derived from a reference virus may comprise a nucleic acid sequence encoding one or more functional elements of the reference virus. A "functional element" may be, for example, a transcriptional regulator (e.g., a promoter/enhancer), a regulator of post-transcriptional processing, a translational regulator, a regulator of post-transcriptional processing, a response element, a repetitive sequence, or a viral protein.

The oncolytic virus can be a natural oncolytic virus or a modified oncolytic virus. The modified oncolytic virus can be genetically modified, such as by modifying one or more genes to enhance its tumor selectivity and/or preferentially replicate in dividing cells. Genetic modifications can include modifications to genes involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, virulence, lysis, and spread, or by integrating exogenous genes, including exogenous immune-regulatory genes, exogenous selection genes, and exogenous reporter genes. The modified oncolytic virus can also be modified at the amino acid level, such as by inserting, deleting, or substituting one or more amino acids. The modified oncolytic virus can also have altered protein expression levels and activity compared to the wild-type virus.

In certain embodiments, the oncolytic virus of the present application is selected from vesicular stomatitis virus (VSV). VSV is a prototypical non-segmented, negative-stranded RNA virus with an 11 kb genome encoding five proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and large polymerase protein (L). Specifically, for example, the vesicular stomatitis virus can be the Indiana strain of VSV, subtype MuddSummer, whose M protein gene sequence is shown in SEQ ID NO: 3, and the M protein amino acid sequence is shown in SEQ ID NO: 4.

In certain embodiments, the M protein can be natural and unmodified, can be naturally mutated, or can be artificially mutated.

For example, VSV can be wild-type and unmodified.

For example, the VSV M protein can be modified as follows: M51R.

For example, the VSV M protein can be modified as follows: M51R and △L111.

For example, the VSV M protein can be modified as follows: M51R, V221F, and S226R.

For example, the VSV M protein can be modified as follows: M51R, ΔL111, V221F, and S226R.

In certain embodiments, the nucleotide sequence encoding the VSV M protein (M51R, V221F, and S226R) is shown in SEQ ID NO: 5, and the amino acid sequence of the VSV M protein (M51R, V221F, and S226R) is shown in SEQ ID NO: 6.

In certain embodiments, the nucleotide sequence encoding the VSV M protein (M51R, ΔL111, V221F, and S226R) is shown in SEQ ID NO: 7, and the amino acid sequence of the VSV M protein (M51R, ΔL111, V221F, and S226R) is shown in SEQ ID NO: 8.

In certain embodiments, the mutation site of the basal protein M of the bronchiolitis virus can also be a single-site mutation, for example: ΔM51; M51A; ΔL111; V221F; S226R.

In certain embodiments, the mutation site of the basal protein M of the bronchiolitis virus may be a combination of multiple mutation sites, for example: M51R and V221F; M51R and S226R; ΔL111 and V221F; ΔL111 and S226R; V221F and S226R; M51R, ΔL111 and V221F; M51R, ΔL111 and S226R; ΔL111, V221F and S226R.

In certain embodiments, the mutation sites of the basal protein M of the bronchiolitis virus can also be mutations at consecutive sites, for example: M51-54A; ΔM51-54; ΔM51-57.

In certain embodiments, the mutation site of the basal protein M of the bronchiolitis virus can also be a combination of consecutive site mutations and single site mutations, for example: M51-54A, V221F, and S226R; ΔM51-54, V221F, and S226R; ΔM51-57, V221F, and S226R.

Modified immune effector cells in the composition

Immune effector cells generally refer to immune cells that participate in immune responses and perform effector functions. The immune effector cells described in this application can be any known type of immune effector cells and can be obtained by conventional biological methods. They include, but are not limited to: lymphocytes, such as T cells, B cells, and NK cells; and bone marrow cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, neutrophils, and dendritic cells.

In certain embodiments, the T cells include, but are not limited to, helper CD4+ T cells, regulatory CD4+ T cells, killer CD8+ T cells, memory T cells, natural killer T cells, mucosal-associated stationary T cells, and γδ T cells.

For example, the T cells can be human peripheral blood T cells.

In this application, the immune effector cells can be autologous cells or allogeneic cells. The immune effector cells can be obtained by in vitro expansion. The technology for large-scale in vitro expansion and culture of immune effector cells is known.

In this application, the immune checkpoints may include PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, BTLA, VISTA, TIGIT, B7-H2, B7-H3, B7-H4, and B7-H6.

In certain embodiments, the modulation of the expression and/or activity of the immune checkpoint may include modulation of an immune checkpoint, such as modulation of PD-1, modulation of PD-L1, modulation of CTLA-4, modulation of LAG-3, modulation of TIM-3, modulation of BTLA, modulation of VISTA, modulation of TIGIT, modulation of B7-H2, modulation of B7-H3, modulation of B7-H4, and modulation of B7-H6.

In certain embodiments, modulation of the expression and/or activity of the immune checkpoints may include modulation of a combination of two or more immune checkpoints, such as: PD-1 and PD-L1; PD-1 and CTLA-4; PD-1 and LAG3; PD-L1 and CTLA-4; LAG-3 and CTLA-4; TIM-3 and CTLA-4; VISTA and CTLA-4; PD-1, CTLA-4, and PD-L1.

In certain embodiments, the modulation of the expression of the immune checkpoint comprises upregulation, downregulation, and/or deletion of the expression of the immune checkpoint compared to unmodified immune effector cells.

In certain embodiments, the modulation of the immune checkpoint activity comprises an increase, decrease, and/or loss of the immune checkpoint activity compared to unmodified immune effector cells.

In certain embodiments, the modulation of the expression and/or activity of the immune checkpoint may be upregulation, downregulation, or deletion of the expression and/or activity of the immune checkpoint; or upregulation of the expression and/or activity of one or more of the adjusted immune checkpoints and downregulation and/or deletion of the expression and/or activity of the remaining adjusted immune checkpoints. For example, upregulation of PD-1 expression and/or activity and downregulation of CTLA-4 expression and/or activity, upregulation of PD-L1 expression and/or activity, downregulation of CTLA4 expression and/or activity, and deletion of LAG-3 expression and/or activity.

In this application, the regulation of the immune checkpoint includes the regulation of the immune checkpoint gene level, transcription level and/or translation level.

In some implementations, one or more of these methods may be used for regulation.

In certain embodiments, the gene-level regulation method includes, for example, gene editing, overexpression, point mutagenesis, and homologous recombination. The gene editing method includes, for example, CRISPR/Cas9, transcription activator-like nucleases (TALENs), zinc finger nucleases (ZFNs), and base editing (BE).

In one embodiment, the immune checkpoint is adjusted to reduce the expression of the PD-1 molecule on T cells. The specific method is to use CRISPR/Cas9 gene editing technology to knock out the PDCD1 gene in human peripheral blood T cells, thereby reducing the expression of the PD-1 molecule.

In certain embodiments, the CRISPR/Cas9 gene editing technology can include a designed sgRNA sequence. The sgRNA can bind to the Cas9 protein to identify and edit the target gene.

In certain embodiments, the immune effector cell knocks out a target sequence of the PD-1 gene selected from SEQ ID NO: 1.

In certain embodiments, the immune effector cell knocks out a target sequence of the CTLA-4 gene selected from SEQ ID NO: 2.

Compositions, pharmaceutical compositions, and kits

In this application, a composition is provided, comprising: (a) the oncolytic virus; and (b) the modified immune effector cell, wherein the expression level and/or activity of the immune checkpoint in the modified immune effector cell is modulated compared to that in an unmodified immune effector cell.

In this application, the composition may comprise a modified oncolytic virus and a modified immune cell. For example, the modified oncolytic virus comprises a modified VSV, wherein the matrix protein M of the VSV comprises amino acid mutations M51R, V221F, and S226R compared to the amino acid sequence set forth in SEQ ID NO: 4, and the modified immune cell comprises a PD-1 gene-knockout T cell. In one embodiment, in the composition, the matrix protein M of the modified oncolytic virus comprises the amino acid sequence set forth in SEQ ID NO: 6, and the modified immune cell comprises a PD-1 gene-knockout T cell.

In this application, the composition may comprise a modified oncolytic virus and a modified immune cell. For example, the modified oncolytic virus comprises a modified VSV, wherein the matrix protein M of the VSV comprises the amino acid mutations M51R, ΔL111, V221F, and S226R compared to the amino acid sequence set forth in SEQ ID NO: 4, and the modified immune cell comprises a PD-1 gene-knockout T cell. In one embodiment, in the composition, the matrix protein M of the modified oncolytic virus comprises the amino acid sequence set forth in SEQ ID NO: 8, and the modified immune cell comprises a PD-1 gene-knockout T cell.

This application also provides a pharmaceutical composition comprising: (a) at least one oncolytic virus and/or a pharmaceutically acceptable carrier; and (b) at least one modified immune effector cell and/or a pharmaceutically acceptable carrier.

This application also provides a kit comprising one or more selected from the following groups: (a) the at least one oncolytic virus and a means for preparing the at least one modified immune effector cell, and/or a pharmaceutically acceptable carrier; (b) the at least one modified immune effector cell and a means for preparing the at least one oncolytic virus, and/or a pharmaceutically acceptable carrier; and (c) a means for preparing the at least one oncolytic virus, a means for preparing the at least one modified immune effector cell, and a pharmaceutically acceptable carrier.

In certain embodiments, the means for preparing the at least one modified immune effector cell in the kit refers to any individual who can obtain the modified immune effector cell by using the means, such as an sgRNA sequence designed for use in obtaining PD-1 molecule-deleted T cells. The means for preparing the at least one oncolytic virus in the kit refers to any individual who can obtain the oncolytic virus by using the means, such as plasmids or primers for each mutation site.

In certain embodiments, the composition, pharmaceutical composition and kit can be formulated into a drug for clinical use and can include a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The composition, pharmaceutical composition and kit can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal administration, which may include injection or infusion. Suitable formulations may include viruses and/or cells in a sterile or isotonic medium. The composition, pharmaceutical composition and kit can also be formulated into a fluid, including a gel form. Fluid preparations can be formulated for administration to a selected area of the human or animal body by injection or infusion (e.g., via a catheter).

Methods, Applications, and Uses

This application provides methods for preparing the composition, the pharmaceutical composition, and the reagent kit.

In certain embodiments, the composition, the pharmaceutical composition, and the components in the reagent kit may be in the same container or placed in different containers.

In certain embodiments, the oncolytic virus can be formulated for intratumoral administration.

In certain embodiments, the oncolytic virus and the immune effector cells can be formulated for intravenous administration.

In certain embodiments, the immune effector cells can be formulated for local administration.

This application also provides a method for treating tumors, comprising administering the composition, the pharmaceutical composition, and/or the kit to a subject in need thereof.

In certain embodiments, one or more of the composition, the pharmaceutical composition, and the kit can be administered to a subject in need thereof.

In certain embodiments, the oncolytic virus and the modified immune cells are administered simultaneously. Simultaneous administration can be administered as a mixture of the components or separately. Administration can be performed in the same manner, such as into the same vein or other blood vessel, or in different manners, such as simultaneous intravenous and intratumoral administration.

In certain embodiments, the oncolytic virus and the modified immune cells are administered sequentially. The order of administration may be the oncolytic virus first, followed by the immune effector cells; or the immune effector cells first, followed by the oncolytic virus. Administration may be in the same manner or in different manners. Each component may be administered once or in multiple doses. In certain embodiments, sequential administration may be at any time interval, including minutes, hours, days, weeks, months, or years. In certain embodiments, sequential administration refers to administration separated by at least 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months.

In certain embodiments, the amount administered is a therapeutically effective amount.

This application also provides the use of the oncolytic virus in combination with the oncolytic virus and the modified immune effector cells. This refers to the oncolytic virus used in combination with the modified immune effector cells.

This application also provides the use of the modified immune effector cells in combination with the oncolytic virus and the modified immune cells. That is, the modified immune cells are used in combination with the oncolytic virus and the modified immune cells.

On the other hand, this application also provides the use of the composition, the pharmaceutical composition, and the reagent kit in the preparation of drugs for treating tumors.

On the other hand, the present application also provides the oncolytic virus and the modified immune effector cell for use in treating tumors.

In certain embodiments, the tumor comprises a solid tumor and/or a hematological tumor.

In certain embodiments, the tumor includes, but is not limited to, head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.

Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and uses of this application, and are not intended to limit the scope of the invention.

Embodiment

Example 1: Construction of a stomatitis virus mutant and demonstration of its effects

1. Site-directed mutagenesis of the Indiana strain of bronchiolitis virus (BSV) was performed according to the method in Table 1 to obtain three groups of mutant strains. The group without gene mutation was designated JBS000 and served as a control.

The specific method for constructing mutant strains is a conventional technique in this field, and the method is as follows:

(1) Construction of plasmids. Using the pVSV-XN2 plasmid as a template, different mutation sites as described in Table 1 were introduced by PCR. The plasmid and primers for each mutation site were used for PCR. The PCR products were then subjected to 1% agarose gel electrophoresis and subsequently recovered using a gel recovery kit to obtain plasmids with different mutations in the M matrix protein.

(2) Virus rescue. BHK-21 cells were infected with the poxvirus vTF7-3 expressing T7 RNA polymerase at an MOI of 5. After 1 hour of infection, the BHK-21 cells were rinsed once with DPBS buffer. A plasmid transfection premix was then prepared, specifically including: pBS-N, pBS-P, pBS-L, and the mutant plasmid prepared in step (1). Among them, pBS-N, pBS-P, and pBS-L refer to expression plasmids that cloned the VSV N, VSV P, and VSV L protein genes, respectively, and express the N, P, and L proteins required for virus rescue, respectively. Plasmid transfection was performed according to the method described in the instructions for use of lipofectamine 2000. After 4 hours, the culture medium was replaced with fresh DMEM complete medium containing 10% fetal bovine serum. After 48 hours, the supernatant was aspirated and the poxvirus was removed using a 0.22μm filter membrane. Add the filtered fluid to fresh BHK-21 cells; observe the cells daily for signs of cell pathology. When the cells show signs of pathology, collect the supernatant. After confirmation by RT-PCR, purify the virus using a viral plaque assay. This will yield a mutant strain.

(3) M protein gene sequencing. Viral genomic RNA was extracted using a Trizol reagent kit, and reverse transcription was performed using random primers. The reverse transcribed cDNA was PCR-generated using primers designed for the M protein gene sequence. The primer sequences were: 5'-AAAAAAGTAACAGATATCAC-3' (SEQ ID NO: 9); 5'-ACATTTTTCCAGTTTCCTTTTTGG-3' (SEQ ID NO: 10). The product was recovered after 1% agarose gel electrophoresis and sent to a sequencing company for sequencing.

2. Demonstration of the in vitro cell invasion ability of different mutant strains.

The in vitro cell invasion ability of different mutant strains was demonstrated. 200 pfu of each of JBS000, JBS001, JBS002, JBS003, and JBS004 were added to MEF (human fibroblast) and LLC (murine non-small cell lung cancer) cell cultures, respectively. The tissue culture infective dose (TCID50) produced by each mutant strain was measured. The specific testing method is as follows:

(1) Add 3 mL of cell suspension to each well of a 6-well culture plate to a cell count of 4 × 10 ⁵ cells/well. Incubate at 37°C, 5% CO 2 for 16 h.

(2) Add 200 pfu of each of the viruses JBS000, JBS001, JBS002, JBS003, and JBS004 to each well, and set up two wells with normal cells as controls. After 24 hours, collect 100 μL of the cell supernatant.

(3) In a 96-well culture plate, add 100 μL of Vero cell suspension to each well to make the cell count reach 1×10 ⁴ cells/mL. Incubate at 37°C and 5% CO ₂ for 16 h.

(4) In a 1.5 mL EP tube, dilute the supernatant collected in step (2) in 10-fold increments, from 10 ^-1 to 10 ^-11 , for a total of 11 titers.

(5) Inoculate the diluted supernatant into the 96-well culture plate in step (3), inoculating one column for each dilution (8 wells in total), and inoculating 100 μL per well. Set up one column for the normal cell control group.

(6) After 48 hours, observe the fluorescence of cells in each well. If there is fluorescence, the well is marked as infected.

(7) Calculate TCID50 according to the Karber method.

The results, shown in Figure 1, demonstrate that the VSV mutant strain significantly increased its ability to replicate and proliferate in lung cancer cells (LLC) in vitro, while its ability to replicate and infect normal fibroblasts (MEFs) was significantly reduced. Therefore, the mutant strain possesses a specific ability to infect tumor cells.

3. Demonstrate the in vitro cell-killing ability of different mutants. Equal amounts of each mutant were used to infect different cells in vitro. Cell viability was assessed using the MTT assay 24 hours later. The specific method is as follows:

(1) Add 100 μL of cell suspension to each well of a 96-well culture plate to a cell count of 1 × 10 ⁴ cells/well. Incubate at 37°C, 5% CO ₂ for 16 h. The cell types tested are: LLC, MEF, and HeLa (human tumor cells).

(2) Dilute JBS000, JBS001, JBS002, JBS003, and JBS004 to MOIs (multiplicity of infection) of 0.001, 0.01, 0.1, and 1.0, respectively. Inoculate 100 μL of each dilution gradient into 4 wells and incubate at 37°C, 5% CO ₂ for 40 h.

(3) Discard the supernatant from the 96-well culture plate, add fresh DMED medium, and then add 5 mg/mL MTT solution, 20 μL/well. Incubate at 37°C, 5% _CO2 for 4 h.

(4) Centrifuge the 96-well plate at 2500 g/min for 5 minutes at room temperature. Then, use a 1 mL disposable sterile syringe to gently aspirate the supernatant.

(5) Add DMSO to each well, 100 μL/well, and incubate at 37°C for 10 minutes.

(6) Use a multifunctional enzyme marker, shake for 2 minutes, and measure the OD value of each well at a wavelength of 570nm.

The results, shown in Figure 2, demonstrate that all mutants exhibited robust tumor cell-killing activity. With the exception of JBS000, none exhibited significant killing activity against MEF cells. This indicates that, in vitro, all attenuated strains, with the exception of JBS000, exhibited specific killing activity against tumor cells and had no significant effect on normal cells.

4. Testing the difficulty of clearing different mutant strains from cells

The test was performed using the IFN-β indicator. Cells were cultured according to steps (1) and (2) of step 3 above and mutant strains were added. Subsequently, the cells of each tissue were disrupted, and total RNA was extracted from each cell using TRIzol (Invitrogen). The RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with DNA Eraser (Takara) reverse transcription reagent kit, and stained with LightCycler 480SYBR Green I Master (Roche) dye. The Ct value of each gene was detected on the LightCycler 480 quantitative PCR instrument. The relative expression of the target genes IFN-β and VSV-G was calculated using the ΔΔCt method.

The results are shown in Figure 3. They demonstrate that in LLC cell lines, all mutant strains, except JBS000, increased IFN-β expression levels. Furthermore, in MEF cells, all viruses upregulated IFN-β expression levels.

Example 2: CRISPR/Cas9 technology to eliminate T cell PD-1 molecules

1. Construction of PD-1-sgRNA-CRISPR/Cas9 plasmid

Based on the structure of the human PD-1 molecule encoding gene, PDCD1, two sgRNAs were designed targeting its second exon. A fusion plasmid carrying sgRNAs that recognize the human PDCD1 gene and the Cas9 enzyme was constructed. An example of the fusion plasmid components is shown in Figure 4.

2. Detection of PD-1 molecule expression on the surface of human peripheral blood T cells

Fresh human peripheral blood (PBMCs) were separated by gradient centrifugation, and 1×10 ⁶ PBMCs were used for flow cytometry analysis of PD-1 expression on the T cell surface. The results showed that PD-1 was expressed on the surface of human peripheral blood T cells, as shown in Figure 5, indicating that human peripheral blood T cells can be used as a tool for analyzing the effects of PDCD1 gene deletion.

3. PD-1-sgRNA-CRISPR/Cas9 system downregulates PD-1 expression on the surface of human peripheral blood T cells

The PD-1-sgRNA-CRISPR/Cas9 plasmid was electroporated into freshly isolated human peripheral blood PBMCs. The electroporated PBMCs were activated with CD3 and CD28 antibodies and cultured at 37°C, 5% _CO₂ . PD-1 expression on T cells was assessed 24 hours and 8 days after electroporation.

The results showed that while PD-1 expression on the cell surface of the PDCD1 knockout group was not significantly downregulated 24 hours after electroporation, it was significantly downregulated on the eighth day of culture compared to the negative control group, as shown in Figure 5. This indicates that knockout of the PDCD1 gene using CRISPR/Cas9 technology can lead to a sustained downregulation of PD-1 expression on the surface of human peripheral blood T cells. In Figure 5, the unedited group refers to cells without gene knockout, and the edited group refers to cells with gene knockout.

Example 3: CRISPR/Cas9 technology to eliminate T cell PD-1 molecules and CTLA-4 molecules

1. Construction of PD-1 and CTLA-4 dual gene knockout vector

The gRNA sequence was designed based on the human PD-1 and CTLA-4 gene sequences. The pU6gRNA-CMV-Cas9-GFP expression vector was digested with Bbs I and then recovered and ligated to the oligo doublet formed with the PD-1 oligo sequence to construct a PD-1 gene knockout vector containing a single gRNA.

Using the pU6gRNA-CMV-Cas9 expression vector as a template, the expression vector was linearized with primers, with a cleavage point located between the gRNA and CMV promoter sequences. Hind III and EcoR I restriction sites were added to both segments. Next, using the pU6gRNA-CMV-Cas9-GFP expression vector as a template, the gRNA expression unit was cloned using primers and ligated into pUC19. The CTLA-4 oligo sequence was then ligated into the intermediate vector using Bbs I restriction enzymes. The linearized pU6gRNA-CMV-Cas9-GFP expression vector and the intermediate vector were then digested with Hind III and EcoR I, recovered, and ligated to create the knockout vector pU6gRNA-Cas9-GFP, which can express gRNAs with two different target sequences.

2. The pU6gRNA-Cas9-GFP system downregulates the expression of PD-1 and CTLA-4 on the surface of human peripheral blood T cells.

Freshly isolated human peripheral blood T cells were electroporated with the pU6gRNA-Cas9-GFP plasmid, activated with CD3 and CD28 antibodies, and cultured at 37°C in 5% CO2. Eight days after electroporation, cells were harvested and flow cytometry was performed to assess the expression of PD-1 and CTLA-4 molecules on the T cell surface.

The results showed that both gRNA and Cas9 fusion electroporation plasmids downregulated the expression of PD-1 and CTLA-4 molecules on the surface of T cells, indicating that gRNA can effectively knock down the expression of PD-1 and CTLA-4 genes in T cells.

Example 4: In vitro tumoricidal assay of modified VSV

Demonstration of an in vitro anti-tumor experiment using oncolytic viruses. Tumor cells were infected with JBS003 oncolytic virus, and the CCK8 assay was used to measure the tumor cell killing rate 48 hours later. The specific method is as follows:

(1) Add 100 μL of A549 tumor cell line (human non-small cell lung cancer) suspension to each well of a 96-well culture plate to a cell count of 4 × 10 ³ cells/well. Incubate at 37°C, 5% CO ₂ for 16 h.

(2) The oncolytic virus was diluted to five gradients of 0.001, 0.01, 0.1, 1.0, and 10.0 according to the multiplicity of infection (MOI); 100 μL of oncolytic virus was inoculated into each well, and 4 replicates were inoculated into each dilution gradient. The cells were cultured at 37°C and 5% CO2 for 48 h.

(3) Discard the supernatant from the 96-well culture plate, add fresh DMED medium (100 μL/well), and then add CCK8 solution (10 μL/well). Incubate at 37°C, 5% CO2 for 4 hours.

(4) Using a multifunctional enzyme marker, shake for 2 minutes, measure the OD value of each well at a wavelength of 450 nm and calculate the tumor killing rate.

(5) Tumor killing rate calculation formula: Tumor cell killing rate (%) = (OD value of tumor cell control group - OD value of experimental group) / OD value of tumor cell control group × 100%.

The results of in vitro anti-tumor experiments using oncolytic viruses are shown in Figure 6. These results indicate that oncolytic viruses have a certain degree of anti-tumor activity.

Example 5: In vitro tumor killing assay of PD-1-deficient T cells

After 48 hours of co-culture of PD-1-deficient T cells with tumor cells, the CCK8 assay was used to measure the tumor cell-killing rate of the PD-1-deficient T cells. The specific method is as follows:

(1) Add 100 μL of A549 tumor cell line (human non-small cell lung cancer) suspension to each well of a 96-well culture plate to a cell count of 4 × 10 ³ cells/well. Incubate at 37°C, 5% CO ₂ for 16 h.

(2) Dilute the PD-1-deficient T cells to four gradients of 1:1, 5:1, 10:1, and 20:1 according to the effector-target ratio (E:T); inoculate 100 μL of PD-1-deficient T cells into each well, and inoculate 4 replicate wells for each dilution gradient. Incubate at 37°C, 5% CO2 for 48 h.

(3) Discard the supernatant and lymphocytes in the 96-well culture plate, add fresh DMED medium (100 μL/well), and then add CCK8 solution (10 μL/well). Incubate at 37°C, 5% CO2 for ₄ h.

(4) Using a multifunctional enzyme marker, shake for 2 minutes, measure the OD value of each well at a wavelength of 450 nm and calculate the tumor killing rate.

(5) Tumor killing rate calculation formula: Tumor cell killing rate (%) = (OD value of tumor cell control group - OD value of experimental group) / OD value of tumor cell control group × 100%.

The in vitro anti-tumor effects of PD-1-deficient T cells are shown in Figure 8. The results indicate that PD-1-deficient T cells have superior anti-tumor activity.

Example 6: Tumor killing assay of modified VSV and PD-1 deleted T cells in combination therapy for melanoma (transplanted tumor)

96 C57BL/6 mice with no significant differences were selected and subcutaneously inoculated with 2×10 ⁶ B16-F10-NY-ESO-1 melanoma cells. On day 9 after inoculation, when the transplanted tumors had grown to approximately 100 ^mm³ , all mice were divided into 12 groups: a control group (PBS group) received an intratumoral injection of 50 μL PBS, and the remaining 11 treatment groups received intratumoral inoculations of JBS000, JBS001, JBS002, JBS003, JBS004, PD-1 knockout T cells, JBS000 and PD-1 knockout T cells, JBS001 and PD-1 knockout T cells, JBS002 and PD-1 knockout T cells, JBS003 and PD-1 knockout T cells, and JBS004 and PD-1 knockout T cells. Each group received ^10⁻⁻ pfu (pfu) of each drug every two days for a total of three doses. From the start of drug administration to the end of the experiment, the volume of transplanted tumors was recorded every 2 days. Volume (mm ³ ) = (longest diameter × shortest diameter ² )/2.

The results showed that compared with the control group, each treatment group had a certain therapeutic effect on melanoma.

Example 7: Tumor killing assay of modified VSV and PD-1 deleted T cells in the treatment of fibrosarcoma (transplanted tumor)

96 C57BL/6 mice with no significant differences were selected and subcutaneously inoculated with 2×10 ⁶ MCA-205-NY-ESO-1 fibrosarcoma cells. On day 9 after inoculation, when the transplanted tumors had grown to approximately 100 ^mm³ , all mice were divided into 12 groups: a control group (PBS group) received an intratumoral injection of 50 μL PBS, and the remaining 11 treatment groups received intratumoral inoculations of JBS000, JBS001, JBS002, JBS003, JBS004, PD-1 knockout T cells, JBS000 and PD-1 knockout T cells, JBS001 and PD-1 knockout T cells, JBS002 and PD-1 knockout T cells, JBS003 and PD-1 knockout T cells, and JBS004 and PD-1 knockout T cells. Each group received ^10⁻⁻ pfu (pfu) of each drug every two days for a total of three doses. From the start of drug administration to the end of the experiment, the volume of transplanted tumors was recorded every 2 days. Volume (mm ³ ) = (longest diameter × shortest diameter ² )/2.

The results showed that compared with the control group, each treatment group had a certain therapeutic effect on fibrosarcoma.

Example 8: Tumor killing assay of modified VSV and T cells with PD-1 and CTLA-4 deleted in the treatment of melanoma (transplanted tumor)

Mice were treated according to the method of Example 5 and subcutaneously inoculated with 2×10 ⁶ B16-F10-NY-ESO-1 melanoma cells. Treatment was performed when the transplanted tumors grew to approximately 100 mm ³ in size. All mice were divided into 10 groups: the control group (PBS group) was injected intratumorally with 50 μL PBS, and the remaining 9 groups were treated with intratumoral inoculation of JBS000, JBS001, JBS002, JBS003, JBS004, PD-1 and CTLA-4 deleted T cells, JBS000 and PD-1 and CTLA-4 deleted T cells, JBS001 and PD-1 and CTLA-4 deleted T cells, JBS002 and PD-1 and CTLA-4 deleted T cells, JBS003 and PD-1 and CTLA-4 deleted T cells, and JBS004 and PD-1 and CTLA-4 deleted T cells. Each group consisted of 8 animals, and the drug was administered once every 2 days for a total of 3 doses, with a single inoculation dose of 10 ⁸ pfu/animal. Tumor volume was recorded every 2 days from the start of drug administration until the end of the experiment.

The results showed that compared with the control group, each treatment group had a certain therapeutic effect on melanoma.

Example 9: Tumor killing assay of modified VSV and T cells with PD-1 and CTLA-4 molecules deleted in the treatment of fibrosarcoma (transplanted tumor)

Mice were treated according to the method of Example 5 and subcutaneously inoculated with 2×10 ⁶ MCA-205-NY-ESO-1 fibrosarcoma cells. Treatment was performed when the transplanted tumors grew to approximately 100 mm ³ in size. All mice were divided into 12 groups: the control group (PBS group) was injected intratumorally with 50 μL PBS, and the remaining 11 groups were treated with intratumoral inoculation of JBS000, JBS001, JBS002, JBS003, JBS004, PD-1 and CTLA-4 deleted T cells, JBS000 and PD-1 and CTLA-4 deleted T cells, JBS001 and PD-1 and CTLA-4 deleted T cells, JBS002 and PD-1 and CTLA-4 deleted T cells, JBS003 and PD-1 and CTLA-4 deleted T cells, and JBS004 and PD-1 and CTLA-4 deleted T cells. Each group consisted of 8 animals, and the drug was administered once every 2 days for a total of 3 doses, with a single inoculation dose of 10 ⁸ pfu/animal. Tumor volume was recorded every 2 days from the start of drug administration until the end of the experiment.

The results showed that compared with the control group, each treatment group had a certain therapeutic effect on fibrosarcoma.

Example 10: Evaluation of the efficacy of combined therapy with modified VSV and PD-1 molecule-deficient T cells

Several qualified subjects were selected and divided into two groups. The dosing sequence was different. The dosing strategy is shown in Figure 7. VSV-OVV-01 represents the modified oncolytic virus used in this application. The specific dosing strategy is as follows:

Group A (PD-1 knockout T cells, Figure 3A): PD-1 knockout T cells were administered with a fixed dose (total cell number 3.6× ¹⁰⁹ ~4.4× ¹⁰⁹ or 1.8× ¹⁰⁹ ~2.2× ¹⁰⁹ ) starting from day 1 of week 1 (W1D1, W stands for week, D stands for day). The dose was divided into three doses at 20%, 30%, and 50% of the total dose, with a one-day interval between each dose. Starting from W1D1, each cycle was 4 weeks. If there was no disease progression, intolerable toxicity, death, withdrawal from the study, loss to follow-up, termination of the study by the sponsor, or the subject started new cancer treatment, the drug was continued. Four cycles; VSV-OVV-01 is administered at a fixed dose, the clinically effective dose or maximum dose in Phase 1, starting on Week 2 Day 1 and administered every two weeks. If there are no: disease progression, intolerable toxicity, death, study withdrawal, loss of visit, study termination by the sponsor, or the subject starting new cancer treatment, then the drug will be administered for eight consecutive cycles. Safety and efficacy follow-up will be conducted regularly from the start of dosing to the end of the trial, at the following time points:

Security follow-up

Safety visits were conducted at baseline and within 7 days before each medication dose (W2D1, W4D1, W5D1, W6D1, W8D1, W9D1, W10D1, W12D1, W13D1, W14D1, and W16D1); and at each efficacy visit (baseline, W6D7, W12D7, and W20D7). Baseline, W13D1, and W12D7 were conducted at the same time point, resulting in a total of 13 safety visits.

Treatment follow-up

The baseline, W6D7, W12D7, and W20D7 tests were performed once each, with a time window of -7 days.

(A. First use T cells with PD-1 knockout, B. First use VSV-OVV-01)

Group B (VSV first, Figure 3B): OVV-01 was administered at a fixed dose, i.e., the clinically effective dose or the highest dose in the first phase. The first dose was administered on W1D1, the second dose was administered on W4D1, and then every 2 weeks for 7 consecutive doses, for a total of 8 doses. T cells with PD-1 knockout were administered starting on W5D1 at a fixed dose (total cell count of 3.6× ¹⁰⁹ to 4.4× ¹⁰⁹ or 1.8× ¹⁰⁹ to 2.2× ¹⁰⁹ cells), administered three times at 20%, 30%, and 50% of the total dose, with one day between each administration. Starting on W5D1, each four-week cycle was considered one. If there were no: disease progression, intolerable toxicity, death, withdrawal from the study, loss to follow-up, termination of the study by the sponsor, or initiation of new cancer treatment, the drug was administered for four consecutive cycles. From the start of dosing to the end of the trial, regular safety and efficacy follow-up visits were required at the following time points:

Security follow-up

Safety visits were conducted at baseline and within 7 days before each medication dose (W4D1, W5D1, W6D1, W8D1, W9D1, W10D1, W12D1, W13D1, W14D1, W16D1, and W17D1); and at each efficacy visit (baseline, W6D7, W12D7, and W20D7). Baseline, W13D1, and W12D7 were conducted at the same time point, resulting in a total of 14 safety visits.

Treatment follow-up

The baseline, W6D7, W12D7, and W20D7 tests were performed once each, with a time window of -7 days.

The results showed that the progression-free survival of subjects treated with the combination therapy increased by 30% to 50% compared to those treated with either drug alone.

Example 11: In vitro tumor-killing ability of a modified oncolytic virus combined with PD-1-deficient T cells

A549 tumor cells were infected in vitro with JBS003 oncolytic virus at an MOI of 10. PD-1-depleted T cells with varying effector-to-target ratios were also added. Cell viability was assessed using the CCK8 assay 48 hours later. The specific method is as follows:

(1) Add 100 μL of A549 tumor cell line (human non-small cell lung cancer) suspension to each well of a 96-well culture plate to a cell count of 4 × 10 ³ cells/well. Incubate at 37°C, 5% CO ₂ for 16 h.

(2) Dilute the oncolytic virus to a multiplicity of infection (MOI) of 10; remove the original culture medium in the 96-well plate and inoculate 100 μL of oncolytic virus into each well.

(3) At the same time, the PD-1-depleted T cells were diluted to four gradients of 1:1, 5:1, 10:1, and 20:1 according to the effector-target ratio (E:T); 100 μL of PD-1-depleted T cells were inoculated into each well, and 4 replicate wells were inoculated for each dilution gradient.

(4) After culturing at 37°C, 5% _CO₂ for 48 h, discard the supernatant and PD-1-depleted T cells in the 96-well culture plate, add fresh DMED medium (100 μL/well), and then add CCK8 solution (10 μL/well). Incubate at 37°C, 5% _CO₂ for 4 h.

(5) Using a multifunctional enzyme marker, shake for 2 minutes, measure the OD value of each well at a wavelength of 450 nm and calculate the tumor killing rate.

(6) Tumor killing rate calculation formula: Tumor cell killing rate (%) = (OD value of tumor cell control group - OD value of experimental group) / OD value of tumor cell control group × 100%.

Figure 8 shows the tumor-killing effects of the modified oncolytic virus combined with PD-1-deleted T cells. The figure shows the use of the modified oncolytic virus alone, PD-1-deleted T cells alone, and the combination of the modified oncolytic virus and PD-1-deleted T cells. The results demonstrate that the combination of the modified oncolytic virus and PD-1-deleted T cells exhibits very strong anti-tumor activity. The modified oncolytic virus and PD-1-deleted T cells exert a synergistic effect, significantly enhancing the anti-tumor effect.

Claims (34)

A composition comprising: (a) an oncolytic virus, wherein the oncolytic virus is bronchiolitis virus and the bronchiolitis virus matrix protein M is modified; and (b) a modified immune effector cell, wherein the expression and/or activity of an immune checkpoint is modulated in the modified immune effector cell compared to an unmodified immune effector cell. The composition as described in claim 1, wherein the modification of the bronchiolitis virus matrix protein M comprises mutations at more than one site. The composition of claim 1, wherein the mutation site of the bronchiolitis virus matrix protein M is selected from one or more of the following groups: M51, L111, V221, and S226. The composition of claim 1, wherein the mutation site of the basal protein M of the buccal stomatitis virus is selected from the following single-site mutations: a) M51R; b) ΔL111; c) V221F; and d) S226R. The composition of claim 1, wherein the combination of mutation sites in the basal protein M of the bronchiolitis virus is selected from any one of the following groups: a) M51R and ΔL111; b) M51R and V221F; c) M51R and S226R; d) ΔL111 and V221F; e) ΔL111 and S226R; f) V221F and S226R; g) M51R, ΔL111, and V221F; h) M51R, ΔL111, and S226R; i) M51R, V221F, and S226R; j) ΔL111, V221F, and S226R; and k) M51R, ΔL111, V221F, and S226R. The composition of claim 5, wherein the combination of mutation sites selected from M51R, V221F, and S226R in the basal protein M of stomatitis virus comprises the amino acid sequence shown in SEQ ID NO: 6. The composition of claim 5, wherein the combination of mutation sites selected from M51R, ΔL111, V221F, and S226R in the basal protein M of stomatitis virus comprises the amino acid sequence shown in SEQ ID NO: 8. The composition of claim 1, wherein the immune effector cells are selected from the group consisting of T cells, NK cells, B cells, and macrophages. The composition as described in claim 1, wherein the immune effector cells comprise autologous cells and allogeneic cells. The composition according to claim 1, comprising immune effector cells expanded in vitro. The composition as described in claim 1, wherein the number of the immune checkpoints is more than one. The composition of claim 1, wherein the immune detection point is selected from one or more of the following groups: PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, BTLA, VISTA, TIGIT, B7-H2, B7-H3, B7-H4 and B7-H6. The composition of claim 1, wherein the immune checkpoint is PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, BTLA, VISTA, TIGIT, B7-H2, B7-H3, B7-H4, or B7-H6. The composition of claim 1, wherein the immune checkpoint is a combination selected from any one of the following groups: a) PD-1 and CTLA-4; b) PD-1 and PD-L1; c) PD-L1 and CTLA4; d) LAG-3 and CTLA-4; e) TIM-3 and CTLA-4; f) VISTA and CTLA-4; and g) PD1, CTLA-4, and PD-L1. The composition of claim 1, wherein the regulation of the expression level of the immune checkpoint comprises upregulation, downregulation and/or deletion of the expression level of the immune checkpoint compared to unmodified immune effector cells. The composition of claim 1, wherein the modulation of the immune checkpoint activity comprises an increase, decrease, and/or loss of the immune checkpoint activity compared to unmodified immune cells. The composition of claim 1, wherein the regulation of the immune checkpoint comprises the regulation of immune checkpoint gene levels, transcription levels and/or translation levels. The composition of claim 17, wherein the regulation of gene expression level comprises gene editing, overexpression, point mutation and/or homologous recombination. The composition of claim 18, wherein the gene editing is selected from one or more of the following groups: CRISPR/Cas9, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN) and monobase editor (BE). The composition of claim 19, wherein the gene editing comprises a designed sequence of sgRNA. The composition of claim 1, wherein the immune effector cell comprises a knockout target sequence for the PD-1 gene selected from SEQ ID NO: 1. The composition of claim 1, wherein the immune effector cell knocks out a target sequence of the CTLA-4 gene selected from SEQ ID NO: 2. A pharmaceutical composition comprising: (a) at least one oncolytic virus and/or a pharmaceutically acceptable carrier in the composition of any one of claims 1 to 22; and (b) at least one immune effector cell and/or a pharmaceutically acceptable carrier in the composition of any one of claims 1 to 22. A kit comprising one or more selected from the following groups: a) at least one oncolytic virus in the composition of any one of claims 1 to 22 or the pharmaceutical composition of claim 23 and a means for preparing at least one modified immune effector cell in the composition of any one of claims 1 to 22 or the pharmaceutical composition of claim 23, and/or a pharmaceutically acceptable carrier; b) at least one modified immune effector cell in the composition of any one of claims 1 to 22 or the pharmaceutical composition of claim 23. and means for preparing at least one oncolytic virus in the composition of any one of claims 1 to 22 or the pharmaceutical composition of claim 23, and/or a pharmaceutically acceptable carrier; and, c) means for preparing at least one oncolytic virus in the composition of any one of claims 1 to 22 or the pharmaceutical composition of claim 23 and means for preparing at least one modified immune effector cell in the composition of any one of claims 1 to 22 or the pharmaceutical composition of claim 23, and/or a pharmaceutically acceptable carrier. A method for preparing the composition according to any one of claims 1 to 22, the pharmaceutical composition according to claim 23, and the test kit according to claim 24. An in vitro use of the oncolytic virus in the composition of any one of claims 1 to 22 or the pharmaceutical composition of claim 23 in combination with the oncolytic virus and the modified immune effector cell. An in vitro use of the modified immune effector cell in the composition of any one of claims 1 to 22 or the pharmaceutical composition of claim 23 in combination with the oncolytic virus and the modified immune cell. Use of the composition according to any one of claims 1 to 22, the pharmaceutical composition according to claim 23, and/or the kit according to claim 24 in the preparation of a medicament for treating tumors. The use according to claim 28, wherein the oncolytic virus is administered to a subject in need thereof via an intravenous and/or intratumoral route, and wherein the immune effector cells are administered to a subject in need thereof via an intravenous and/or local administration route. The use according to claim 28, wherein the oncolytic virus and the modified immune cells are administered simultaneously, and the dosage administered is a therapeutically effective amount. The use as described in claim 28, wherein the oncolytic virus and the modified immune cells are administered separately, and the dosage administered is a therapeutically effective amount. The use according to claim 28, wherein the oncolytic virus and the modified immune cells are administered once or multiple times. The use according to claim 28, wherein the tumor comprises a solid tumor and/or a hematological tumor. The use according to claim 28, wherein the tumor comprises one or more selected from the group consisting of head and neck cancer, melanoma, soft tissue sarcoma, breast cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, liver cancer, cervical cancer, neuroblastoma, synovial sarcoma, and round cell liposarcoma.