WO2022057904A1 - 溶瘤病毒与经改造的免疫细胞联合治疗肿瘤 - Google Patents

溶瘤病毒与经改造的免疫细胞联合治疗肿瘤 Download PDF

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WO2022057904A1
WO2022057904A1 PCT/CN2021/119114 CN2021119114W WO2022057904A1 WO 2022057904 A1 WO2022057904 A1 WO 2022057904A1 CN 2021119114 W CN2021119114 W CN 2021119114W WO 2022057904 A1 WO2022057904 A1 WO 2022057904A1
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cells
virus
composition
oncolytic virus
immune
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PCT/CN2021/119114
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English (en)
French (fr)
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周国庆
邓涛
喻堃
杨何
张凡
张苏宏
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成都美杰赛尔生物科技有限公司
上海荣瑞医药科技有限公司
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Priority to CN202180062625.9A priority Critical patent/CN116802278A/zh
Publication of WO2022057904A1 publication Critical patent/WO2022057904A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/766—Rhabdovirus, e.g. vesicular stomatitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464484—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/464488—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/57—Skin; melanoma
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of biomedicine, in particular to the combined treatment of tumors with oncolytic viruses and modified immune cells.
  • Immune effector cells are cells involved in the immune response in the human body.
  • Immune checkpoints are some inhibitory molecules in the immune system. They regulate the intensity or breadth of the immune response through ligand-receptors to avoid damage and destruction of the body caused by excessive responses. It is one of the negative feedback regulation mechanisms of the normal body.
  • immune checkpoints have become the main reason for inducing tumor immune tolerance, resulting in low efficiency of the body's anti-tumor immune response.
  • Virus treatment of cancer has developed rapidly in the past two decades.
  • One of the biggest advances in virus treatment is to use the difference between tumor cells and normal cells to modify certain viral structures so that they can be selectively replicated in tumor cells. , and finally achieve the purpose of killing tumor cells.
  • These modified viruses are called oncolytic viruses, which are derived from herpesviruses, poxviruses, and the like.
  • oncolytic viruses which are derived from herpesviruses, poxviruses, and the like.
  • it has been found that some wild-type viruses also have the function of selectively replicating in cells and thus oncolytic.
  • the efficacy of using oncolytic virus alone to treat tumors is sometimes not very satisfactory.
  • oncolytic virus is used in combination with chemotherapeutic drugs to treat tumors, since the chemotherapeutic drugs kill normal cells equally, it will bring strong side effects.
  • the application provides a composition comprising: (a) an oncolytic virus; and (b) a modified immune effector cell, wherein the modified immune effector cell is in phase with the unmodified immune effector cell ratio, the expression and/or activity of immune checkpoints are adjusted.
  • 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 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 accepted carrier.
  • the present application also provides methods for preparing the composition, the pharmaceutical composition, and the kit.
  • the present application also provides the application of the oncolytic virus in the combination of the oncolytic virus and the modified immune effector cells, the modified immune effector cells in the oncolytic virus and the modified immune effector cells.
  • Application of modified immune cells in combination The present application also provides a method of treating a tumor, comprising administering the composition, the pharmaceutical composition and/or the kit to a subject in need thereof.
  • the present application also provides applications of the composition, the pharmaceutical composition, and the kit in preparing a medicament for treating tumors.
  • the combination of oncolytic virus and modified immune effector cells effectively exerts the synergistic effect of the two, enhances the lethality of tumors, and improves the therapeutic effect of tumors.
  • the application provides a composition comprising: (a) an oncolytic virus; and (b) a modified immune effector cell, wherein the modified immune effector cell is combined with an immune effector without the modification
  • the expression and/or activity of immune checkpoints is modulated compared to effector cells.
  • the oncolytic virus comprises a native oncolytic virus and an engineered oncolytic virus.
  • the oncolytic virus is selected from the group consisting of vesicular stomatitis virus (VSV), poxvirus, herpes simplex virus, measles virus, Semliki Forest virus, polio Viruses, Reovirus, Seneca Valley Virus, Echo-type Enterovirus, Coxsackie Virus, Newcastle Disease Virus and Maraba Virus.
  • VSV vesicular stomatitis virus
  • poxvirus herpes simplex virus
  • measles virus Semliki Forest virus
  • polio Viruses Reovirus
  • Seneca Valley Virus Seneca Valley Virus
  • Echo-type Enterovirus Coxsackie Virus
  • Newcastle Disease Virus Newcastle Disease Virus
  • Maraba Virus Maraba Virus
  • the vesicular stomatitis virus comprises matrix protein M, nucleocapsid protein N, phosphoprotein P, large polymerase protein L and/or glycoprotein G.
  • the matrix protein M of the vesicular stomatitis virus is engineered.
  • the modification of the vesicular stomatitis virus matrix protein M comprises mutations at more than one site.
  • the mutation site of the vesicular stomatitis virus matrix protein M is selected from one or more of the group consisting of M51, L111, V221 and S226.
  • the mutation site of the vesicular stomatitis virus matrix protein M is selected from the following group of mutations at a single site:
  • the combination of mutation sites of the vesicular stomatitis virus matrix protein M is selected from any of the following groups:
  • the vesicular stomatitis virus matrix protein M of the combination of mutation sites selected from M51R, V221F and S226R comprises the amino acid sequence shown in SEQ ID NO:6.
  • the vesicular stomatitis virus matrix protein M of the combination of mutation sites selected from M51R, ⁇ L111, V221F and S226R comprises the amino acid sequence shown in SEQ ID NO:8.
  • the immune effector cells are selected from the group consisting of T cells, B cells, NK cells, and macrophages.
  • the immune effector cells comprise autologous cells and allogeneic cells.
  • the number of immune checkpoints is more than one.
  • the immune checkpoint is selected from one or more of the group consisting of PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, BTLA, VISTA, TIGIT, B7 -H2, B7-H3, B7-H4 and B7-H6.
  • 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 immune checkpoint is a combination selected from the group consisting of:
  • the modulation of the expression of the immune checkpoint comprises up-regulation, down-regulation and/or deletion of the expression of the immune checkpoint compared to unmodified immune effector cells.
  • the modulation of immune checkpoint activity comprises an increase, decrease and/or absence of immune checkpoint activity compared to unmodified immune cells.
  • the modulation of immune checkpoints comprises regulation of immune checkpoint gene expression levels, transcriptional levels and/or translational levels.
  • the modulation of the level of gene expression comprises gene editing, overexpression, point mutation and/or homologous recombination.
  • the gene editing method is selected from one or more of the group consisting of CRISPR/Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and single base editing (BE).
  • CRISPR/Cas9 transcription activator-like effector nucleases
  • TALENs transcription activator-like effector nucleases
  • ZFNs zinc finger nucleases
  • BE single base editing
  • the gene editing comprises an sgRNA of a designed sequence.
  • the immune effector cell knocks out a target sequence for the PD-1 gene selected from the group consisting of SEQ ID NO:1.
  • the immune effector cell knocks out a target sequence for the CTLA-4 gene selected from the group consisting of SEQ ID NO:2.
  • the present application also provides a pharmaceutical composition
  • a pharmaceutical composition comprising: (a) at least one oncolytic virus and/or a pharmaceutically acceptable carrier; and (b) at least one Modified immune effector cells and/or pharmaceutically acceptable carriers.
  • the application also provides a kit comprising one or more selected from the group consisting of:
  • the present application also provides methods for preparing the composition, the pharmaceutical composition, and the kit.
  • the present application also provides the application of the oncolytic virus in the combined use of the oncolytic virus and the modified immune effector cells.
  • the present application also provides the use of the modified immune effector cells in the combined use of the oncolytic virus and the modified immune cells.
  • 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.
  • the oncolytic virus is administered to a subject in need thereof by intravenous and/or intratumoral routes, wherein the immune effector cells are administered to a subject in need thereof by intravenous and/or topical administration subject administration.
  • the oncolytic virus and the modified immune cells are administered concurrently in a therapeutically effective amount.
  • the oncolytic virus and the modified immune cells are administered separately in a therapeutically effective amount.
  • the oncolytic virus and the modified immune cells are administered in one or more administrations.
  • the present application also provides applications of the composition, the pharmaceutical composition, and the kit in the preparation of a medicament for treating tumors.
  • the tumors described herein comprise solid tumors and/or hematological tumors.
  • the tumor is selected from one or more of 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 Carcinoma, neuroblastoma, synovial sarcoma, round cell liposarcoma.
  • 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 ability of each modified oncolytic virus to kill LLC cells and Hela cells and MEF cells in vitro;
  • Figure 3 shows a schematic diagram of the effect of each modified oncolytic virus on the expression level of IFN- ⁇ in LLC cells and MEF cells in vitro;
  • Figure 4 shows a schematic diagram of the CRISPR/Cas9 fusion plasmid element
  • Figure 5 shows the expression of PD-1 molecule on the modified surface of human peripheral blood T cells detected by flow cytometry
  • Figure 6 shows the in vitro anti-tumor effect detection of the modified oncolytic virus
  • Figure 7 shows the T cell therapy strategy of modified oncolytic virus combined with knockout of PD-1 molecule
  • Figure 8 shows the tumor-killing effect of the engineered oncolytic virus combined with PD-1 knockout T cells.
  • composition comprises at least two components.
  • the components in the composition can be mixed and placed separately, or they can be placed separately.
  • the components of the composition may be administered simultaneously or separately.
  • the separate administration can be prioritized. For example, one component of the composition is administered before the other component of the composition.
  • a composition comprises the oncolytic virus and the modified immune effector cells.
  • the term "oncolytic virus” generally refers to a virus capable of replicating in and killing tumor cells.
  • the virus is engineered to increase selectivity for tumor cells.
  • Oncolytic viruses include, but are not limited to, vesicular stomatitis virus, pox virus, herpes simplex virus, measles virus, Semliki Forest virus, poliovirus, reovirus, Seneca Valley virus, echogenic enterovirus tract virus, Coxsackie virus, Newcastle disease virus and Maraba virus.
  • the oncolytic virus is vesicular stomatitis virus.
  • a preferred vesicular stomatitis virus may be a MuddSummer subtype strain of vesicular stomatitis virus with site-directed mutation of the matrix protein M gene.
  • modified refers to an altered state or structure of a molecule or cell of the invention.
  • Molecules can be modified in many ways, including chemically, structurally, and functionally.
  • Cells can be modified by introducing nucleic acids.
  • Immune effector cell is used interchangeably with “immune cell” and generally refers to cells that play a role in an immune response.
  • Immune effector cells include: lymphocytes, such as T and B cells, natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • T cells refers to thymus-derived cells. It is involved in various cell-mediated immune responses, including thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • T cell populations include, but are not limited to, helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; CD8+ T cells), CD4+CD8+ T cells, CD4-CD8 - T cells or any other subset of T cells.
  • 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, if desired, can be further isolated by positive or negative selection techniques.
  • Immune checkpoints 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, SIRP ⁇ , CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, arginase, CD73 and A2aR (see eg WO 2012/177624) .
  • the term also encompasses biologically active protein fragments, as well as nucleic acids encoding fragments of full-length immune checkpoint proteins and biologically active proteins thereof. In certain embodiments, the terms also encompass any and fragments of the homology descriptions provided in accordance with this application.
  • the term “reduced expression” is used interchangeably with “reduced expression”, “down-regulated expression” and generally refers to the expression of a molecule in a modified cell compared to the expression of the molecule in a wild-type cell decreased expression.
  • the term “increased expression” is used interchangeably with “increased expression”, “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 .
  • the reduction in expression can be a reduction in the expression of the protein by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
  • the decrease in the amount of molecular expression can be assessed by measuring the decrease in the level of RNA encoded by the gene of interest and/or the decrease in the expression level of the molecule.
  • the term “reduced activity” is used interchangeably with “reduced activity”, “down-regulated activity” and generally refers to a decrease in the amount of activity of a molecule in modified cells compared to the amount of activity in wild-type cells .
  • the term “increased activity” is used interchangeably with “increased activity”, “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.
  • the decreased activity level or decreased expression level can be a decrease in the activity of the molecule by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
  • the amount of synthesis of the molecule is not reduced, but the amino acid sequence is modified such that the activity of the molecule is directly or indirectly reduced.
  • deletion generally refers to the loss of the expression level and/or activity of the molecule of interest.
  • the deletion can be at the gene level, or at the transcriptional or translational level, and in certain embodiments, the deletion can mean that the expression and activity of the molecule of interest cannot be detected.
  • modulate is used interchangeably with “modulate” and generally refers to a change or change in the expression level and/or activity of a molecule of interest. Modulation can be an increase or decrease in the magnitude of a particular activity or function of the molecule of interest. In certain embodiments, activities and functions include, but are not limited to, binding characteristics, enzymatic activity, cellular receptor activation, and signal transduction.
  • naturally occurring generally refers to naturally occurring, unmodified.
  • a polypeptide or polynucleotide sequence that is native to an organism, including a virus can be isolated from a natural source and has not been intentionally or otherwise modified in the laboratory.
  • engineered generally refers to changes in natural molecules and the like by artificial means. Modifications may be by modification, mutation, synthesis, etc. of the molecule. "Engineered” can be distinguished from naturally occurring. For example: if a cell or organism is manipulated such that its genetic information is altered (e.g. to introduce new genetic material that did not previously exist, e.g. by transformation, matching, somatic hybridization, transfection, transduction, or other mechanisms, or to alter or remove previously existing genetic material, such as by substitution or deletion mutation), then it is considered “engineered”.
  • large polymerase protein L is used interchangeably with “L protein” and generally refers to the vesicular stomatitis virus RNA polymerase protein.
  • the VSV L gene encodes the RNA poly E protein, and the L protein is involved in the processes of initiation, elongation, methylation, capping, and poly A tail formation.
  • nucleocapsid protein N is used interchangeably with “N protein” and generally refers to the vesicular stomatitis virus nucleoprotein.
  • the N protein effectively protects viral RNA from digestion by various nucleases.
  • N protein is group-specific, shared by many types and subtypes, has high antigenicity, and can stimulate the body to produce non-neutralizing antibodies.
  • phosphoprotein P also known as "P protein” generally refers to the vesicular stomatitis virus phosphoprotein.
  • the P gene is highly variable phosphorylated.
  • the P, L, and N protein-RNA complexes are necessary for transcriptase activity.
  • matrix protein M is used interchangeably with “M protein” and generally refers to the vesicular stomatitis virus matrix protein.
  • Matrix protein M is an important virulence factor for VSV and a protein in vesicular stomatitis virus known to interfere with the innate immune response of mice.
  • glycoprotein G is used interchangeably with “G protein” and generally refers to the glycoprotein of vesicular stomatitis virus.
  • M51R refers to the mutation of methionine M at position 51 to arginine R
  • ⁇ L111 refers to the deletion of leucine at position 111.
  • mutation generally refers to a change in the nucleotide or amino acid sequence of a wild-type molecule, eg, a change in the DNA and/or amino acid sequence of the extracellular domain of wild-type CTLA4. Mutations in DNA can change codons, resulting in changes in the amino acid sequence. DNA changes can include substitutions, deletions, insertions, alternative splicing or truncations. Amino acid changes may include substitutions, deletions, insertions, additions, truncations, or processing or cleavage errors of the protein. Alternatively, mutations in the nucleotide sequence can result in silent mutations in the amino acid sequence as well understood in the art.
  • nucleotide codons encode the same amino acid. Examples include the nucleotide codons CGU, CGG, CGC and CGA encoding the amino acid arginine (R); or the codons GAU and GAC encoding the amino acid aspartic acid (D).
  • a protein can be encoded by one or more nucleic acid molecules that differ in their specific nucleotide sequence, yet still encode protein molecules having the same sequence.
  • single base editing generally refers to single base conversion gene editing techniques.
  • the technology is based on the complex formed by dCas9 without nuclease activity or Cas9n with single-stranded DNA nickase activity (Cas9 nickase), cytosine deaminase, uracil glycosylase inhibitor (UGI) and sgRNA.
  • Cas9 nickase single-stranded DNA nickase activity
  • cytosine deaminase cytosine deaminase
  • UFI uracil glycosylase inhibitor
  • sgRNA single base editing
  • point mutation has a broad and a narrow concept.
  • Generalized point mutations can be base substitutions, base insertions, or base deletions.
  • Narrow sense point mutations also known as single base changes, can be single base substitutions. Base substitutions can be further divided into transitions and transversions.
  • 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.
  • autologous cells generally refers to cells derived from an individual and later reintroduced into the same individual.
  • the individual may be a human or an animal.
  • allogeneic cells generally refers to cells derived from one individual and then introduced into another individual.
  • the individual may be a human or an animal.
  • in vitro expansion generally refers to the administration of certain nutrients and stimulating factors to promote cell proliferation in an environment outside the body.
  • transcription generally refers to the process of producing a nucleic acid copy of an initial nucleic acid. Transcription generally involves the production of RNA copies of DNA nucleic acids, and can sometimes also involve the production of DNA copies of starting RNA nucleic acids (eg, reverse transcription).
  • translation refers to the process at the ribosome, where mRNA chains control the assembly of amino acid sequences to generate proteins or peptides.
  • overexpression is also referred to as “overexpressed” or “overexpressed”, and generally refers to the expression of a gene at a level higher than the level of gene expression in normal cells.
  • gene editing generally refers to directing or effecting a change (eg, deletion) of one or more nucleic acids at or near a targeted genomic DNA site.
  • TALEN transcription factor-like responsive nuclease
  • TALEN transcription factor-like responsive nuclease
  • TALEN also refers to one or both members of a pair of TALENs that are engineered to function together to cleave DNA at the same site.
  • TALENs working together can refer to left and right TALENs, which relate to the chirality of DNA or TALEN-pairs.
  • TALs The code of TALs has been reported (PCT Publication WO 2011/072246), in which each DNA-binding repeat is responsible for recognizing one base pair in the DNA target sequence. Residues can be assembled to target DNA sequences. Briefly, target sites for TALEN binding were determined, creating fusion molecules comprising a nuclease and a series of RVDs that recognize the target site. After binding, the nuclease cuts the DNA so that cellular repair machinery can operate to make genetic modifications at the cut end.
  • TALEN refers to proteins comprising transcriptional activator-like (TAL) effector-binding domains and nuclease domains, including monomeric TALENs that are themselves functional and other TALENs that require dimerization with another monomeric TALEN.
  • TALENs have been shown to induce genetic modifications in immortalized human cells using two major eukaryotic DNA repair pathways, non-homologous end joining (NHEJ) and homology-mediated repair.
  • NHEJ non-homologous end joining
  • TALENs are usually used in pairs, but monomeric TALENs are also known.
  • TALENs Genetic modification using TALENs or other tools can, for example, be selected from the group consisting of insertions, deletions, insertions of exogenous nucleic acid fragments, and substitutions.
  • a DNA target site is identified and a TALEN pair is created that can specifically bind to that site.
  • TALENs are delivered into cells or embryos as proteins, mRNAs, or via vectors encoding TALENs.
  • TALENs cleave DNA to form double-strand breaks, which are then repaired, often resulting in the formation of indels, or incorporation of sequences or polymorphisms contained in the accompanying exogenous nucleic acid into which the insertion is made into the chromosome, or serve as a template for repairing breaks using modified sequences.
  • This template-driven repair is a useful method of altering chromosomes, providing efficient changes to the cell's chromosomes.
  • Zinc finger nuclease also known as “ZFN” generally refers to an artificial restriction enzyme produced by fusion of a zinc finger DNA-binding domain with a DNA-cleavage domain.
  • Zinc finger domains can be engineered to target desired DNA sequences, thus enabling zinc finger nucleases to target unique sequences within complex genomes. Using endogenous DNA repair machinery, these reagents can be used to alter the genomes of advanced organisms.
  • ZFNs can be used in gene inactivation methods.
  • the zinc finger DNA-binding domain has approximately 30 amino acids and folds into a stable structure. Each zinc finger binds primarily to a triplet within the DNA substrate. Amino acid residues at key positions contribute to most sequence-specific interactions with DNA sites. These amino acids can be altered while maintaining the necessary structure for the remaining amino acids. By concatenating several domains, it is possible to bind to longer DNA sequences. Other functions, such as non-specific FokI cleavage domain (N), transcriptional activator domain (A), transcriptional repressor domain (R), and methylase (M) can be fused with ZFP to form ZFN, zinc finger, respectively Transcription activator (ZFA), zinc finger transcription repressor (ZFR) and zinc finger methylase (ZFM). Materials and methods for making genetically modified animals using zinc finger zinc finger nucleases are disclosed, for example, in patents US8106255; US2012/0192298; US2011/0023159 and US2011/0281306.
  • knockout generally refers to the abolition of gene expression of one or more genes.
  • kit generally refers to a collection of the components, which may or may not be packaged together or may not be packaged together.
  • the components of the kit may be contained in separate containers (ie, kits with separate parts), may be provided in a single container, or may not be placed in a container.
  • the kit may contain instructions for carrying out the method. Instructions are available as user manuals in paper or electronic form. For example, the manual may contain instructions for explaining the results obtained when the above-described methods are performed using the kits of the present application.
  • tool generally refers to elements, components, reagents, molecules, etc., required to be able to prepare a molecule of interest.
  • the term "subject” generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease, especially a person in need of the oncolytic virus expression vector subject.
  • the subject may include a cancer patient.
  • the subject is a human or non-human mammal.
  • Non-human mammals can include any mammalian species other than humans, such as livestock animals (eg, cows, pigs, sheep, chickens, rabbits, or horses), or rodents (eg, rats and mice), Or primates (eg, gorillas and monkeys), or domestic animals (eg, 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 hybrid of various races. A human “subject” can be an elderly, adult, adolescent, child, or infant.
  • the term "pharmaceutical composition” generally refers to a formulation in a form that allows the biological activity of the active ingredient to be effective, and which does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is to be administered.
  • these formulations may contain the active ingredient of the drug together with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a pharmaceutical composition for parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered by various means, eg intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • a pharmaceutically acceptable adjuvant generally means, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications, Adjuvants with reasonable benefit/risk ratios.
  • a pharmaceutically acceptable adjuvant can be one that is approved by a regulatory agency (eg, US Food and Drug Administration, China Food and Drug Administration, or European Medicines Agency) or listed in a generally recognized pharmacopeia (eg, US Pharmacopeia, Those adjuvants of the Chinese Pharmacopoeia or the European Pharmacopoeia for use in animals, more particularly in humans.
  • pharmaceutically acceptable adjuvants may be aluminum-based adjuvants, mineral salt adjuvants, tonicity active adjuvants, bacterial derived adjuvants, emulsification adjuvants, liposome adjuvants, cytokine adjuvants , carbohydrate adjuvants and DNA and RNA oligomer adjuvants and others.
  • the terms “effective amount” or “effective dose” are used interchangeably and generally refer to an amount sufficient to obtain, or at least partially obtain, the desired therapeutic effect.
  • the term “therapeutically effective amount” generally refers to an amount sufficient to cure or at least partially prevent the disease and its complications in an already afflicted subject. An effective amount for this use will depend on the severity of the infection and the general state of the patient's own immune system.
  • the term “combination” is also referred to as "co-administration” and generally refers to administration before, after, or at the same time as another pharmaceutical ingredient.
  • the two or more pharmaceutical components used in combination can be administered by the same mode of administration, or by different modes of administration, either simultaneously or sequentially.
  • one therapeutic agent in combination can be the oncolytic virus and the second therapeutic agent can be the modified immune effector cells.
  • Combinations can also include a third or even more therapeutic agent.
  • treating generally refers to eliminating or ameliorating a disease, or one or more symptoms associated with a disease.
  • treatment generally refers to the administration of one or more drugs to a patient suffering from the disease to bring about elimination or remission of the disease.
  • "treating" may be administering the composition in the presence or absence of other drugs after the onset of symptoms of a particular disease.
  • the term "carrier” generally refers to a component of a pharmaceutical formulation, other than the active component, that is not toxic to a subject.
  • the pharmaceutically acceptable carriers in the compositions, pharmaceutical compositions or kits of the present application may include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, water Phase vehicles (eg, Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose or Lactated Ringer's Injection), non-aqueous vehicles (eg, non-aqueous plant-derived injections) volatile oils, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial substances, isotonic substances (eg, sodium chloride or dextrose), buffers (eg, phosphate or citrate buffers), anti-microbial Oxidizing agents (eg, sodium bisulfate), anesthetics (eg, procaine hydrochloride), suspending/
  • water Phase vehicles
  • tumor generally refers to any new pathological tissue proliferation.
  • Tumors may be benign, such as hemangioma, glioma, teratoma, etc., or malignant, such as adenocarcinoma, sarcoma, glioblastoma, astrocytoma, neuroblastoma, retinoblastoma tumor etc.
  • these tissues can be isolated from readily available sources by methods well known to those skilled in the art.
  • Cas9 refers to the enzyme responsible for DNA cleavage from the bacterial Type II CRISPR/Cas system. Cas9 also includes the wild-type protein and its functional and non-functional mutants. In certain embodiments, the Cas9 can be Cas9 of Streptococcus pyogenes.
  • CTLA-4 also known as cytotoxic T lymphocyte antigen-4
  • CTLA-4 generally refers to an immunosuppressive receptor belonging to the CD28 family.
  • CTLA-4 is expressed exclusively on T cells in vivo and binds two ligands, CD80 and CD86 (also known as B7-1 and B7-2, respectively).
  • CD80 and CD86 also known as B7-1 and B7-2, respectively.
  • the complete hCTLA-4 sequence can be found under GenBank Accession No. AAB59385.
  • the amino acid sequence of human CTLA-4 can be set forth in SEQ ID NO:12.
  • LAG-3 also known as “CD223” generally refers to lymphocyte activation gene 3.
  • human LAG-3 refers to the human sequence LAG-3, such as the complete amino acid sequence of human LAG-3 having Genbank accession number NP_002277.
  • PD-1 generally refers to programmed cell death 1, also referred to as “programmed cell death 1", “CD279”, “cluster of differentiation 279", “PD1”, “PDCD1”.
  • PD-1 is normally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs) and is involved in apoptosis.
  • PD-1 usually contains an extracellular IgV domain, a transmembrane domain and an intracellular domain.
  • PD-1 binds two ligands, PD-L1 and PD-L2.
  • the human PD-1 amino acid sequence can be found under GenBank Accession No. NP_005009.2.
  • the human PD-1 amino acid sequence can be set forth in SEQ ID NO:11.
  • 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 under GenBank accession number Q9NZQ7.
  • TIGIT generally refers to a T cell immune receptor having Ig and ITIM domains. It can be from any vertebrate source, including any native TIGIT of mammals such as primates (eg, humans) and rodents (eg, mice and rats). TIGIT is also known in the art as DKFZp667A205, FLJ39873, V set and immunoglobulin domain containing protein 9, V set and transmembrane domain containing protein 3, VSIG9, VSTM3, and WUCAM. The amino acid sequence of a human TIGIT can be found in UniProt Accession No. Q495A1.
  • TIM-3 generally refers to molecule 3 containing T cell immunoglobulin and mucin domains.
  • the natural ligand for TIM-3 is Galectin 9 (Gal9).
  • the term "BTLA” generally refers to B and T lymphocyte attenuating agents.
  • the BTLA can be human BTLA.
  • VISTA generally refers to T cell activation V domain Ig inhibitory proteins.
  • VISTA can 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 No. NP_071436.
  • the oncolytic virus can be obtained from any virus member currently identified.
  • the oncolytic virus replicates higher in dividing cells and has a propensity to kill cells compared to non-dividing cells.
  • Oncolytic viruses described in this application include engineered viruses with oncolytic effects and wild-type viruses with oncolytic effects, including but not limited to: vesicular stomatitis virus, pox virus, retrovirus, herpes simplex virus, Measles virus, Semliki Forest virus, Poliovirus, Reovirus, Seneca Valley virus, Echo type enterovirus, Coxsackie virus, Newcastle disease virus, Maraba virus, Alpha virus, Slow Virus, influenza virus, Sinbis virus, myxoma virus, rhabdovirus, picornavirus, parvovirus, etc.
  • the oncolytic virus may also be derived from, for example, herpes simplex virus, vaccinia virus, vesicular stomatitis virus, autonomous parvovirus, myxoma virus, Newcastle disease Kagu virus, measles virus, retrovirus, influenza virus, Sindbis virus or pox virus.
  • An oncolytic virus wherein "derived from” a reference virus comprises the nucleic acid sequence or amino acid sequence possessed by the reference virus.
  • an oncolytic virus "derived from” a reference virus comprises one or more genes possessed by the reference virus.
  • an oncolytic virus "derived from” a reference virus encodes one or more proteins encoded by the reference virus.
  • an oncolytic virus derived from a reference virus may comprise nucleic acid sequences encoding one or more functional elements of the reference virus.
  • a "functional element” can be, for example, a transcriptional regulator (eg, a promoter/enhancer), a post-transcriptionally processed regulator, a translational regulator, a post-transcriptional processed regulator, a response element, a repeat sequence, or a viral protein.
  • the oncolytic virus can be a natural oncolytic virus or an engineered oncolytic virus.
  • the modified oncolytic virus may be an oncolytic virus modified at the genetic level, such as modified by one or more genes, so as to improve its tumor selectivity and/or preferentially replicate in dividing cells.
  • the modification at the gene level can be the modification of genes involved in DNA replication, nucleic acid metabolism, host tropism, surface attachment, virulence, lysis and diffusion processes, or the modification of integration of exogenous genes, including exogenous genes. Exogenous immune regulatory genes, exogenous screening genes, exogenous reporter genes, etc.
  • the engineered oncolytic virus can also be an oncolytic virus engineered at the amino acid level, such as one or more amino acid insertions, deletions, and substitutions.
  • the engineered oncolytic virus can also be an oncolytic virus with altered protein expression levels and activities compared to wild type.
  • the oncolytic virus of the present application is selected from the group consisting of vesicular stomatitis virus (VSV), a prototypical non-segmented negative-strand RNA virus whose 11 kb genome encodes 5 proteins: Nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and large polymerase protein (L).
  • VSV vesicular stomatitis virus
  • N Nucleocapsid protein
  • P phosphoprotein
  • M matrix protein
  • G glycoprotein
  • L large polymerase protein
  • the vesicular stomatitis virus can be the vesicular stomatitis virus Indiana strain
  • the VSV MuddSummer subtype the gene sequence encoding the M protein is shown in SEQ ID NO: 3
  • the amino acid sequence of the M protein is shown in SEQ ID NO: 3 ID NO: 4.
  • the M protein can be natural unmodified, naturally mutated, or artificially mutated.
  • VSV can be wild type, unmodified.
  • VSV M protein can be modified as follows: M51R.
  • VSV M protein can be modified as follows: M51R and ⁇ L111.
  • VSV M protein can be modified as follows: M51R, V221F and S226R.
  • VSV M protein can be modified as follows: M51R, AL111, V221F and S226R.
  • the nucleotide sequence encoding the VSVM protein (M51R, V221F and S226R) is set forth in SEQ ID NO: 5, and the amino acid sequence of the VSVM protein (M51R, V221F and S226R) is set forth in SEQ ID NO: 6 shown.
  • the nucleotide sequence encoding the VSV M protein (M51R, AL111, V221F, and S226R) is set forth in SEQ ID NO: 7
  • the amino acid sequence of the VSV M protein (M51R, AL111, V221F, and S226R) is set forth in SEQ ID NO: 8.
  • the mutation site of the vesicular stomatitis virus matrix protein M can also be a single site mutation, for example: ⁇ M51; M51A; ⁇ L111; V221F; S226R.
  • the mutation site of the vesicular stomatitis virus matrix protein M can also be a combined mutation of multiple sites, for example: M51R and V221F; M51R and S226R; ⁇ L111 and V221F; ⁇ L111 and S226R; V221F and S226R; M51R, ⁇ L111 and V221F; M51R, ⁇ L111 and S226R; ⁇ L111, V221F and S226R.
  • the mutation site of the vesicular stomatitis virus matrix protein M can also be a mutation of a continuous site, for example: M51-54A; ⁇ M51-54; ⁇ M51-57.
  • the mutation site of the vesicular stomatitis virus matrix protein M can also be a combination of continuous site and single site mutation, for example: M51-54A, V221F and S226R; ⁇ M51-54, V221F and S226R; ⁇ M51-57, V221F and S226R.
  • Immune effector cells usually refer to immune cells that participate in immune responses and perform effector functions.
  • the immune effector cells described in this application can be known various types of immune effector cells, and can be obtained by conventional biological methods, including but not limited to: lymphocytes, such as T cells, B cells, and NK cells; Bone marrow cells such as monocytes, macrophages, eosinophils, mast cells, basophils, neutrophils and dendritic cells.
  • 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, mucosa-associated constant T cells, ⁇ T cells cell.
  • the T cells can be human peripheral blood T cells.
  • the immune effector cells can be autologous cells or allogeneic cells.
  • the immune effector cells can be immune effector cells expanded in vitro. Techniques for large-scale in vitro expansion of immune effector cells are known.
  • 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.
  • the adjustment of the expression and/or activity of the immune checkpoint may include the adjustment of an immune checkpoint, for example, the adjustment of PD-1, the adjustment of PD-L1, the adjustment of CTLA-4, the adjustment of LAG -3 adjustment, TIM-3 adjustment, BTLA adjustment, VISTA adjustment, TIGIT adjustment, B7-H2 adjustment, B7-H3 adjustment, B7-H4 adjustment and B7-H6 adjustment.
  • an immune checkpoint for example, the adjustment of PD-1, the adjustment of PD-L1, the adjustment of CTLA-4, the adjustment of LAG -3 adjustment, TIM-3 adjustment, BTLA adjustment, VISTA adjustment, TIGIT adjustment, B7-H2 adjustment, B7-H3 adjustment, B7-H4 adjustment and B7-H6 adjustment.
  • the modulation of immune checkpoint expression and/or activity may include modulation of a combination of two to more immune checkpoints, eg: 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.
  • said modulation of immune checkpoint expression comprises up-regulation, down-regulation and/or deletion of said immune checkpoint expression compared to unmodified immune effector cells.
  • the modulation of immune checkpoint activity comprises an increase, decrease and/or absence of immune checkpoint activity compared to unmodified immune effector cells.
  • the adjustment of the expression and/or activity of the immune checkpoint may be up-regulation, down-regulation or deletion of the expression and/or activity of the immune checkpoint; it may also be one or more of the adjusted immune checkpoints.
  • Up-regulation of checkpoint expression and/or activity and down-regulation and/or absence of remaining adjusted immune checkpoint expression and/or activity For example: up-regulation of PD-1 expression and/or activity and down-regulation of CTLA-4 expression and/or activity, up-regulation of PD-L1 expression and/or activity, and down-regulation of CTLA4 expression and/or activity and loss of LAG-3 expression and/or activity.
  • the regulation of immune checkpoints includes regulation of immune checkpoint gene levels, transcriptional levels and/or translational levels.
  • one of these methods can be used for modulation, or more than two methods can be used for modulation.
  • the method of regulation at the gene level includes, for example, gene editing, overexpression, point mutation, homologous recombination.
  • gene editing methods include, for example: CRISPR/Cas9, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) and single base editing (BE).
  • the adjustment of the immune checkpoint is that the expression of PD-1 molecule in T cells is reduced, and the specific method is to use CRISPR/Cas9 gene editing technology to knock out the PDCD1 gene of human peripheral blood T cells, and reduce the expression of PD-1 in T cells. Expression of PD-1 molecules.
  • the CRISPR/Cas9 gene editing technology may comprise an sgRNA of the designed sequence.
  • the sgRNA can bind to Cas9 protein to recognize and edit the target gene.
  • the immune effector cell knocks out a target sequence for the PD-1 gene selected from the group consisting of SEQ ID NO:1.
  • the immune effector cell knocks out a target sequence for the CTLA-4 gene selected from the group consisting of SEQ ID NO:2.
  • compositions comprising, pharmaceutical compositions and kits
  • composition comprising: (a) the oncolytic virus; and (b) the modified immune effector cells, wherein the modified immune effector cells are associated with unmodified immune effector cells. Compared with the modified immune effector cells, the expression and/or activity of immune checkpoints are adjusted.
  • the composition may comprise an engineered oncolytic virus and a modified immune cell, eg, the engineered oncolytic virus comprises an engineered VSV whose matrix protein M and SEQ ID Compared with the amino acid sequence shown in NO:4, the modified immune cells comprise amino acid mutations of M51R, V221F and S226R, and the modified immune cells comprise PD-1 knockout T cells.
  • the matrix protein M of the engineered oncolytic virus comprises the amino acid sequence shown in SEQ ID NO: 6, and the modified immune cell comprises PD-1 Knockout T cells.
  • the composition may comprise an engineered oncolytic virus and a modified immune cell, eg, the engineered oncolytic virus comprises an engineered VSV whose matrix protein M and SEQ ID Compared with the amino acid sequence shown in NO:4, the modified immune cells comprise amino acid mutations of M51R, ⁇ L111, V221F and S226R, and the modified immune cells comprise PD-1 knockout T cells.
  • the matrix protein M of the engineered oncolytic virus comprises the amino acid sequence shown in SEQ ID NO: 8
  • the modified immune cell comprises PD-1 Knockout T cells.
  • a pharmaceutical composition comprising: (a) at least one oncolytic virus and/or a pharmaceutically acceptable carrier; and (b) at least one Modified immune effector cells and/or a pharmaceutically acceptable carrier.
  • kits comprising one or more selected from the group consisting of: (a) the at least one oncolytic virus and the preparation of the at least one modified immune A means for effector cells, and/or a pharmaceutically acceptable carrier; (b) the at least one modified immune effector cell and the means for preparing the at least one oncolytic virus, and/or pharmaceutically acceptable and, (c) means for producing said at least one oncolytic virus and means for producing said at least one modified immune effector cell and a pharmaceutically acceptable carrier.
  • the means for preparing the at least one modified immune effector cell in the kit means that any individual can obtain the modified immune effector cell by using the tool, such as obtaining PD -1 sgRNA of the designed sequence used in molecular knockout T cells.
  • the means for preparing the at least one oncolytic virus in the kit means that any individual can obtain the oncolytic virus by using the means. For example, plasmids, primers for each mutation site.
  • compositions, pharmaceutical compositions, and kits can be formulated as a medicament for clinical use, and can include a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant.
  • the compositions, pharmaceutical compositions and kits can be formulated for topical, parenteral, systemic, intracavitary, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, dermal intrathecal, oral or transdermal routes of administration, which may include injection or infusion. Suitable formulations may contain virus and/or cells in sterile or isotonic medium.
  • the compositions, pharmaceutical compositions and kits can also be formulated in fluid, including gel form. Fluid formulations can be formulated for administration by injection or infusion (eg, through a catheter) to selected areas of the human or animal body.
  • preparation methods of the composition, the pharmaceutical composition, and the kit are provided.
  • compositions, the pharmaceutical composition, and the components of the kit may be in the same container, or may be placed in different containers.
  • the oncolytic virus can be formulated for intratumoral administration.
  • the oncolytic virus and the immune effector cells can be formulated for intravenous administration.
  • the immune effector cells can be formulated for topical administration.
  • a method for treating tumors comprises administering the composition, the pharmaceutical composition and/or the kit to a subject in need thereof.
  • one or more of the composition, the pharmaceutical composition, and the kit can be administered to a subject in need thereof.
  • the oncolytic virus and the modified immune cells are administered simultaneously. Simultaneous administration can be performed by mixing the components together or separately. Administration may be in the same manner, eg, in the same vein or other vessel, or in a different manner, eg, concurrently with intravenous administration and intratumoral administration.
  • the oncolytic virus and the modified immune cells are administered sequentially.
  • the order of administration may be that the oncolytic virus is administered first, and then the immune effector cells are administered; or the immune effector cells may be administered first, and then the oncolytic virus is administered. It can be administered in the same way, or it can be administered in a different way.
  • Each component can be administered at one time or divided into multiple administrations.
  • sequential administration can be administered at any time interval, including minutes, hours, days, weeks, months, or years.
  • administering sequentially refers to 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 separated by one of day, 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 apply.
  • the dose administered is a therapeutically effective amount.
  • the application of the oncolytic virus in the combined use of the oncolytic virus and the modified immune effector cells is also provided. That is, the oncolytic virus used for the combined use of the oncolytic virus and the modified immunization.
  • the use of the modified immune effector cells in the combined use of the oncolytic virus and the modified immune cells is also provided. That is, the modified immune cells used in combination with the oncolytic virus and the modified immunity.
  • the present application also provides applications of the composition, the pharmaceutical composition, and the kit in the preparation of a medicament for treating tumors.
  • the present application also provides the oncolytic virus and the modified immune effector cells for use in the treatment of tumors.
  • the tumor comprises a solid tumor and/or a hematological tumor.
  • 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.
  • Example 1 Construction and effect display of vesicular stomatitis virus mutants
  • mutant strain is a conventional technique in the field, and the method is as follows:
  • Plasmids (1) Construction of plasmids. Using pVSV-XN2 plasmid as a template, different mutation sites as described in Table 1 were introduced by PCR method. The plasmids and the primers for each mutation site were subjected to PCR together, and the PCR products were subjected to 1% agarose gel electrophoresis, followed by gel-tipping recovery using a gel recovery kit to obtain plasmids with different mutations of the M matrix protein.
  • pBS-N, pBS-P, and pBS-L refer to the expression plasmids that clone 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 carried out according to the method described in the instruction manual of lipofectamine 2000. After 4 h, fresh DMEM complete medium containing 10% fetal bovine serum was replaced. After 48 h, the supernatant was aspirated, and the pox virus was removed with a 0.22 ⁇ m filter. The filtrate was added to fresh BHK-21 cells; the cytopathic conditions were observed every day, and the supernatant was collected when the cells showed pathological changes. After the success of RTPCR identification was confirmed, the virus was purified by virus plaque experiment. That is to obtain mutant strains.
  • the viral genomic RNA was extracted with Trizol kit, reverse transcription reaction was performed with random primers, and the reverse transcribed cDNA was PCR-reduced with primers designed for the M protein gene sequence.
  • the primer sequences are: 5'-AAAAAAGTAACAGATATCAC-3' (SEQ ID NO: 9); 5'-ACATTTTTCCAGTTTCCTTTTTGG-3' (SEQ ID NO: 10).
  • the product was recovered by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing.
  • step (2) In a 1.5 mL EP tube, the supernatant harvested in step (2) was serially diluted by 10 times, 10 -1 to 10 -11 , with a total of 11 titers.
  • IFN- ⁇ indicator Tested with IFN- ⁇ indicator.
  • Cells were cultured according to steps (1) and (2) of step 3 above and mutant strains were added. Then each group of cells was disrupted, total RNA was extracted from each cell with TRIzol (Invitrogen), reverse transcribed into cDNA using PrimeScript RT Reagent Kit with DNA Eraser (Takara) reverse transcription kit, and LightCycler 480SYBR Green I Master (Roche) dye was used Staining was performed, and the Ct value of each gene was detected on a LightCycler 480 quantitative PCR instrument. The relative expression levels of target genes IFN- ⁇ and VSV-G were calculated by ⁇ Ct method.
  • Example 2 Knockout of T cell PD-1 molecule by CRISPR/Cas9 technology
  • fusion plasmid carrying the sgRNA and Cas9 enzyme that recognizes the human PDCD1 gene was constructed. Examples of fusion plasmid elements are shown in Figure 4.
  • the PD-1-sgRNA-CRISPR/Cas9 system down-regulates the expression of PD-1 molecule on the surface of human peripheral blood T cells
  • the PD-1-sgRNA-CRISPR/Cas9 plasmid was electroporated into freshly isolated human peripheral blood PBMC cells, and the electroporated PBMC cells were activated with CD3 and CD28 antibodies and cultured at 37°C under 5% CO 2 for 24 h after electroporation On the 8th day, the cultured cells were taken to detect the expression of PD-1 molecule on the surface of T cells.
  • Example 3 Knockout of PD-1 and CTLA-4 molecules in T cells by CRISPR/Cas9 technology
  • gRNA sequences were designed.
  • the pU6gRNA-CMV-Cas9-GFP expression vector was digested with BbsI, and the oligo double-strand formed by the PD-1 oligo sequence was connected after recovery to construct a PD-1 gene knockout vector containing one gRNA.
  • the expression vector was linearized with primers, the breakpoint was located between the gRNA and CMV promoter sequences, and HindIII and EcoRI enzyme cleavage sites were added to the two segments. Then, using the pU6gRNA-CMV-Cas9-GFP expression vector as a template, the gRNA expression unit was cloned with primers and linked into pUC19, and then cut into CTLA-4 oligo sequence by BbsI to obtain an intermediate vector.
  • the linearized pU6gRNA-CMV-Cas9-GFP expression vector and the intermediate vector were digested with HindIII and EcoRI, and the two were connected after recovery to obtain a knockout vector pU6gRNA-Cas9-GFP that can express two gRNAs with different target sequences.
  • the pU6gRNA-Cas9-GFP system down-regulates the expression of PD-1 molecule and CTLA-4 on the surface of human peripheral blood T cells
  • the pU6gRNA-Cas9-GFP plasmid was electroporated into freshly isolated human peripheral blood T cells, activated with CD3 and CD28 antibodies, and cultured at 37°C with 5% CO2. The cells were taken for flow cytometry on the 8th day after electroporation. Expression of PD-1 and CTLA-4 molecules on the cell surface.
  • A549 tumor cell line human non-small cell lung cancer
  • 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 ⁇ L of oncolytic virus was inoculated in each well, and each dilution gradient was inoculated with 4 replicates. Wells were incubated at 37°C under 5% CO2 for 48h.
  • MOI multiplicity of infection
  • tumor cell killing rate (%) (OD value of tumor cell control group ⁇ OD value of experimental group)/OD value of tumor cell control group ⁇ 100%.
  • Example 5 In vitro tumor lethality detection of T cells knocked out of PD-1 molecule
  • A549 tumor cell line human non-small cell lung cancer
  • the PD-1 knockout 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); PD-1 was inoculated in each well 100 ⁇ L of knockout T cells were inoculated into 4 duplicate wells for each dilution gradient, and cultured at 37°C under 5% CO2 for 48h.
  • E:T effector target ratio
  • tumor cell killing rate (%) (OD value of tumor cell control group ⁇ OD value of experimental group)/OD value of tumor cell control group ⁇ 100%.
  • Example 6 Detection of tumor lethality in combined treatment of melanoma (transplantation tumor) with engineered VSV and PD-1 molecule knockout T cells
  • mice with no significant difference were selected and inoculated subcutaneously with 2 ⁇ 10 6 B16-F10-NY-ESO-1 melanoma cells.
  • the control group PBS group
  • the other 11 groups were treated groups, which were inoculated with JBS000 intratumorally.
  • Example 7 Detection of tumor lethality in combined treatment of fibrosarcoma (transplanted tumor) with engineered VSV and PD-1 molecule knockout T cells
  • mice 96 C57BL/6 mice with no significant difference were selected and inoculated subcutaneously with 2 ⁇ 10 6 MCA-205-NY-ESO-1 fibrosarcoma cells.
  • the control group PBS group
  • the other 11 groups were treated groups, which were inoculated with JBS000 intratumorally.
  • Example 8 Detection of tumor lethality in combined treatment of melanoma (transplanted tumor) with modified VSV and T cells knocking out PD-1 and CTLA-4 molecules
  • mice were treated according to the method of Example 5, and 2 ⁇ 10 6 B16-F10-NY-ESO-1 melanoma cells were subcutaneously inoculated. When the volume of the transplanted tumor grows to about 100mm 3 , the treatment will be carried out. All mice were divided into 10 groups: the control group (PBS group) was injected with 50 ⁇ L of PBS, and the other 9 groups were treatment groups, which were inoculated with JBS000, JBS001, JBS002, JBS003, JBS004, knockout PD-1 and CTLA respectively.
  • PBS group the control group
  • the other 9 groups were treatment groups, which were inoculated with JBS000, JBS001, JBS002, JBS003, JBS004, knockout PD-1 and CTLA respectively.
  • Example 9 Detection of tumor lethality in combined treatment of fibrosarcoma (transplanted tumor) with engineered VSV and T cells knocking out PD-1 and CTLA-4 molecules
  • mice were treated according to the method of Example 5, and 2 ⁇ 10 6 MCA-205-NY-ESO-1 fibrosarcoma cells were subcutaneously inoculated. When the volume of the transplanted tumor grows to about 100mm 3 , the treatment will be carried out. All mice were divided into 12 groups: the control group (PBS group) was injected with 50 ⁇ L of PBS, and the other 11 groups were treatment groups, which were inoculated with JBS000, JBS001, JBS002, JBS003, JBS004, knocked out PD-1 and CTLA respectively.
  • PBS group the control group
  • the other 11 groups were treatment groups, which were inoculated with JBS000, JBS001, JBS002, JBS003, JBS004, knocked out PD-1 and CTLA respectively.
  • Example 10 Evaluation of the effect of combined therapy with engineered VSV and PD-1 knockout T cells
  • VSV-OVV-01 represents the modified oncolytic virus in this application.
  • the specific dosing strategy is as follows:
  • Group A (use PD-1 knockout T cells first, Figure 7A): PD-1 knockout T cells were treated with a fixed dose from the first day of week 1 (W1D1, W indicates week, D indicates day) (total amount of 3.6 ⁇ 10 9 to 4.4 ⁇ 10 9 or 1.8 ⁇ 10 9 to 2.2 ⁇ 10 9 cells), 20%, 30%, and 50% of the total dose were divided into 3 times, each time interval 1-day dosing, starting at W1D1, every 4 weeks, if not occurring: disease progression, unacceptable toxicity, death, study withdrawal, loss to follow-up, sponsor termination of study or subject initiation of new
  • continuous medication is used for 4 cycles; VSV-OVV-01 adopts a fixed dose, that is, the clinically effective dose or the highest dose in the first stage, starting from W2D1, once every 2 weeks, if it does not occur: disease progression, In the event of unacceptable toxicity, death, withdrawal from the study, loss to follow-up, termination of the study by the sponsor, or initiation of new tumor therapy by the subject, the drug
  • VSV first, Figure 7B OVV-01 was administered at a fixed dose, that is, the clinically effective dose or the highest dose in the first phase, the first dose at W1D1, the second dose at W4D1, and then every 1 time in 2 weeks, 7 times in a row, a total of 8 doses.
  • T cells knocking out PD-1 molecules were administered from W5D1, using a fixed dose (total 3.6 ⁇ 10 9 to 4.4 ⁇ 10 9 or 1.8 ⁇ 10 9 to 2.2 ⁇ 10 9 cells), respectively, with a total dose of 20 %, 30%, and 50% are divided into 3 doses, and each dose is administered at an interval of 1 day. Starting from W5D1, every 4 weeks is a cycle.
  • Example 11 In vitro tumor killing ability of engineered oncolytic virus combined with PD-1 molecule knockout T cells
  • the specific method is as follows:
  • A549 tumor cell line human non-small cell lung cancer
  • the oncolytic virus was diluted to 10 according to the multiplicity of infection (MOI); the original medium in the 96-well plate was removed, and 100 ⁇ L of the oncolytic virus was inoculated in each well.
  • MOI multiplicity of infection
  • the PD-1 knockout 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); PD-1 was seeded in each well. 1 100 ⁇ L of knockout T cells, each dilution gradient was seeded in 4 replicate wells.
  • 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 tumor killing results of the engineered oncolytic virus combined with PD-1 knockout T cells are shown in Figure 8, which shows the use of the engineered oncolytic virus alone, PD-1 knockout T cells alone, and Combined use of engineered oncolytic virus and PD-1 knockout T cells; the results show that the combination of engineered oncolytic virus and PD-1 knockout T cells shows very strong anti-tumor ability , the engineered oncolytic virus synergized with PD-1 knockout T cells, significantly enhancing the antitumor effect.

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Abstract

一种溶瘤病毒和经修饰的免疫效应细胞联合治疗肿瘤，提供了包含溶瘤病毒和经修饰的免疫效应细胞的组合物、药物组合物及试剂盒。还提供了所述组合物、药物组合物及试剂盒的制备方法及在治疗肿瘤和制备治疗肿瘤药物中的用途。

Description

溶瘤病毒与经改造的免疫细胞联合治疗肿瘤 技术领域
本申请涉及生物医药领域，具体的涉及溶瘤病毒与经改造的免疫细胞联合治疗肿瘤。
背景技术
免疫效应细胞是人体内参与免疫应答的细胞，免疫检查点是免疫系统中存在的一些抑制性分子，通过配体-受体调控免疫应答的强度或广度，避免过度应答引起机体的损伤和破坏，是正常机体负反馈调节机制之一。但在肿瘤的发生和过程中，免疫检查点却成为诱导肿瘤免疫耐受的主要原因，导致机体抗肿瘤免疫应答的效率低下。
病毒治疗癌症的方法近二十年来发展迅速，目前病毒治疗最大的进展之一是利用肿瘤细胞与正常细胞的差异，对某些病毒结构进行改造，使其能被选择性地在肿瘤细胞中复制，最终达到杀死肿瘤细胞的目的。这些被改造后的病毒称为溶瘤病毒，其来源于疱疹病毒、痘病毒等。目前发现某些野生型病毒也具有在细胞中选择性复制从而溶瘤的功能。然而，使用溶瘤病毒单独治疗肿瘤的疗效有时并不十分理想，溶瘤病毒与化疗药物联合使用治疗肿瘤时，由于化疗药物对正常细胞同等杀伤，会带来强烈的副作用。
因此，目前在肿瘤和/或癌症的免疫治疗中，仍然需要更加有效的治疗方案及药物。
发明内容
本申请提供了一种组合物，其包含：(a)溶瘤病毒；以及(b)经修饰的免疫效应细胞，其中所述经修饰的免疫效应细胞与未经所述修饰的免疫效应细胞相比，免疫检查点的表达量和/或活性被调整。本申请还提供了一种药物组合物，其包含：(a)所述的至少一种溶瘤病毒和/或药学上可接受的载剂；以及(b)所述的至少一种免疫效应细胞和/或药学上可接受的载剂。本申请还提供了一种试剂盒，其包含所述溶瘤病毒、制备所述溶瘤病毒的工具、所述经修饰的免疫效应细胞、制备所述免疫效应细胞的工具和/或药学上可接受的载剂。另一方面，本申请还提供了所述组合物、所述药物组合物、所述试剂盒的制备方法。本申请还提供了所述溶瘤病毒在所述溶瘤病毒和所述经修饰的免疫效应细胞的联用中的应用，所述经修饰的免疫效应细胞在所述溶瘤病毒和所述经修饰的免疫细胞联用中的应用。本申请还提供了一种治疗肿瘤的方法，其包含向有需要的受试者施用所述组合物、所述药物组合物和/或所述试剂盒。本申请还提供了所述组合物、所述药物组合物、所述试剂盒在制备治疗肿瘤的药物中的应用。 溶瘤病毒与经修饰的免疫效应细胞联用，有效发挥了两者的协同作用，增强了肿瘤的杀伤力，提高了肿瘤的治疗效果。
一方面，本申请提供了一种组合物，其包含：(a)溶瘤病毒；以及(b)经修饰的免疫效应细胞，其中所述经修饰的免疫效应细胞与未经所述修饰的免疫效应细胞相比，免疫检查点的表达量和/或活性被调整。
在某些实施方式中，所述溶瘤病毒包含天然的溶瘤病毒和经过改造的溶瘤病毒。
在某些实施方式中，所述溶瘤病毒选自下组：水疱性口炎病毒(vesicular stomatitis virus,VSV)、痘病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、脊髓灰质炎病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒。
在某些实施方式中，所述水疱性口炎病毒包含基质蛋白M、核衣壳蛋白N、磷蛋白P、大聚合酶蛋白L和/或糖蛋白G。
在某些实施方式中，所述水疱性口炎病毒的基质蛋白M经过改造。
在某些实施方式中，所述水疱性口炎病毒基质蛋白M的改造包含一个以上位点的突变。
在某些实施方式中，所述水疱性口炎病毒基质蛋白M的突变位点选自下组中的一个或多个：M51、L111、V221和S226。
在某些实施方式中，所述水疱性口炎病毒基质蛋白M的突变位点选自下组单个位点的突变：
a)M51R；
b)ΔL111；
c)V221F；以及
d)S226R。
在某些实施方式中，所述水疱性口炎病毒基质蛋白M的突变位点的组合选自下组中的任一组：
a)M51R且ΔL111；
b)M51R且V221F；
c)M51R且S226R；
d)ΔL111且V221F；
e)ΔL111且S226R；
f)V221F且S226R；
g)M51R、ΔL111且V221F；
h)M51R、ΔL111且S226R；
i)M51R、V221F且S226R；
j)ΔL111、V221F且S226R；以及，
k)M51R、ΔL111、V221F且S226R。
在某些实施方式中，所述突变位点组合选自M51R、V221F且S226R的水疱性口炎病毒基质蛋白M包含如SEQ ID NO：6所示的氨基酸序列。
在某些实施方式中，所述突变位点组合选自M51R、ΔL111、V221F且S226R的水疱性口炎病毒基质蛋白M包含如SEQ ID NO：8所示的氨基酸序列。
在某些实施方式中，所述免疫效应细胞选自下组：T细胞、B细胞、NK细胞和巨噬细胞。
在某些实施方式中，所述免疫效应细胞包含自体细胞和异体细胞。
在某些实施方式中，所述免疫检查点的数量为一个以上。
在某些实施方式中，所述免疫检查点选自下组中的一个或多个：PD-1、PD-L1、CTLA-4、LAG-3、TIM-3、BTLA、VISTA、TIGIT、B7-H2、B7-H3、B7-H4和B7-H6。
在某些实施方式中，所述免疫检查点为PD-1、PD-L1、CTLA-4、LAG-3、TIM-3、BTLA、VISTA、TIGIT、B7-H2、B7-H3、B7-H4或B7-H6。
在某些实施方式中，所述免疫检查点为选自下组的组合：
a)PD-1和CTLA-4；
b)PD-1和PD-L1；
c)PD-L1和CTLA4；
d)LAG-3和CTLA-4；
e)TIM-3和CTLA-4；
f)VISTA和CTLA-4；以及
g)PD1、CTLA-4和PD-L1。
在某些实施方式中，所述免疫检查点表达量的调整包含与未经修饰的免疫效应细胞相比，所述免疫检查点表达量的上调、下调和/或缺失。
在某些实施方式中，所述免疫检查点活性的调整包含与未经修饰的免疫细胞相比，所述免疫检查点活性的增加、降低和/或缺失。
在某些实施方式中，所述免疫检查点的调整包含免疫检查点基因表达水平、转录水平和/或翻译水平的调控。
在某些实施方式中，所述基因表达水平的调控包含基因编辑、过表达、点突变和/或同源 重组。
在某些实施方式中，所述基因编辑方法选自下组的一种或多种：CRISPR/Cas9、类转录激活因子效应核酸酶(TALEN)、锌指核酸酶(ZFN)和单碱基编辑(BE)。
在某些实施方式中，所述基因编辑包含设计序列的sgRNA。
在某些实施方式中，所述免疫效应细胞敲除针对选自SEQ ID NO：1的PD-1基因的靶序列。
在某些实施方式中，所述免疫效应细胞敲除针对选自SEQ ID NO：2的CTLA-4基因的靶序列。
另一方面，本申请还提供了药物组合物，其包含：(a)所述的至少一种溶瘤病毒和/或药学上可接受的载剂；以及(b)所述的至少一种经修饰的免疫效应细胞和/或药学上可接受的载剂。
另一方面，本申请还提供了试剂盒，其包含选自下组中的一种或多种：
a)所述的至少一种溶瘤病毒和制备所述的至少一种经修饰的免疫效应细胞的工具，和/或药学上可接受的载剂；
b)所述的至少一种经修饰的免疫效应细胞和制备所述的至少一种溶瘤病毒的工具，和/或药学上可接受的载剂；以及，
c)制备所述的至少一种溶瘤病毒的工具和制备所述的至少一种经修饰的免疫效应细胞的工具，和/或药学上可接受的载剂；
另一方面，本申请还提供了所述组合物、所述药物组合物、所述试剂盒的制备方法。
另一方面，本申请还提供了所述溶瘤病毒在所述溶瘤病毒和所述经修饰的免疫效应细胞的联用中的应用。
另一方面，本申请还提供了所述经修饰的免疫效应细胞在所述溶瘤病毒和所述经修饰的免疫细胞联用中的应用。
另一方面，本申请还提供了一种治疗肿瘤的方法，其包含向有需要的受试者施用所述组合物、所述药物组合物、所述试剂盒的制备方法。
在某些实施方式中，所述溶瘤病毒通过静脉内和/或瘤内途径向有需要的受试者施用，其中所述免疫效应细胞通过静脉内和/或局部给药方式向有需要的受试者施用。
在某些实施方式中，所述溶瘤病毒和所述经修饰的免疫细胞同时施用，施用剂量为治疗有效量。
在某些实施方式中，所述溶瘤病毒和所述经修饰的免疫细胞分别施用，施用剂量为治疗 有效量。
在某些实施方式中，所述溶瘤病毒和所述经修饰的免疫细胞一次施用或多次施用。
另一方面，本申请还提供了所述组合物、所述药物组合物、所述试剂盒在制备治疗肿瘤的药物中的应用。
在某些实施方式中，本申请所述肿瘤包含实体瘤和/或血液瘤。
在某些实施方式中，所述肿瘤选自下组中的一种或多种：头颈部癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤、滑膜肉瘤、圆细胞型脂肪肉瘤。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是各经改造的溶瘤病毒在体外的LLC细胞和MEF细胞内复制能力示意图；
图2显示的是各经改造的溶瘤病毒对体外的LLC细胞、Hela细胞核MEF细胞杀伤能力示意图；
图3显示的是各经改造的溶瘤病毒对体外LLC细胞和MEF细胞内IFN-β表达水平影响示意图；
图4显示的是CRISPR/Cas9融合质粒元件示意图；
图5显示的是流式细胞术检测人外周血T细胞经修饰后表面PD-1分子的表达；
图6显示的是经改造的溶瘤病毒的体外抗肿瘤效果检测；
图7显示的是经改造的溶瘤病毒联合敲除PD-1分子的T细胞治疗策略；
图8显示的是经改造的溶瘤病毒联合敲除PD-1分子的T细胞的肿瘤杀伤效果检测。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书 所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“组合物”包含至少两种组分。在本申请中，所述组合物中的各组分可以混合放置，也可以分开放置。在本申请中，所述组合物中的各组分可以同时施用，也可以分别施用。所述分别施用可以区分先后顺序。例如，先施用组合物中的一个组分，再施用组合物中的另一个组分。在一个具体的实施方案中，组合物包含所述溶瘤病毒和所述经修饰的免疫效应细胞。
在本申请中，术语“溶瘤病毒”通常指能够在肿瘤细胞中复制并杀死肿瘤细胞的病毒。在某些实施方案中，所述病毒被改造以提高肿瘤细胞的选择性。溶瘤病毒包括但不限于水疱性口炎病毒、痘病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、脊髓灰质炎病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒。在优选的实施方案中，溶瘤病毒使水疱性口炎病毒。优选的水疱性口炎病毒可以是水疱性口炎病毒MuddSummer亚型株，其基质蛋白M基因进行定点基因突变。
在本申请中，术语“经修饰的”是指本发明的分子或细胞的改变的状态或结构。可以以许多方式——包括化学地、结构上和功能上——修饰分子。可以通过引入核酸来修饰细胞。
在本申请中，术语“免疫效应细胞”可与“免疫细胞”互换使用，通常指在免疫应答中起作用的细胞。免疫效应细胞包括：淋巴细胞，如T细胞和B细胞、天然的杀伤细胞；骨髓细胞，如单核细胞、巨噬细胞、嗜酸性细胞、肥大细胞、嗜碱性细胞和粒细胞。
在本申请中，术语“T细胞”是指胸腺衍生的细胞。其参与各种细胞介导的免疫反应，包括胸腺细胞、初始T淋巴细胞、不成熟的T淋巴细胞、成熟的T淋巴细胞、静息T淋巴细胞或活化的T淋巴细胞。在某些实施方案中，T细胞群包括但不限于辅助性T细胞(HTL；CD4+T细胞)、细胞毒性T细胞(CTL；CD8+T细胞)、CD4+CD8+T细胞、CD4-CD8-T细胞或者T细胞的任何其它亚群。在某些实施方案中，T细胞群包括但不限于表达下述标志物中的一种或多种的T细胞：CD3、CD4、CD8、CD27、CD28、CD45RA、CD45RO、CD62L、CD127、CD197和HLA-DR，并且若需要，可以通过阳性或阴性选择技术进一步分离。
在本申请中，术语“免疫检查点”通常指免疫细胞表面上的一组分子，其通过下调或抑制肿瘤免疫应答来调整免疫应答。免疫检查点包括但不限于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、精氨酸酶、CD73和 A2aR(参见例如WO 2012/177624)。所述术语还涵盖生物活性蛋白质片段，以及编码全长免疫检查点蛋白及其生物活性蛋白质的片段的核酸。在某些实施方案中，所述术语还涵盖根据本申请提供的同源性描述的任何和片段。
在本申请中，术语“表达量降低”可以与“表达量减少”、“表达量下调”互换使用，通常指与在野生型细胞中分子的表达量相比，经修饰的细胞中的分子表达量的下降。术语“表达量升高”可以与“表达量增加”、“表达量上调”互换使用，通常指与在野生型细胞中分子的表达量相比，经修饰的细胞中的分子表达量的上升。在某些实施方案中，表达量的下降可以是蛋白的表达量下降了至少10％、20％、30％、40％、50％、60％、70％、80％、90％或更大。在某些实施方案中，通过测量由目标基因编码的RNA水平的下降和/或分子表达水平的下降，可以评估分子表达量的下降。
在本申请中，术语“活性降低”可以与“活性减少”、“活性下调”互换使用，通常指与野生型细胞中的活性量相比，在经修饰的细胞中分子的活性量的下降。术语“活性升高”可以与“活性增加”、“活性上调”互换使用，通常指与野生型细胞中的活性量相比，在经修饰的细胞中分子的活性量的上升。降低的活性水平或降低的表达水平可以是分子的活性下降了至少10％、20％、30％、40％、50％、60％、70％、80％、90％或更大。在某些实施方案中，分子的合成的量没有下降，但是氨基酸序列经过修饰，使得分子活性直接地或间接的下降。
在本申请中，术语“缺失”通常指目的分子表达量和/或活性的失去。缺失可以是基因水平的缺失，也可以是转录水平或翻译水平的缺失，在某些实施方案中，缺失可以指检测不到目的分子的表达和活性。
在本申请中，术语“调整”可以与“调节”互换使用，通常指目的分子的表达量和/或活性中的变化或改变。调整可以是目的分子的特定活性或功能量级中的增加或减少。在某些实施方案中，活性和功能包括但不限于结合特征、酶促活性、细胞受体激活和信号转导。
在本申请中，术语“天然的”通常是指天然存在的，未经修饰改造的。例如，存在于生物(包括病毒)中的天然的多肽或多核苷酸序列，可分离自天然来源，并且还没有在实验室被有意修饰或以其它方式修饰。
在本申请中，术语“经过改造的”通常指通过人工手段对天然的分子等加以改变。改变的方式可以是对所述分子进行修饰、突变、合成等。“经过改造的”可以区别于天然存在的。例如：如果细胞或生物体经操作使得其基因信息改变(例如引入先前不存在的新基因材 料，例如通过转型、匹配、体细胞杂交、转染、转导或其它机制，或改变或移除先前存在的基因材料，例如通过取代或缺失突变)，那么其被视为“经过改造的”。
在本申请中，术语“大聚合酶蛋白L”可以与“L蛋白”互换使用，通常指水疱性口炎病毒RNA聚合酶蛋白。VSV L基因编码RNA poly E蛋白，L蛋白涉及起始、延伸、甲基化、戴帽、聚A尾形成等过程。
在本申请中，术语“核衣壳蛋白N”可以与“N蛋白”互换使用，通常指水疱性口炎病毒核蛋白。N蛋白可有效保护病毒RNA免受各种核酸酶的消化。N蛋白呈群特异性，为许多型和亚型所共有，有高的抗原性，可以刺激机体产生非中和抗体。
在本申请中，术语“磷蛋白P”又称“P蛋白”，通常指水疱性口炎病毒磷酸蛋白。P基因呈高度不一的磷酸化。P、L、N蛋白-RNA复合物对转录酶活性的发挥是必需的。
在本申请中，术语“基质蛋白M”可以与“M蛋白”互换使用，通常指水疱性口炎病毒基质蛋白。基质蛋白M是VSV重要的毒力因子，也是水疱性口炎病毒中已知可干扰小鼠天然免疫应答的蛋白。
在本申请中，术语“糖蛋白G”可以与“G蛋白”互换使用，通常指水疱性口炎病毒的糖蛋白。
在本申请中，术语“M51R”指的是第51位甲硫氨酸M突变为精氨酸R；术语“ΔL111”指的是第111位亮氨酸缺失。
在本申请中，术语“突变”通常是指野生型分子的核苷酸或氨基酸序列中的变化，例如，野生型CTLA4细胞外结构域的DNA和/或氨基酸序列中的变化。DNA中的突变可以改变密码子，从而导致氨基酸序列中的变化。DNA变化可以包括置换、缺失、插入、可变剪接或截短。氨基酸变化可以包括置换、缺失、插入、添加、截短、或蛋白质的加工或切割错误。可替代地，核苷酸序列中的突变可以导致如本领域充分理解的氨基酸序列中的沉默突变。在那点上，某些核苷酸密码子编码相同的氨基酸。例子包括编码氨基酸精氨酸(R)的核苷酸密码子CGU、CGG、CGC和CGA；或编码氨基酸天冬氨酸(D)的密码子GAU和GAC。因此，蛋白质可以由在其具体核苷酸序列方面不同，但仍编码具有相同序列的蛋白质分子的一种或多种核酸分子编码。
在本申请中，“单碱基编辑(BE)”通常指单碱基转换的基因编辑技术。该技术基于无核酸酶活性的dCas9或有单链DNA切口酶活性的Cas9n(Cas9 nickase)、胞嘧啶脱氨酶、尿嘧啶糖基化酶抑制子(UGI)以及sgRNA形成的复合体，在不引起双链DNA断裂的情况下，实现在一定的活性窗口内C到T到G到A的单碱基精准编辑。
在本申请中，术语“点突变”具有广义和狭义的概念。广义点突变可以是碱基替换、碱基插入或碱基缺失。狭义点突变也称单碱基改变，其可以是单碱基替换。碱基替换又可以分为转换和颠换。
同源重组在本申请中，术语“同源重组”通常指由两个基因组基因座共同拥有的保守区处的核苷酸序列交换。同源重组包括对称同源重组和不对称同源重组。不对称同源重组也称不等重组。
在本申请中，术语“自体细胞”通常指来源于一个个体，后来被重新引入同一个体的细胞。所述个体可以是人，也可以是动物。
在本申请中，术语“异体细胞”通常指来源于一个个体，然后被引入另一个个体的细胞。所述个体可以是人，也可以是动物。
在本申请中，术语“体外扩增”通常指在机体外部的环境下，给予一定的营养和刺激因子促使细胞增殖。
在本申请中，术语“转录”通常指产生起始核酸的核酸拷贝的过程。转录通常涉及DNA核酸的RNA拷贝的生成，并且有时还可以涉及起始RNA核酸的DNA拷贝的生成(例如，反转录)。
在本申请中，术语“翻译”涉及在核糖体处的过程，其中mRNA链控制氨基酸序列的组装以生成蛋白或肽。
在本申请中，术语“过表达”又称“超表达”或“过表达的”，通常指基因以高于正常细胞中基因表达水平的水平表达。
在本申请中，术语“基因编辑”通常指在所靶向的基因组DNA位点处或其附近指导或实现一个或多个核酸的改变(例如缺失)。
在本申请中，术语“类转录因子效应核酸酶”又称“TALEN”，通常指较宽泛的含义，包括可以在没有来自另一TALEN的辅助下切割双链DNA的单体TALEN。术语“TALEN”还指被工程化设计为一起起作用以在相同位点处切割DNA的一对TALEN的一个或两个成员。一起工作的TALENs可以指左TALEN和右TALEN，其涉及DNA或TALEN-对的手性。
已经报道了TAL的密码(PCT公开WO 2011/072246)，其中每个DNA结合重复序列负责识别DNA靶序列中的一个碱基对。残基可经组装，以靶向DNA序列。简而言之，确定TALEN结合的靶位点，创建包含核酸酶和识别靶位点的一系列RVD的融合分子。结合后，核酸酶切割DNA，以便细胞修复机构可以操作，以在切割端进行基因修饰。术语 TALEN指的是包含转录激活因子样(TAL)效应物结合结构域和核酸酶结构域的蛋白质，包括本身具有功能的单体TALEN以及需要与另一单体TALEN二聚化的其它TALEN。当两个单体TALEN相同时，二聚化可得到同型二聚体TALEN，或者，当两个单体TALEN不同时，二聚化可得到异型二聚体TALEN。已经表明，TALEN利用两个主要的真核DNA修复通路，非同源末端连接(NHEJ)和同源介导修复，在永生人类细胞中诱导基因修饰。TALEN通常成对使用，但也已知有单体TALEN。
采用TALEN或其它工具开展的基因修饰可以，例如，选自由插入，缺失，外源核酸片段的插入，和取代组成的组。一般来说，鉴定处DNA靶位点，并创建能够与该位点特异性结合的TALEN对。例如，作为蛋白质、mRNA，或通过编码TALEN的载体，将TALEN递送到细胞或胚胎内。TALEN切割DNA以形成双链断裂，然后对该双链断裂进行修复，这通常导致形成插入缺失，或并入在伴随的外源核酸中所包含的序列或多态性，该外源核酸被插入到染色体中，或起到使用经修饰的序列修复断裂的模板的作用。这种模板驱动的修复是改变染色体的有用的方法，为细胞染色体提供有效的改变。
在本申请中，术语“锌指核酸酶”又称“ZFN”，通常指通过锌指DNA-结合结构域与DNA-切割结构域融合产生的人工限制酶。锌指结构域可以经工程改造，以靶向期望的DNA序列，这样使锌指核酸酶能够靶向复杂基因组内的唯一序列。利用内源DNA修复机构，这些试剂可用于改变高级有机体的基因组。在某些实施方案中，ZFN可在基因失活方法中使用。
锌指DNA结合结构域具有大约30个氨基酸，并折叠成稳定的结构。每个锌指主要与DNA底物内的三联体结合。关键位置处的氨基酸残基有助于与DNA位点的大多数序列特异性相互作用。这些氨基酸可以发生改变，同时使剩余氨基酸保持必要的结构。通过几个结构域串联，可以与更长的DNA序列结合。其它功能，如非特异性FokⅠ切割结构域(N)、转录激活因子结构域(A)、转录抑制因子结构域(R)和甲基酶(M)可以与ZFP融合，以分别形成ZFN、锌指转录激活因子(ZFA)、锌指转录抑制因子(ZFR)和锌指甲基酶(ZFM)。采用锌指锌指核酸酶制造基因修饰的动物的材料和方法在，例如，专利US8106255；US2012/0192298；US2011/0023159和US2011/0281306中进行了公开。
在本申请中，术语“敲除”通常指一种或多种基因的基因表达被清除。
在本申请中，术语“试剂盒”通常是指所述组分的集合，所述组分可或可不包装，可或可不包装在一起。试剂盒的组分可包含在分开的容器中(即具有分开部分的试剂盒)，可在单个容器内提供，可不放置在容器内。另外，试剂盒可以包含实施所述方法的说明书。说明 书可通过纸或电子形式的用户手册提供。例如，手册可包含用于解释当使用本申请的试剂盒实施上述方法时得到的结果的说明。
在本申请中，术语“工具”通常指能够制备目的分子所需要的元件、组分、试剂、分子等。
在本申请中，术语“受试者”通常是指需要诊断、预后、改善、预防和/或治疗疾病的人或非人动物(包括哺乳动物)，特别是需要所述溶瘤病毒表达载体的受试者。在一些实施方式中，所述受试者可以包括癌症患者。在某些实施方式中，所述受试者是人或非人哺乳动物。非人哺乳动物可以以包括任何除人之外的哺乳动物物种，例如家畜动物(例如，牛、猪、羊、鸡、兔或马)，或啮齿类动物(例如，大鼠和小鼠)，或灵长类动物(例如，大猩猩和猴子)，或家养动物(例如，狗和猫)。“受试者”可以是雄性或者雌性，也可以是不同年龄阶段。人类“受试者”可以是高加索人、非洲人、亚洲人、闪族人，或其他种族，或各种族的杂合体。人类“受试者”可以是老年、成年、青少年、儿童或者婴儿。
在本申请中，术语“药物组合物”通常指以允许活性成分的生物学活性有效的形式的制剂，并且其不含有对所述制剂待施用的受试者有不可接受的毒性的另外成分。在某些实施方案中，这些制剂可以包含药物的活性组分以及药学上可接受的载剂。在某些实施方式中，所述药物组合物包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中的药物组合物。药物组合物可以通过不同方式给药，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
在本申请中，术语“药学上可接受”通常是指在合理医学判断的范围内，适宜用于与人和动物的组织接触而无过度的毒性、刺激、过敏反应或者其他问题或并发症，具有合理的收益/风险比的佐剂。在一些实施方式中，药学上可接受的佐剂可以指由管理机构批准(如美国食品药品管理局、中国食品药品管理局或欧洲药品局)或者列于普遍认可的药典中(如美国药典、中国药典或欧洲药典)的用于动物(更特别地用于人)的那些佐剂。在本申请中，药学上可接受的佐剂可以是基于铝的佐剂、矿物盐佐剂、张力活性佐剂、细菌来源的佐剂、乳化佐剂、脂质体佐剂、细胞因子佐剂、糖类佐剂和DNA和RNA寡聚体佐剂及其他。
在本申请中，术语“有效量”或“有效剂量”可互换使用，通常指足够获得或至少部分获得预期疗效的量。术语“治疗有效量”通常是指在一个已经患病的受试者中足够治愈或至少部分阻止该病及其并发症的量。对于这种用途有效的量将依赖于感染的严重程度和病人自身免疫系统的整体状态。
在本申请中，术语“联用”又称“共同施用”，通常指在另一个药物成分之前，或之后，或同时给药。联用的两个或多个药物成分可以是通过相同的给药方式给药，也可以是通过不同给药方式给药，可以是同时施用，也可以是先后施用。例如，联用的一种治疗剂可以是所述溶瘤病毒，第二种治疗剂可以是所述经修饰的免疫效应细胞。联用也可以包含第三种甚至更多种治疗剂。
在本申请中，术语“治疗”通常指消除或改善疾病，或与疾病相关的一种或多种症状。在某些实施方案中，治疗通常指向患有这种疾病的患者施用一种或多种药物使得疾病消除或缓解。在某些实施方案中，“治疗”可以是在特定疾病的症状发作后，在其他药物存在或不存在的情况下施用所述组合物。
在本申请中，术语“载剂”通常指药物制剂中除了活性组分以外，对受试者无毒的组分。在某些实施方案中，本申请的组合物、药物组合物或试剂盒中的药学上可接受的载剂可以包括但不限于，例如，药学可接受的液体、凝胶或固体载剂、水相介质(例如，氯化钠注射液、林格氏液注射液、等渗葡萄糖注射液、无菌水注射液、葡萄糖或乳酸林格注射液)、非水相介质(例如，植物来源的不挥发性油、棉花子油、玉米油、芝麻油或者花生油)、抗微生物物质、等渗物质(例如，氯化钠或葡萄糖)、缓冲液(例如，磷酸盐或枸橼酸盐缓冲液)、抗氧化剂(例如，硫酸氢钠)、麻醉剂(例如，盐酸普鲁卡因)、悬浮剂/分散剂(例如，羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮)、螯合剂(例如，EDTA(乙二胺四乙酸)或EGTA(乙二醇双(2-氨基乙基醚)四乙酸))、乳化剂(例如，聚山梨醇酯80(吐温-80))、稀释剂、佐剂、辅料、无毒辅助物质、其他本领域公知的组分或以上的多种组合。适用的组分可包括，例如，填充剂、粘合剂、崩解剂、缓冲液、防腐剂、润滑剂、搅味剂、增稠剂、着色剂或乳化剂。
在本申请中，术语“肿瘤”通常是指任何新的病理性的组织增生。肿瘤可能是良性的，如血管瘤、胶质瘤、畸胎瘤等，也可能是恶性的，如腺癌、肉瘤、胶质母细胞瘤、星形细胞瘤、神经母细胞瘤、视网膜母细胞瘤等。用于研究时，可通过本领域技术人员熟知的方法从易于获得的资源中将这些组织分离出来。
在本申请中，术语“Cas9”或“Cas9分子”是指来自细菌II型CRISPR/Cas系统的负责DNA切割的酶。Cas9还包括野生型蛋白质及其功能性和非功能性突变体。在某些实施方案中，Cas9可以为酿脓链球菌的Cas9。
在本申请中，术语“CTLA-4”又称细胞毒性T淋巴细胞抗原-4，通常指属于CD28家族的免疫抑制受体。CTLA-4在体内专门表达于T细胞上，并结合两种配体，CD80和CD86 (也分别称为B7-1和B7-2)。完整hCTLA-4序列可见于GenBank登录号AAB59385下。例如，人CTLA-4的氨基酸序列可如SEQ ID NO:12所示。
术语“LAG-3”也称“CD223”，通常指淋巴细胞活化基因3。术语“人类LAG-3”指人类序列LAG-3，如具有Genbank登录号NP_002277的人类LAG-3的完整氨基酸序列。
在本申请中，术语“PD-1”通常是指程序性细胞死亡1，也称为“程序性死亡1”、“CD279”、“分化簇279”、“PD1”、“PDCD1”。PD-1通常在T细胞、B细胞、自然杀伤T细胞、活化的单核细胞和树突细胞(DC)上表达，并参与细胞凋亡。PD-1通常包含一个细胞外IgV结构域，跨膜区和胞内结构域。PD-1可结合两种配体，PD-L1和PD-L2。人PD-1氨基酸序列可见于GenBank登录号NP_005009.2下。例如，人PD-1氨基酸序列可如SEQ ID NO:11所示。
在本申请中，术语“PD-L1”通常指程序性死亡配体1，又称为分化簇274(CD274)或B7同源物1(B7-H1)。PD-L1是PD-1的两种细胞表面糖蛋白配体之一。PD-1受体/PD-L1配体复合物可以下调T细胞活化和细胞因子分泌。完整hPD-L1序列可见于GenBank登录号Q9NZQ7下。
在本申请中，术语“TIGIT”通常指具有Ig和ITIM域的T细胞免疫受体。其可以来自任何脊椎动物来源，包括哺乳动物诸如灵长类(例如人)和啮齿类(例如小鼠和大鼠)的任何天然TIGIT。TIGIT在本领域也称作DKFZp667A205，FLJ39873，含有V集和免疫球蛋白域的蛋白9，含有V集和跨膜域的蛋白3，VSIG9，VSTM3，和WUCAM。一种人TIGIT的氨基酸序列可以见UniProt登录号Q495A1。
在本申请中，术语“TIM-3”通常指含有T细胞免疫球蛋白和粘蛋白结构域的分子3。TIM-3的天然配体是半乳糖凝集素9(Gal9)。
在本申请中，术语“BTLA”通常指B和T淋巴细胞衰减剂。在具体实施方式中，BTLA可以为人BTLA。
在本申请中，术语“VISTA”通常指T细胞活化V结构域Ig抑制蛋白。在某些实施方式中，VISTA可以为人VISTA，其通常指具有人氨基酸序列的VISTA蛋白。其氨基酸序列可见于GenBank登录号NP_071436下。
发明详述
组合物中的溶瘤病毒
在本申请中，所述溶瘤病毒可以从目前鉴定的任何病毒成员获得。与非分裂细胞相比，所述溶瘤病毒在分裂细胞中具有更高的复制并具有杀灭细胞的倾向性。本申请所述溶瘤病毒 包括具有溶瘤作用的经改造的病毒和具有溶瘤作用的野生型病毒，其包括但不限于：水疱性口炎病毒、痘病毒、逆转录病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、脊髓灰质炎病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒、马拉巴病毒，α病毒、慢病毒、流感病毒、Sinbis病毒、粘液瘤病毒、弹状病毒、小核糖核酸病毒、细小病毒等。
在某些实施方案中，所述溶瘤病毒也可以是衍生自例如单纯疱疹病毒、牛痘病毒、水疱性口炎病毒、自主细小病毒、粘液瘤病毒、新城疫病毒、呼肠孤病毒、塞内卡谷病毒、麻疹病毒、逆转录病毒、流感病毒、辛德比斯病毒或痘病毒。其中“衍生自”参考病毒的溶瘤病毒包含所述参考病毒拥有的核酸序列或氨基酸序列。在某些实施方案中，“衍生自”参考病毒的溶瘤病毒包含所述参考病毒拥有的一个或多个基因。在某些实施方案中，“衍生自”参考病毒的溶瘤病毒编码所述参考病毒编码的一种或多种蛋白质。
在某些实施方案中，衍生自参考病毒的溶瘤病毒可包含编码所述参考病毒的一个或多个功能元件的核酸序列。“功能元件”可以是例如转录调节子(例如启动子/增强子)、转录后加工的调节子、翻译调节子、转录后加工的调节子、响应元件、重复序列或病毒蛋白。
所述溶瘤病毒可以是天然的溶瘤病毒，也可以是经过改造的溶瘤病毒。其中所述经过改造的溶瘤病毒可以是经过基因水平改造的溶瘤病毒，如经过一个或多个基因的修饰来改造，从而提高其肿瘤选择性和/或在分裂细胞中优先复制。所述基因水平的改造可以为修饰参与DNA复制、核酸代谢、宿主向性、表面附着、毒力、裂解和扩散过程的基因，也可以为整合外源基因的改造，所述外源基因包括外源免疫调节基因、外源筛选基因、外源报告基因等。所述经过改造的溶瘤病毒也可以是经过氨基酸水平改造的溶瘤病毒，如一个或多个氨基酸的插入、缺失、替换。所述经过改造的溶瘤病毒也可以是蛋白表达水平和活性与野生型相比发生改变的溶瘤病毒。
在某些实施方案中，本申请的溶瘤病毒选自水疱性口炎病毒(VSV)，水疱性口炎病毒是一种原型非节段负链RNA病毒，其大小11kb基因组编码5种蛋白：核衣壳蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)和大聚合酶蛋白(L)。具体地，例如，所述水疱性口炎病毒可以为水疱性口炎病毒印第安纳株，VSV MuddSummer亚型，其编码M蛋白的基因序列如SEQ ID NO：3所示，M蛋白的氨基酸序列如SEQ ID NO：4所示。
在某些实施方案中，所述M蛋白可以为天然的未经修饰的，可以为天然突变的，也可以为人工改造发生突变的。
例如，VSV可以为野生型，未经修饰。
例如，VSV M蛋白可以进行如下修饰：M51R。
例如，VSV M蛋白可以进行如下修饰：M51R且ΔL111。
例如，VSV M蛋白可以进行如下修饰：M51R、V221F且S226R。
例如，VSV M蛋白可以进行如下修饰：M51R、ΔL111、V221F且S226R。
在某些实施方案中，编码VSV M蛋白(M51R、V221F且S226R)的核苷酸序列如SEQ ID NO：5所示，VSV M蛋白(M51R、V221F且S226R)的氨基酸序列如SEQ ID NO：6所示。
在某些实施方案中，编码VSV M蛋白(M51R、ΔL111、V221F且S226R)的核苷酸序列如SEQ ID NO：7所示，VSV M蛋白(M51R、ΔL111、V221F且S226R)的氨基酸序列如SEQ ID NO：8所示。
在某些实施方案中，所述水疱性口炎病毒基质蛋白M的突变位点还可以为单个位点的突变，例如：ΔM51；M51A；ΔL111；V221F；S226R。
在某些实施方案中，所述水疱性口炎病毒基质蛋白M的突变位点还可以为多个位点的组合突变，例如：M51R且V221F；M51R且S226R；ΔL111且V221F；ΔL111且S226R；V221F且S226R；M51R、ΔL111且V221F；M51R、ΔL111且S226R；ΔL111、V221F且S226R。
在某些实施方案中，所述水疱性口炎病毒基质蛋白M的突变位点还可以为连续位点的突变，例如：M51-54A；ΔM51-54；ΔM51-57。
在某些实施方案中，所述水疱性口炎病毒基质蛋白M的突变位点还可以为连续位点和单个位点突变的组合，例如：M51-54A、V221F且S226R；ΔM51-54、V221F且S226R；ΔM51-57、V221F且S226R。
组合物中的经修饰的免疫效应细胞
免疫效应细胞通常是指参与免疫应答，行使效应功能的免疫细胞。本申请所述的免疫效应细胞可以为已知的各种类型的免疫效应细胞，并且可以通过常规的生物学方法得到，其包括但不限于：淋巴细胞，如T细胞、B细胞、NK细胞；骨髓细胞，如单核细胞、巨噬细胞、嗜酸性细胞、肥大细胞、嗜碱性细胞、嗜中性粒细胞和树突状细胞。
在某些实施方案中，所述T细胞包括但不限于：辅助CD4+T细胞、调节CD4+T细胞、杀手CD8+T细胞、记忆T细胞、自然杀伤T细胞、粘膜相关恒定T细胞、γδT细胞。
例如，所述T细胞可以为人外周血T细胞。
在本申请中，所述免疫效应细胞可以为自体细胞，也可以为异体细胞。所述免疫效应细 胞可以为经体外扩增得到的免疫效应细胞。免疫效应细胞的大规模体外扩增培养技术是已知的。
在本申请中，所述免疫检查点可以包括PD-1、PD-L1、CTLA-4、LAG-3、TIM-3、BTLA、VISTA、TIGIT、B7-H2、B7-H3、B7-H4和B7-H6。
在某些实施方案中，所述免疫检查点表达量和/或活性的调整可以包括一个免疫检查点的调整，例如：PD-1的调整、PD-L1的调整、CTLA-4的调整、LAG-3的调整、TIM-3的调整、BTLA的调整、VISTA的调整、TIGIT的调整、B7-H2的调整、B7-H3的调整、B7-H4的调整和B7-H6的调整。
在某些实施方案中，所述免疫检查点表达量和/或活性的调整可以包括两个至多个免疫检查点的组合的调整，例如：PD-1和PD-L1；PD-1和CTLA-4；PD-1和LAG3；PD-L1和CTLA-4；LAG-3和CTLA-4；TIM-3和CTLA-4；VISTA和CTLA-4；PD-1、CTLA-4和PD-L1。
在某些实施方案中，其中所述免疫检查点表达量的调整包含与未经修饰的免疫效应细胞相比，所述免疫检查点表达量的上调、下调和/或缺失。
在某些实施方案中，其中所述免疫检查点活性的调整包含与未经修饰的免疫效应细胞相比，所述免疫检查点活性的增加、降低和/或缺失。
在某些实施方案中，其中所述免疫检查点表达量和/或活性的调整可以为所述免疫检查点表达量和/或活性的上调、下调或缺失；也可以为其中一个以上调整的免疫检查点表达量和/或活性的上调以及其余调整的免疫检查点的表达量和/或活性的下调和/或缺失。例如：PD-1的表达量和/或活性的上调和CTLA-4表达量和/或活性的下调，PD-L1的表达量和/或活性的上调、CTLA4的表达量和/或活性的下调和LAG-3的表达量和/或活性的缺失。
在本申请中，所述免疫检查点的调整包含免疫检查点基因水平、转录水平和/或翻译水平的调控。
在某些实施方案中，可以使用其中一种方法来进行调控，也可以使用两种以上方法来进行调控。
在某些实施方案中，所述基因水平的调控方法包括例如：基因编辑、过表达、点突变、同源重组。所述基因编辑方法包括例如：CRISPR/Cas9、类转录激活因子效应核酸酶(TALEN)、锌指核酸酶(ZFN)和单碱基编辑(BE)。
在一个实施方案中，所述免疫检查点的调整为T细胞的PD-1分子的表达被降低，具体方法为使用CRISPR/Cas9基因编辑技术对人外周血T细胞的PDCD1基因进行敲除，降低PD-1分子的表达。
在某些实施方案中，所述CRISPR/Cas9基因编辑技术可以包含设计序列的sgRNA。所述sgRNA可以与Cas9蛋白结合识别剪辑目的基因。
在某些实施方案中，所述免疫效应细胞敲除针对选自SEQ ID NO：1的PD-1基因的靶序列。
在某些实施方案中，所述免疫效应细胞敲除针对选自SEQ ID NO：2的CTLA-4基因的靶序列。
组合物、药物组合物和试剂盒
在本申请中，提供了一种组合物，其包含：(a)所述溶瘤病毒；以及(b)所述经修饰的免疫效应细胞，其中所述经修饰的免疫效应细胞与未经所述修饰的免疫效应细胞相比，其免疫检查点的表达量和/或活性被调整。
在本申请中，所述组合物可以包含经改造的溶瘤病毒和经修饰的免疫细胞，例如，所述经改造的溶瘤病毒包含经改造的VSV，所述VSV的基质蛋白M与SEQ ID NO:4所示的氨基酸序列相比，包含M51R，V221F和S226R的氨基酸突变，且所述经修饰的免疫细胞包含PD-1基因敲除的T细胞。在某个实施方式中，在所述组合物中，所述经改造的溶瘤病毒的基质蛋白M包含SEQ ID NO：6所示的氨基酸序列，且所述经修饰的免疫细胞包含PD-1基因敲除的T细胞。
在本申请中，所述组合物可以包含经改造的溶瘤病毒和经修饰的免疫细胞，例如，所述经改造的溶瘤病毒包含经改造的VSV，所述VSV的基质蛋白M与SEQ ID NO:4所示的氨基酸序列相比，包含M51R，ΔL111，V221F和S226R的氨基酸突变，且所述经修饰的免疫细胞包含PD-1基因敲除的T细胞。在某个实施方式中，在所述组合物中，所述经改造的溶瘤病毒的基质蛋白M包含SEQ ID NO：8所示的氨基酸序列，且所述经修饰的免疫细胞包含PD-1基因敲除的T细胞。
在本申请中，还提供了一种药物组合物，其包含：(a)所述的至少一种溶瘤病毒和/或药学上可接受的载剂；以及(b)所述的至少一种经修饰的免疫效应细胞和/或药学上可接受的载剂。
在本申请中，还提供了一种试剂盒，其包含选自下组中的一种或多种：(a)所述的至少一种溶瘤病毒和制备所述的至少一种修饰的免疫效应细胞的工具，和/或药学上可接受的载剂；(b)所述的至少一种修饰的免疫效应细胞和制备所述的至少一种溶瘤病毒的工具，和/或药学上可接受的载剂；以及，(c)制备所述的至少一种溶瘤病毒的工具和制备所述的至少一种修饰的免疫效应细胞的工具和药学上可接受的载剂。
在某些实施方案中，所述试剂盒中制备所述的至少一种修饰的免疫效应细胞的工具指的是任何个体可以通过使用该所述工具获得所述修饰的免疫效应细胞，例如获得PD-1分子敲除的T细胞使用的设计序列的sgRNA。所述试剂盒中制备所述的至少一种溶瘤病毒的工具指的是任何个体可以通过使用该所述工具获得所述溶瘤病毒。例如质粒、各突变位点的引物。
在某些实施方案中，所述组合物、药物组合物和试剂盒可以被配制成用于临床用途的药物，并且可以包含药学上可接受的载体、稀释剂、赋形剂或佐剂。所述组合物、药物组合物和试剂盒可以被配制用于局部、肠胃外、全身、腔内、静脉内、动脉内、肌肉内、鞘内、眼内、结膜内、瘤内、皮下、皮内、鞘内、口服或透皮给药途径，其可包括注射或输注。合适的制剂可以在无菌或等渗培养基中包含病毒和/或细胞。组合物、药物组合物和试剂盒还可以配制成流体，包括凝胶形式。可配制流体制剂用于通过注射或输注(例如通过导管)施用于人体或动物体的选定区域。
方法、应用、用途
在本申请中，提供了所述组合物、所述药物组合物、所述试剂盒的制备方法。
在某些实施方式中，所述组合物、所述药物组合物、所述试剂盒中的各组分可以在同一个容器内，也可以放置在不同容器内。
在某些实施方案中，所述溶瘤病毒可以被配制用于肿瘤内施用。
在某些实施方案中，所述溶瘤病毒和所述免疫效应细胞可以被配制用于静脉内给药。
在某些实施方式中，所述免疫效应细胞可以被配制用于局部给药。
在本申请中，还提供了一种治疗肿瘤的方法，其包含向有需要的受试者施用所述的组合物、所述的药物组合物和/或所述的试剂盒。
在某些实施方式中，可以向有需要的受试者施用所述组合物、所述药物组合物和所述试剂盒中的一种或多种。
在某些实施方式中，所述溶瘤病毒和所述经修饰的免疫细胞同时施用。同时施用可以是各组分混合在一起施用，也可以是分开施用。可以以相同方式施用，例如施用于同一条静脉或其他血管，也可以是不同方式施用，例如静脉内施用和瘤内施用同时进行。
在某些实施方式中，所述溶瘤病毒和所述经修饰的免疫细胞先后施用。施用的顺序可以是先施用所述溶瘤病毒，再施用所述免疫效应细胞；也可以是先施用所述免疫效应细胞，再施用所述溶瘤病毒。可以以相同方式施用，也可以以不同方式施用。各组分可以一次施用，也可以分多次施用。在某些实施方案中，先后施用可以是以任何时间间隔施用，包括分钟、小时、天、周、月或年。在某些实施方案中，先后施用是指以至少2分钟、5分钟、10分钟、 30分钟、1小时、6小时、8小时、12小时、24小时、36小时、48小时、3天、4天、5天、6天、1周、2周、3周、1个月、6周、2个月、3个月、4个月、5个月或6个月之一的时间间隔分开的施用。
在某些实施方案中，施用的剂量为治疗有效量。
在本申请中，还提供了所述溶瘤病毒在所述溶瘤病毒和所述经修饰的免疫效应细胞的联用中的应用。即用于所述溶瘤病毒和所述经修饰的免疫联合应用的所述溶瘤病毒。
在本申请中，还提供了所述经修饰的免疫效应细胞在所述溶瘤病毒和所述经修饰的免疫细胞联用中的应用。即用于所述溶瘤病毒和所述经修饰的免疫联合应用的所述经修饰的免疫细胞。
另一方面，本申请还提供了所述组合物、所述药物组合物、所述试剂盒在制备治疗肿瘤的药物中的应用。
另一方面，本申请还提供了所述溶瘤病毒和所述经修饰的免疫效应细胞，其用于治疗肿瘤。
在某些实施方案中，所述肿瘤包含实体瘤和/或血液瘤。
在某些实施方案中，所述肿瘤包括但不限于：头颈部癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤、滑膜肉瘤和圆细胞型脂肪肉瘤。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1：水疱性口炎病毒突变株的构建及效果展示
1、按表1的方式，对所述水疱性口炎病毒印第安纳株基质蛋白M进行定点突变，获得3组突变后的突变株。未进行基因突变的组别编号为：JBS000，作为对照。
表1：各组突变情况展示表
Figure PCTCN2021119114-appb-000001
Figure PCTCN2021119114-appb-000002
突变株的具体构建方法为本领域的常规技术，方法如下：
(1)构建质粒。以pVSV-XN2质粒为模板，利用PCR方法引入如表1所述的不同的突变位点。将质粒与各突变位点的引物共同进行PCR，再将PCR产物进行1％琼脂糖凝胶电泳，随后通过凝胶回收试剂盒进行割胶回收，获得M基质蛋白不同突变的质粒。
(2)病毒拯救。按MOI＝5，用表达T7RNA聚合酶的痘病毒vTF7-3感染接种BHK-21细胞，感染1h后，使用DPBS缓冲液漂洗一次BHK-21细胞。随后制备质粒转染预混液，具体包括：pBS-N、pBS-P、pBS-L、步骤(1)制备的突变质粒。其中pBS-N、pBS-P、pBS-L分别指克隆了VSV N、VSV P、VSV L蛋白基因的表达质粒，分别表达病毒挽救所需的N、P和L蛋白。按lipofectamine 2000使用说明书所述的方法进行质粒转染，4h后更换新鲜的含10％胎牛血清的DMEM完全培养基，48h后吸取上清，用0.22μm的滤膜去除痘病毒。将滤液加入到新鲜的BHK-21细胞中；每天观察细胞病变情况，待细胞出现病变情况时收取上清，经RTPCR鉴定确认成功后，用病毒空斑实验纯化病毒。即获得突变株。
(3)M蛋白基因测序。利用Trizol试剂盒抽提病毒基因组RNA，用随机引物进行逆转录反应，用针对M蛋白基因序列设计的引物对逆转录出来的cDNA进行PCR。引物序列为：5'-AAAAAAGTAACAGATATCAC-3'(SEQ ID NO:9)；5'-ACATTTTTCCAGTTTCCTTTTTGG-3'(SEQ ID NO:10)。产物经1％琼脂糖凝胶电泳后回收，并送往测序公司进行测序。
2、不同突变株对细胞的体外侵染能力展示。
不同突变株对细胞的体外侵染能力展示。分别在MEF细胞(人的成纤维细胞)培养液、LLC细胞(鼠源非小细胞肺癌细胞)培养液中，分别加入JBS000、JBS001、JBS002、JBS003、JBS004各200pfu，检测各组突变株产生的半数组织培养感染剂量(Tissue culture infective dose,TCID50)。具体测试方法为：
(1)在6孔培养板中每孔加入细胞悬液3mL，使细胞量达到4×10 5个/孔，共6个孔，37℃、5％CO2条件下培养16h。
(2)在各孔中分别加入病毒JBS000、JBS001、JBS002、JBS003、JBS004各200pfu，设正常细胞对照2个孔。在24h时，取细胞上清100μL。
(3)在96孔培养板中，每孔加入Vero细胞悬液100μL，使细胞量达到1×10 4个/mL，37℃、5％CO 2条件下培养16h。
(4)在1.5mLEP管中将步骤(2)中收获的上清作连续10倍的稀释，10 -1～10 -11，共11个滴度。
(5)将稀释好的上清接种到步骤(3)的96孔培养板中，每一稀释度接种一列(共8孔)，每孔接种100μL。设正常细胞对照组一列。
(6)48h后观察每孔细胞荧光情况，有荧光则记为此孔被感染。
(7)按Karber法计算TCID50。
结果如图1所示，结果显示：VSV突变株在体外肺癌细胞(LLC)中的复制扩增能力显著提高，而在正常的成纤维细胞(MEF)内的复制感染能力均显著下降。因此：突变株对肿瘤细胞具有特异性侵染能力。
3、不同突变株对细胞的体外杀伤能力展示。使用等量各突变株分别体外感染不同细胞，24h后MTT法检测细胞活力。具体方法如下：
(1)在96孔培养板中每孔加入细胞悬液100μL，使细胞量达到1×10 4个/孔，37℃，5％CO 2条件下培养16h。测试的细胞种类为：LLC、MEF、Hela(人肿瘤细胞)。
(2)分别将JBS000、JBS001、JBS002、JBS003、JBS004稀释到MOI(multiplicity of infection，感染复数)分别为0.001、0.01、0.1、1.0，每一稀释梯度接种4个孔，每孔接种100μL，在37℃，5％CO 2条件下培养40h。
(3)弃去96孔培养板中的上清，加入新鲜的DMED培养基，再加入5mg/mL的MTT溶液，20μL/孔。在37℃，5％CO 2条件下培养4h。
(4)将96孔板离心，设置转速2500g/min，室温离心5分钟。随后使用1mL一次性无菌注射器，轻轻吸掉上清。
(5)再向每孔中加入DMSO，100μL/孔，37℃放置10分钟。
(6)使用多功能酶标仪，震荡2分钟，在570nm波长下，测定各孔的OD值。
结果如图2所示，结果显示：所有突变株都展现了良好的肿瘤细胞杀伤的能力，除JBS000外，均对MEF细胞无显著的杀伤。即体外状态下，除JBS000外，各减毒株均对肿瘤细胞具 有特异性杀伤，对正常细胞无显著影响。
4、不同突变株在细胞内清除难易度测试
以IFN-β指标进行测试。按上述步骤3的步骤(1)、(2)培养细胞并加入突变株。随后破碎各组细胞，用TRIzol(Invitrogen)从各细胞中提取总RNA，利用PrimeScript RT Reagent Kit with DNA Eraser(Takara)反转录试剂盒逆转录成cDNA，并用LightCycler 480SYBR Green I Master(Roche)染料进行染色，在LightCycler 480定量PCR仪上检测各个基因的Ct值。用ΔΔCt法计算目的基因IFN-β、VSV-G相对表达量。
结果如图3所示，结果显示：在LLC细胞系中，除JBS000外，所有突变株均可引起IFN-β表达水平的提高；而在MEF细胞中，所有病毒均可上调IFN-β的表达水平。
实施例2：CRISPR/Cas9技术敲除T细胞PD-1分子
1、PD-1-sgRNA-CRISPR/Cas9质粒构建
根据人PD-1分子编码基因PDCD1的结构，针对其第二外显子设计2条sgRNA，构建携带有识别人PDCD1基因的sgRNA及Cas9酶的融合质粒。融合质粒元件示例如图4所示。
2、人外周血T细胞表面表达PD-1分子检测
利用梯度离心分离新鲜的人外周血PBMC，取1×10 6个PBMC细胞流式检测T细胞表面PD-1分子的表达水平，结果显示人外周血T细胞表面表达PD-1分子，如图5所示，表明人外周血T细胞可以作为分析PDCD1基因敲除效果的工具细胞。
3、PD-1-sgRNA-CRISPR/Cas9系统下调人外周血T细胞表面PD-1分子的表达
将PD-1-sgRNA-CRISPR/Cas9质粒电转入新鲜分离的人外周血PBMC细胞，电转后的PBMC细胞用CD3和CD28抗体激活，并在37℃5％CO 2条件下培养，电转后24h和第8天分别取培养细胞检测T细胞表面PD-1分子的表达情况。
结果显示：电转后的24h的PDCD1基因敲除组细胞表面PD-1分子表达下调不明显，培养至第8天时敲除组相较于阴性对照组，细胞表面PD-1分子表达下调显著，如图5所示。表明使用CRISPR/Cas9技术敲低了PDCD1基因，且能使人外周血T细胞表面PD-1分子表达持续下调。图5中，未编辑组指的是未进行基因敲除组的细胞，编辑组指的是进行基因敲除组的细胞。
实施例3：CRISPR/Cas9技术敲除T细胞PD-1分子和CTLA-4分子
1、PD-1和CTLA-4双基因敲除载体的构建
根据人PD-1和CTLA-4基因的序列，设计gRNA序列。用BbsⅠ酶切pU6gRNA-CMV- Cas9-GFP表达载体，回收后与PD-1oligo序列形成的oligo双链连接，构建含有一个gRNA的PD-1基因敲除载体。
以pU6gRNA-CMV-Cas9表达载体为模板，用引物将表达载体线性化，断裂点位于gRNA和CMV启动子序列之间，并在两段添加HindⅢ和EcoRⅠ酶切位点。然后以pU6gRNA-CMV-Cas9-GFP表达载体为模板，用引物克隆gRNA表达单元，并连入pUC19，然后通过BbsⅠ酶切连入CTLA-4oligo序列获得中间载体。用HindⅢ和EcoRⅠ双酶切线性化的pU6gRNA-CMV-Cas9-GFP表达载体和中间载体，回收后将二者连接，获得可以表达两个不同target序列gRNA的敲除载体pU6gRNA-Cas9-GFP。
2、pU6gRNA-Cas9-GFP系统下调人外周血T细胞表面PD-1分子和CTLA-4的表达
将pU6gRNA-Cas9-GFP质粒电转入新鲜分离的人外周血T细胞，用CD3和CD28抗体激活，并在在37℃5％CO2条件下培养，电转后第8天取细胞进行流式检测T细胞表面PD-1和CTLA-4分子的表达情况。
结果显示：gRNA及Cas9融合电转质粒均使T细胞表面PD-1分子和CTLA-4分子表达下调，表明gRNA均能有效敲低T细胞中PD-1和CTLA-4基因的表达。
实施例4：经改造的VSV的体外肿瘤杀伤力检测
溶瘤病毒体外抗肿瘤实验展示。使用JBS003溶瘤病毒感染肿瘤细胞，48h后使用CCK8法检测肿瘤细胞的杀伤率。具体方法如下：
(1)在96孔培养板中每孔加入A549肿瘤细胞株(人非小细胞肺癌)悬液100μL，使细胞量达到4×10 3个/孔，37℃，5％CO 2条件下培养16h。
(2)将溶瘤病毒按感染复数(multiplicity of infection，MOI)稀释至0.001、0.01、0.1和1.0和10.0共五个梯度；每孔接种溶瘤病毒100μL，每一稀释梯度均接种4个复孔，在37℃，5％CO2条件下培养48h。
(3)弃去96孔培养板中的上清，加入新鲜的DMED培养基(100μL/孔)，再加入CCK8溶液(10μL/孔)。在37℃，5％CO2条件下培养4h。
(4)使用多功能酶标仪，震荡2分钟，在450nm波长下，测定各孔的OD值并计算肿瘤杀伤率。
(5)肿瘤杀伤率计算公式：肿瘤细胞杀伤率(％)＝(肿瘤细胞对照组OD值－实验组OD值)/肿瘤细胞对照组OD值×100％。
溶瘤病毒的体外抗肿瘤实验结果如图6所示，结果显示，溶瘤病毒具有一定的抗肿瘤能力。
实施例5：敲除PD-1分子的T细胞的体外肿瘤杀伤力检测
使用PD-1敲除的T细胞与肿瘤细胞混合培养48小时后，CCK8法检测PD-1敲除的T细胞对肿瘤细胞杀伤率。具体方法如下：
(1)在96孔培养板中每孔加入A549肿瘤细胞株(人非小细胞肺癌)悬液100μL，使细胞量达到4×10 3个/孔，37℃，5％CO 2条件下培养16h。
(2)将PD-1敲除的T细胞按效靶比(E:T)分别稀释至1:1、5:1、10:1和20:1共四个梯度；每孔接种PD-1敲除的T细胞100μL，每一稀释梯度均接种4个复孔，在37℃，5％CO2条件下培养48h。
(3)弃去96孔培养板中的上清和淋巴细胞，加入新鲜的DMED培养基(100μL/孔)，再加入CCK8溶液(10μL/孔)。在37℃，5％CO 2条件下培养4h。
(4)使用多功能酶标仪，震荡2分钟，在450nm波长下，测定各孔的OD值并计算肿瘤杀伤率。
(5)肿瘤杀伤率计算公式：肿瘤细胞杀伤率(％)＝(肿瘤细胞对照组OD值－实验组OD值)/肿瘤细胞对照组OD值×100％。
PD-1分子敲除的T细胞的体外抗肿瘤结果如图8的部分结果所示，结果显示，PD-1分子敲除的T细胞具有较好的抗肿瘤能力。
实施例6：经改造的VSV和敲除PD-1分子的T细胞联合治疗黑色素瘤(移植瘤)的肿瘤杀伤力检测
选择差异不显著的96只C57BL/6小鼠，分别皮下接种2×10 6个B16-F10-NY-ESO-1黑色素瘤细胞。接种第9天，待移植瘤体积长至100mm 3左右时，将所有小鼠分为12组：对照组(PBS组)瘤内注射50μL PBS，其余11组为治疗组，分别进行瘤内接种JBS000、JBS001、JBS002、JBS003、JBS004、敲除PD-1的T细胞、JBS000和敲除PD-1的T细胞、JBS001和敲除PD-1的T细胞、JBS002和敲除PD-1的T细胞、JBS003和敲除PD-1的T细胞、JBS004和敲除PD-1的T细胞。每2天给药1次，共给药3次，单次接种量均为10 8pfu/只。自开始给药至实验终点，每2天记录一次移植瘤体积，体积(mm 3)＝(长径×短径 2)/2。
结果显示：与对照组相比，各治疗组对黑色素瘤均有一定的治疗效果。
实施例7：经改造的VSV和敲除PD-1分子的T细胞联合治疗纤维肉瘤(移植瘤)的肿瘤杀伤力检测
选择差异不显著的96只C57BL/6小鼠，分别皮下接种2×10 6个MCA-205-NY-ESO-1纤 维肉瘤细胞。接种第9天，待移植瘤体积长至100mm 3左右时，将所有小鼠分为12组：对照组(PBS组)瘤内注射50μL PBS，其余11组为治疗组，分别进行瘤内接种JBS000、JBS001、JBS002、JBS003、JBS004、敲除PD-1的T细胞、JBS000和敲除PD-1的T细胞、JBS001和敲除PD-1的T细胞、JBS002和敲除PD-1的T细胞、JBS003和敲除PD-1的T细胞、JBS004和敲除PD-1的T细胞。每2天给药1次，共给药3次，单次接种量均为10 8pfu/只。自开始给药至实验终点，每2天记录一次移植瘤体积，体积(mm 3)＝(长径×短径 2)/2。
结果显示：与对照组相比，各治疗组对纤维肉瘤均有一定的治疗效果。
实施例8：经改造的VSV和敲除PD-1分子及CTLA-4分子的T细胞联合治疗黑色素瘤(移植瘤)的肿瘤杀伤力检测
按照实施例5的方法对小鼠进行处理，皮下接种2×10 6个B16-F10-NY-ESO-1黑色素瘤细胞。待移植瘤体积长至100mm 3左右时进行处理。将所有小鼠分为10组：对照组(PBS组)瘤内注射50μL PBS，其余9组为治疗组，分别进行瘤内接种JBS000、JBS001、JBS002、JBS003、JBS004、敲除PD-1及CTLA-4的T细胞、JBS000和敲除PD-1及CTLA-4的T细胞、JBS001和敲除PD-1及CTLA-4的T细胞、JBS002和敲除PD-1及CTLA-4的T细胞、JBS003和敲除PD-1及CTLA-4的T细胞、JBS004和敲除PD-1及CTLA-4的T细胞。每组8只动物，每2天给药1次，共给药3次，单次接种量均为10 8pfu/只。自开始给药至实验终点，每2天记录一次移植瘤体积。
结果显示：与对照组相比，各治疗组对黑色素瘤均有一定的治疗效果。
实施例9：经改造的VSV和敲除PD-1分子及CTLA-4分子的T细胞联合治疗纤维肉瘤(移植瘤)的肿瘤杀伤力检测
按照实施例5的方法对小鼠进行处理，皮下接种2×10 6个MCA-205-NY-ESO-1纤维肉瘤细胞。待移植瘤体积长至100mm 3左右时进行处理。将所有小鼠分为12组：对照组(PBS组)瘤内注射50μL PBS，其余11组为治疗组，分别进行瘤内接种JBS000、JBS001、JBS002、JBS003、JBS004、敲除PD-1及CTLA-4的T细胞、JBS000和敲除PD-1及CTLA-4的T细胞、JBS001和敲除PD-1及CTLA-4的T细胞、JBS002和敲除PD-1及CTLA-4的T细胞、JBS003和敲除PD-1及CTLA-4的T细胞、JBS004和敲除PD-1及CTLA-4的T细胞。每组8只动物，每2天给药1次，共给药3次，单次接种量均为10 8pfu/只。自开始给药至实验终点，每2天记录一次移植瘤体积。
结果显示：与对照组相比，各治疗组对纤维肉瘤均有一定的治疗效果。
实施例10：经改造的VSV和敲除PD-1分子T细胞联合治疗的效果评估
选择经评估合格的受试者数人，分为2组，采取不同先后顺序给药，给药策略如图7所示，VSV-OVV-01表示的是本申请中经改造的溶瘤病毒，具体给药策略如下：
A组(先用敲除PD-1分子的T细胞，图7A)：敲除PD-1分子的T细胞从第1周第1天(W1D1，W表示周，D表示天)开始用固定剂量(总量3.6×10 9～4.4×10 9或1.8×10 9～2.2×10 9细胞数)给药，分别以总剂量的20％、30％、50％的方式分3次，每次间隔1天给药，从W1D1开始，每4周为一个周期，如未发生：疾病进展、出现不可耐受的毒性反应、死亡、退出研究、失访、申办方终止研究或受试者开始新的肿瘤治疗，则连续用药4个周期；VSV-OVV-01采用固定剂量，即第一阶段的临床有效剂量或最高剂量，从W2D1开始给药，每2周1次，如未发生：疾病进展、出现不可耐受的毒性反应、死亡、退出研究、失访、申办方终止研究或受试者开始新的肿瘤治疗，则连续用药8次。自开始给药至实验终点，需定期进行安全性随访以及疗效随访，时间点如下：
安全性随访
在基线、每次用药前7天内(W2D1、W4D1、W5D1、W6D1、W8D1、W9D1、W10D1、W12D1、W13D1、W14D1和W16D1)各进行一次；同时在每次疗效访视时(基线、W6D7、W12D7和W20D7)各进行一次；其中基线、W13D1与W12D7为同一时间点，因此，共进行安全性访视13次。
疗效随访
基线、W6D7、W12D7和W20D7各进行1次，时间窗为-7d。
(A.先用敲除PD-1分子的T细胞，B.先用VSV-OVV-01)
B组(先用VSV，图7B)：OVV-01采用固定剂量给药，即第一阶段的临床有效剂量或最高剂量，在W1D1第1次给药，在W4D1第2次给药，之后每2周1次，连续7次，共给药8次。敲除PD-1分子的T细胞从W5D1开始给药，采用固定剂量(总量3.6×10 9～4.4×10 9或1.8×10 9～2.2×10 9细胞数)，分别以总剂量的20％、30％、50％的方式分3次，每次间隔1天给药，从W5D1开始，每4周为一个周期，如未发生：疾病进展、出现不可耐受的毒性反应、死亡、退出研究、失访、申办方终止研究或受试者开始新的肿瘤治疗，则连续用药4个周期。自开始给药至实验终点，需定期进行安全性随访以及疗效随访，时间点如下：
安全性随访
在基线、每次用药前7天内(W4D1、W5D1、W6D1、W8D1、W9D1、W10D1、W12D1、W13D1、W14D1、W16D1和W17D1)各进行一次；同时在每次疗效访视时(基线、W6D7、 W12D7和W20D7)各进行一次；其中基线、W13D1与W12D7为同一时间点，因此，共进行安全性访视14次。
疗效随访
基线、W6D7、W12D7和W20D7各进行1次，时间窗为-7d。
结果显示：联合用药下受试者无进展生存期与单药使用相比增加30％到50％以上。
实施例11：经改造的溶瘤病毒和敲除PD-1分子的T细胞联用的体外肿瘤杀伤能力
使用MOI＝10的JBS003溶瘤病毒体外感染A549肿瘤细胞，同时加入不同效靶比的PD-1敲除T细胞，48h后CCK8法检测细胞活力。具体方法如下：
(1)在96孔培养板中每孔加入A549肿瘤细胞株(人非小细胞肺癌)悬液100μL，使细胞量达到4×10 3个/孔，37℃，5％CO 2条件下培养16h。
(2)将溶瘤病毒按感染复数(multiplicity of infection，MOI)稀释至10；去除96孔板中原来培养基，每孔接种溶瘤病毒100μL。
(3)同时将PD-1敲除的T细胞按效靶比(E:T)分别稀释至1:1、5:1、10:1和20:1共四个梯度；每孔接种PD-1敲除的T细胞100μL，每一稀释梯度均接种4个复孔。
(4)在37℃，5％CO 2条件下培养48h后，弃去96孔培养板中的上清和PD-1敲除的T细胞，加入新鲜的DMED培养基(100μL/孔)，再加入CCK8溶液(10μL/孔)。在37℃，5％CO 2条件下培养4h。
(5)使用多功能酶标仪，震荡2分钟，在450nm波长下，测定各孔的OD值并计算肿瘤杀伤率。
(6)肿瘤杀伤率计算公式：肿瘤细胞杀伤率(％)＝(肿瘤细胞对照组OD值－实验组OD值)/肿瘤细胞对照组OD值×100％。
经改造的溶瘤病毒联合PD-1敲除的T细胞的肿瘤杀伤结果如图8所示，图中显示了单独使用经改造的溶瘤病毒、单独使用PD-1敲除的T细胞、以及联合使用经改造的溶瘤病毒和PD-1敲除的T细胞；结果显示，在经改造的溶瘤病毒和PD-1敲除的T细胞联合用药中，显示出了非常强的抗肿瘤能力，经改造的溶瘤病毒与PD-1敲除的T细胞发挥协同作用，显著提高了抗肿瘤的作用。

Claims (39)

  1. 一种组合物，其包含：
    (a)溶瘤病毒；以及，
    (b)经修饰的免疫效应细胞，
    其中所述经修饰的免疫效应细胞与未经所述修饰的免疫效应细胞相比，免疫检查点的表达量和/或活性被调整。
  2. 根据权利要求1所述的组合物，其中所述溶瘤病毒包含天然的溶瘤病毒和经过改造的溶瘤病毒。
  3. 根据权利要求1-2中任一项所述的组合物，其中所述溶瘤病毒选自下组：水疱性口炎病毒(vesicular stomatitis virus,VSV)、痘病毒、单纯疱疹病毒、麻疹病毒、塞姆利基森林病毒、脊髓灰质炎病毒、呼肠孤病毒、塞内卡谷病毒、埃可型肠道病毒、柯萨奇病毒、新城疫病毒和马拉巴病毒。
  4. 根据权利要求3所述的组合物，其中所述水疱性口炎病毒包含基质蛋白M、核衣壳蛋白N、磷蛋白P、大聚合酶蛋白L和/或糖蛋白G。
  5. 根据权利要求4所述的组合物，其中所述水疱性口炎病毒的基质蛋白M经过改造。
  6. 根据权利要求4-5中任一项所述的组合物，其中所述水疱性口炎病毒基质蛋白M的改造包含一个以上位点的突变。
  7. 根据权利要求4-6中任一项所述的组合物，其中所述水疱性口炎病毒基质蛋白M的突变位点选自下组中的一个或多个：M51、L111、V221和S226。
  8. 根据权利要求4-7中任一项所述的组合物，其中所述水疱性口炎病毒基质蛋白M的突变位点选自下组单个位点的突变：
    a)M51R；
    b)ΔL111；
    c)V221F；以及，
    d)S226R。
  9. 根据权利要求4-8中任一项所述的组合物，其中所述水疱性口炎病毒基质蛋白M的突变位点的组合选自下组中的任一组：
    a)M51R且ΔL111；
    b)M51R且V221F；
    c)M51R且S226R；
    d)ΔL111且V221F；
    e)ΔL111且S226R；
    f)V221F且S226R；
    g)M51R、ΔL111且V221F；
    h)M51R、ΔL111且S226R；
    i)M51R、V221F且S226R；
    j)ΔL111、V221F且S226R；以及，
    k)M51R、ΔL111、V221F且S226R。
  10. 根据权利要求9所述的组合物，其中所述突变位点组合选自M51R、V221F和S226R的水疱性口炎病毒基质蛋白M包含如SEQ ID NO：6所示的氨基酸序列。
  11. 根据权利要求9所述的组合物，其中所述突变位点组合选自M51R、ΔL111、V221F和S226R的水疱性口炎病毒基质蛋白M包含如SEQ ID NO：8所示的氨基酸序列。
  12. 根据权利要求1-11中任一项所述的组合物，其中所述免疫效应细胞选自下组：T细胞、NK细胞、B细胞和巨噬细胞。
  13. 根据权利要求1-12中任一项所述的组合物，其中所述免疫效应细胞包含自体细胞和异体细胞。
  14. 根据权利要求1-13中任一项所述的组合物，其包含经体外扩增得到的免疫效应细胞。
  15. 根据权利要求1-14中任一项所述的组合物，其中所述免疫检查点的数量为一个以上。
  16. 根据权利要求1-15中任一项所述的组合物，其中所述免疫检查点选自下组中的一个或多个：PD-1、PD-L1、CTLA-4、LAG-3、TIM-3、BTLA、VISTA、TIGIT、B7-H2、B7-H3、B7-H4和B7-H6。
  17. 根据权利要求1-16中任一项所述的组合物，其中所述免疫检查点为PD-1、PD-L1、CTLA-4、LAG-3、TIM-3、BTLA、VISTA、TIGIT、B7-H2、B7-H3、B7-H4或B7-H6。
  18. 根据权利要求1-17中任一项所述的组合物，其中所述免疫检查点为选自下组中任一组的组合：
    a)PD-1和CTLA-4；
    b)PD-1和PD-L1；
    c)PD-L1和CTLA4；
    d)LAG-3和CTLA-4；
    e)TIM-3和CTLA-4；
    f)VISTA和CTLA-4；以及
    g)PD1、CTLA-4和PD-L1。
  19. 根据权利要求1-18中任一项所述的组合物，其中所述免疫检查点表达量的调整包含与未经修饰的免疫效应细胞相比，所述免疫检查点表达量的上调、下调和/或缺失。
  20. 根据权利要求1-19中任一项所述的组合物，其中所述免疫检查点活性的调整包含与未经修饰的免疫细胞相比，所述免疫检查点活性的增加、降低和/或缺失。
  21. 根据权利要求1-20中任一项所述的组合物，其中所述免疫检查点的调整包含免疫检查点基因水平、转录水平和/或翻译水平的调控。
  22. 根据权利要求21所述的组合物，其中所述基因表达水平的调控包含基因编辑、过表达、点突变和/或同源重组。
  23. 根据权利要求22所述的组合物，其中所述基因编辑方法选自下组的一种或多种：CRISPR/Cas9、类转录激活因子效应核酸酶(TALEN)、锌指核酸酶(ZFN)和单碱基编辑(BE)。
  24. 根据权利要求22-23中任一项所述的组合物，其中所述基因编辑包含设计序列的sgRNA。
  25. 根据权利要求1-24中任一项所述的组合物，其中所述免疫效应细胞包含敲除针对选自SEQ ID NO：1的PD-1基因的靶序列。
  26. 根据权利要求1-25中任一项所述的组合物，其中所述免疫效应细胞敲除针对选自SEQ ID NO：2的CTLA-4基因的靶序列。
  27. 药物组合物，其包含：(a)权利要求1-26中任一项所述的至少一种溶瘤病毒和/或药学上可接受的载剂；以及(b)权利要求1-26中任一项所述的至少一种免疫效应细胞和/或药学上可接受的载剂。
  28. 试剂盒，其包含选自下组中的一种或多种：
    a)权利要求1-27中任一项所述的至少一种溶瘤病毒和制备权利要求1-27中任一项所述的至少一种修饰的免疫效应细胞的工具，和/或药学上可接受的载剂；
    b)权利要求1-27中任一项所述的至少一种修饰的免疫效应细胞和制备权利要求1-27中任一项所述的至少一种溶瘤病毒的工具，和/或药学上可接受的载剂；以及，
    c)制备权利要求1-27中任一项所述的至少一种溶瘤病毒的工具和制备权利要求1-27中任一项所述的至少一种修饰的免疫效应细胞的工具，和/或药学上可接受的载剂。
  29. 权利要求1-26中任一项所述的组合物、权利要求27所述的药物组合物和权利要求28所述的试剂盒的制备方法。
  30. 权利要求1-29中任一项所述溶瘤病毒在所述溶瘤病毒和所述经修饰的免疫效应细胞的联用中的应用。
  31. 权利要求1-30中任一项所述经修饰的免疫效应细胞在所述溶瘤病毒和所述经修饰的免疫细胞联用中的应用。
  32. 一种治疗肿瘤的方法，其包含向有需要的受试者施用根据权利要求1-26中任一项所述的组合物、根据权利要求27所述的药物组合物和/或根据权利要求28所述的试剂盒。
  33. 根据权利要求32所述的方法，其中所述溶瘤病毒通过静脉内和/或瘤内途径向有需要的受试者施用，其中所述免疫效应细胞通过静脉内和/或局部给药方式向有需要的受试者施用。
  34. 根据权利要求32-33中任一项所述的方法，其中所述溶瘤病毒和所述经修饰的免疫细胞同时施用，施用剂量为治疗有效量。
  35. 根据权利要求32-34中任一项所述的方法，其中所述溶瘤病毒和所述经修饰的免疫细胞分别施用，施用剂量为治疗有效量。
  36. 根据权利要求32-35中任一项所述的方法，其中所述溶瘤病毒和所述经修饰的免疫细胞一次施用或多次施用。
  37. 权利要求1-26中任一项所述的组合物、权利要求27所述的药物组合物、权利要求28所述的试剂盒在制备治疗肿瘤的药物中的应用。
  38. 根据权利要求32-36中任一项所述的方法，或根据权利要求37所述的应用，其中所述肿瘤包括实体瘤和/或血液瘤。
  39. 根据权利要求32-36中任一项所述的方法，或根据权利要求37所述的应用，其中所述肿瘤包含选自下组的一种或多种：头颈部癌、黑色素瘤、软组织肉瘤、乳腺癌、食管癌、肺癌、卵巢癌、膀胱癌、肝癌、宫颈癌、神经母细胞瘤、滑膜肉瘤和圆细胞型脂肪肉瘤。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024012277A1 (zh) * 2022-07-14 2024-01-18 上海荣瑞医药科技有限公司 重组溶瘤病毒及其应用
WO2024012278A1 (zh) * 2022-07-14 2024-01-18 上海荣瑞医药科技有限公司 一种重组溶瘤病毒及其应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017044780A1 (en) * 2015-09-09 2017-03-16 Tvax Biomedical I, Llc Methods for combining adoptive t cell therapy with oncolytic virus adjunct therapy
CN107794269A (zh) * 2017-09-30 2018-03-13 成都美杰赛尔生物科技有限公司 促进基因编辑t细胞活化及扩增的生物膜、制法及应用
CN109069561A (zh) * 2016-01-11 2018-12-21 图恩斯通有限合伙公司 溶瘤病毒和检查点抑制剂组合疗法
CN110461346A (zh) * 2017-03-15 2019-11-15 美国安进公司 溶瘤病毒单独或与检查点抑制剂组合用于治疗癌症的用途
CN110678192A (zh) * 2017-04-21 2020-01-10 新罗杰股份有限公司 溶瘤痘苗病毒与免疫检查点抑制剂联合疗法
CN111065410A (zh) * 2017-09-06 2020-04-24 弗雷德哈钦森癌症研究中心 用于改善过继细胞疗法的方法
CN111286493A (zh) * 2020-05-12 2020-06-16 上海荣瑞医药科技有限公司 一种溶瘤病毒疫苗及其与免疫细胞联合治疗肿瘤的药物
CN111320677A (zh) * 2018-12-14 2020-06-23 苏州奥特铭医药科技有限公司 溶瘤病毒疫苗和过继性免疫细胞联合疗法
CN111467489A (zh) * 2020-05-12 2020-07-31 上海荣瑞医药科技有限公司 一种治疗肿瘤的药物
CN111743923A (zh) * 2019-03-27 2020-10-09 北京康万达医药科技有限公司 包含分离的重组溶瘤腺病毒和免疫细胞的治疗剂及其应用

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017044780A1 (en) * 2015-09-09 2017-03-16 Tvax Biomedical I, Llc Methods for combining adoptive t cell therapy with oncolytic virus adjunct therapy
CN109069561A (zh) * 2016-01-11 2018-12-21 图恩斯通有限合伙公司 溶瘤病毒和检查点抑制剂组合疗法
CN110461346A (zh) * 2017-03-15 2019-11-15 美国安进公司 溶瘤病毒单独或与检查点抑制剂组合用于治疗癌症的用途
CN110678192A (zh) * 2017-04-21 2020-01-10 新罗杰股份有限公司 溶瘤痘苗病毒与免疫检查点抑制剂联合疗法
CN111065410A (zh) * 2017-09-06 2020-04-24 弗雷德哈钦森癌症研究中心 用于改善过继细胞疗法的方法
CN107794269A (zh) * 2017-09-30 2018-03-13 成都美杰赛尔生物科技有限公司 促进基因编辑t细胞活化及扩增的生物膜、制法及应用
CN111320677A (zh) * 2018-12-14 2020-06-23 苏州奥特铭医药科技有限公司 溶瘤病毒疫苗和过继性免疫细胞联合疗法
CN111743923A (zh) * 2019-03-27 2020-10-09 北京康万达医药科技有限公司 包含分离的重组溶瘤腺病毒和免疫细胞的治疗剂及其应用
CN111286493A (zh) * 2020-05-12 2020-06-16 上海荣瑞医药科技有限公司 一种溶瘤病毒疫苗及其与免疫细胞联合治疗肿瘤的药物
CN111467489A (zh) * 2020-05-12 2020-07-31 上海荣瑞医药科技有限公司 一种治疗肿瘤的药物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN CHUN-YU, HUTZEN BRIAN, WEDEKIND MARY F, CRIPE TIMOTHY P: "Oncolytic virus and PD-1/PD-L1 blockade combination therapy", ONCOLYTIC VIROTHERAPY, vol. 7, 1 January 2018 (2018-01-01), pages 65 - 77, XP055796308, DOI: 10.2147/OV.S145532 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024012277A1 (zh) * 2022-07-14 2024-01-18 上海荣瑞医药科技有限公司 重组溶瘤病毒及其应用
WO2024012278A1 (zh) * 2022-07-14 2024-01-18 上海荣瑞医药科技有限公司 一种重组溶瘤病毒及其应用

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