WO2023016572A1

WO2023016572A1 - Modified vaccinia virus and application thereof

Info

Publication number: WO2023016572A1
Application number: PCT/CN2022/112550
Authority: WO
Inventors: 袁明
Original assignee: 深圳市华药康明生物药业有限责任公司
Priority date: 2021-08-13
Filing date: 2022-08-15
Publication date: 2023-02-16
Also published as: CN117957323A

Abstract

Provided is a modified vaccinia virus, containing a gene encoding one or more chemokines, a gene encoding one or more T-cell growth factors, and/or a gene encoding one or more T-cell activation factors. Also provided is a pharmaceutical composition containing the modified vaccinia virus and an immune cell. Further provided are preparation methods for and uses of the modified vaccinia virus and the pharmaceutical composition.

Description

Modified vaccinia virus and its application

technical field

The application relates to the field of biomedicine, in particular to a modified vaccinia virus and its application.

Background technique

Cellular immunotherapy has been proven to be very effective in the treatment of B-cell leukemia, B-cell lymphoma, and multiple myeloma, but it has not yet shown ideal therapeutic effects in the treatment of solid tumors. The main reasons include: 1) unacceptably greater risk of toxicity (derived from potential "off-target" effects, due to the expression of target molecules in many normal tissues); 2) insufficient translocation of T cells into the tumor; 3) Effector functions of T cells are impaired in the tumor microenvironment. Additional concerns relate to the inherent heterogeneity of solid tumors, which predisposes to incomplete tumor targeting of T cells and leads to acquired resistance.

Studies have shown that oncolytic viruses can induce and enhance the production of type I interferon in the tumor microenvironment (TME). In addition, oncolytic viruses can enhance the effector functions of dendritic cells (DC) and T cells, and reduce regulatory T cells (Treg ) and myeloid-derived suppressor cells (MDSC)-induced immunosuppression, which can change the immune phenotype of the tumor from a "cold" to a "hot" state, and the immune hot tumor promotes the entry, expansion and function of T cells.

How to make more effective use of the advantages of oncolytic viruses and promote the effective entry of therapeutic T cells into solid tumors to function is an urgent problem to be solved.

Contents of the invention

The application provides a modified vaccinia virus and its application. Compared to an unmodified vaccinia virus (e.g., a wild-type vaccinia virus), the modified vaccinia virus may have one or more characteristics selected from the group consisting of: 1) effectively attracting T cells into the interior of a solid tumor; 2) 3) significantly promoting the proliferation of therapeutic T cells entering into solid tumors; and 4) significantly promoting the anti-tumor effect of T cells.

In one aspect, the application provides a modified vaccinia virus comprising genes encoding one or more chemokines, and encoding one or more T cell growth factor genes and/or encoding one or more A gene for a T cell activator.

In certain embodiments, the modified vaccinia virus comprises a thymidine kinase (TK)-deficient vaccinia virus.

In certain embodiments, the gene encoding TK in the TK-deficient vaccinia virus comprises one or more mutations.

In certain embodiments, all genes encoding TK are deleted in the TK-deficient vaccinia virus.

In certain embodiments, the modified vaccinia virus comprises a vaccinia virus in which the A46R gene comprises one or more mutations.

In certain embodiments, the modified vaccinia virus comprises a vaccinia virus in which the entire A46R gene has been deleted.

In certain embodiments, the modified vaccinia virus comprises a vaccinia virus deficient in TK and deleted in the A46R gene.

In certain embodiments, the modified vaccinia virus comprises a vaccinia virus in which both the TK gene and the A46R gene have been deleted.

In certain embodiments, the chemokine comprises CXCL9, CXCL10 and/or CXCL11.

In some embodiments, the CXCL9 comprises the amino acid sequence shown in SEQ ID NO:5.

In some embodiments, the CXCL9 comprises the nucleotide sequence shown in SEQ ID NO:6.

In some embodiments, the CXCL10 comprises the amino acid sequence shown in SEQ ID NO:7.

In some embodiments, the CXCL10 comprises the nucleotide sequence shown in SEQ ID NO:8.

In certain embodiments, the T cell growth factor comprises IL-2.

In certain embodiments, the IL-2 comprises the amino acid sequence shown in SEQ ID NO: 11.

In some embodiments, the IL-2 comprises the nucleotide sequence shown in SEQ ID NO: 12.

In certain embodiments, the T cell activator comprises IL-21.

In certain embodiments, the IL-21 comprises the amino acid sequence shown in SEQ ID NO:9.

In some embodiments, the IL-21 comprises the nucleotide sequence shown in SEQ ID NO:10.

In certain embodiments, the modified vaccinia virus comprises genes encoding CXCL9, CXCL10, IL-2, and IL-21, respectively.

In certain embodiments, in the modified vaccinia virus, the gene encoding the chemokine is located at the location of the TK gene of the modified vaccinia virus.

In certain embodiments, the TK gene of the modified vaccinia virus is replaced by the gene encoding a chemokine.

In certain embodiments, the gene encoding T cell growth factor and/or the gene encoding T cell activator is located at the position where the A46R gene of the modified vaccinia virus is located.

In certain embodiments, the A46R gene of the modified vaccinia virus is replaced by the gene encoding T cell growth factor and/or the gene encoding T cell activator.

In certain embodiments, in the modified vaccinia virus, a gene encoding a chemokine is inserted at the position where the TK gene is deleted, and a gene encoding a T cell activator and/or a T cell encoding factor is inserted at the position where the A46R gene is deleted. growth factor genes.

In certain embodiments, in the modified vaccinia virus, the gene encoding CXCL9 and the gene encoding CXCL10 are inserted at the position where the TK gene is deleted, and the gene encoding IL-21 and the gene encoding IL-1 are inserted at the position where the A46R gene is deleted. 2 genes.

In certain embodiments, the modified vaccinia virus is Lister.

In another aspect, the present application also provides a nucleic acid molecule encoding the modified vaccinia virus.

On the other hand, the present application also provides a cell comprising the modified vaccinia virus, and/or, the nucleic acid molecule.

In certain embodiments, the cells comprise host cells.

In certain embodiments, the cells comprise tumor cells.

On the other hand, the present application also provides a pharmaceutical composition, which comprises the modified vaccinia virus, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition is formulated for systemic administration, and/or, is formulated for intravenous administration.

On the other hand, the present application also provides a pharmaceutical combination, which comprises: 1) the modified vaccinia virus, and 2) immune effector cells.

In certain embodiments, the immune effector cells comprise modified immune effector cells.

In certain embodiments, the immune effector cells comprise T cells.

In certain embodiments, the immune effector cells comprise human T cells.

On the other hand, the present application also provides the modified vaccinia virus, the nucleic acid molecule, the cell, the pharmaceutical composition, and/or the use of the pharmaceutical combination in the preparation of a drug, the drug For the prevention and/or treatment of diseases and/or conditions.

In certain embodiments, the disease and/or condition comprises a tumor.

In certain embodiments, the tumor comprises solid tumors and/or non-solid tumors.

On the other hand, the present application also provides a method for improving the efficiency of the modified vaccinia virus in causing immune effector cells to enter the tumor, which comprises: making the modified vaccinia virus contain a protein encoding one or more chemokines. Genes and genes encoding one or more T cell growth factor genes and/or genes encoding one or more T cell activating factors.

In certain embodiments, the modified vaccinia virus comprises one or more mutations comprising the A46R gene and/or the TK gene.

In certain embodiments, the A46R gene and/or the TK gene of the modified vaccinia virus are all deleted.

In certain embodiments, the modified vaccinia virus comprises genes encoding CXCL9, CXCL10, IL-2 and/or IL-21, respectively.

In certain embodiments, the modified vaccinia virus inserts the gene encoding CXCL9 and the gene encoding CXCL10 at the position where the TK gene is deleted, and inserts the gene encoding IL-21 and the gene encoding IL-2 at the position where the A46R gene is deleted .

In certain embodiments, the modified vaccinia virus is a Listeria strain.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1 shows a schematic diagram of the structure of the TK shuttle carrier.

Figure 2 shows a schematic diagram of the structure of the A46R shuttle vector.

Figure 3 shows a schematic diagram of the recombinant structure of the TKcxcl virus.

Figure 4 shows a schematic diagram of the TKcxcl virus genome.

Figure 5 shows a schematic representation of the recombination of the modified vaccinia virus (Onco-T) described in this application.

Figure 6 shows a schematic diagram of the viral genome of the modified vaccinia virus (Onco-T) described in this application.

Fig. 7 shows the identification results of deletion of TK gene and A46R gene of the modified vaccinia virus (Onco-T) described in the present application.

Figure 8 shows the results of ELISA detection of IL-21 gene and IL-2 gene insertions of the modified vaccinia virus (Onco-T) described in the present application.

Figure 9 shows the effect of foreign protein expressed by the modified vaccinia virus (Onco-T) described in this application on T cell chemotaxis.

Figure 10 shows the effect of foreign protein expressed by the modified vaccinia virus (Onco-T) described in this application on the proliferation of T cells.

Detailed ways

The implementation of the invention of the present application will be described by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In the present application, the term "modified vaccinia virus" generally refers to a virus belonging to the Poxviridae family. The modified vaccinia virus may be a virus belonging to the subfamily Chordopoxviridae. The modified vaccinia virus may also be a virus belonging to the genus Orthopoxvirus. In the present application, the modified vaccinia virus may include modified vaccinia virus (Vaccinia virus), vaccinia virus (cowpox virus), canary pox virus (Canarypox virus), mousepox virus (Ectromelia virus) or mucus virus Tumor virus (Myxoma virus). In the present application, the modified vaccinia virus may include a modified vaccinia virus (Vaccinia virus), which may be a DNA virus (for example, its genome may be a linear double-stranded DNA). The modified vaccinia virus has a relatively large genome (for example, more than 25kb); foreign genes can be relatively stable in the modified vaccinia virus; the host range is wide; and it may not be integrated into the genome of the host cell. The modified vaccinia virus may comprise a Lister strain.

In this application, the term "mutation" generally refers to a difference in the amino acid or nucleic acid sequence of a specific protein or nucleic acid (gene, RNA) relative to the wild-type protein or nucleic acid, respectively.

In this application, the term "TK deficient" generally refers to an altered function, activity or gene compared to the TK of wild-type vaccinia virus. For example, the TK deficiency may refer to decreased or absent TK activity. For example, the TK deficiency may refer to a mutation in a gene encoding TK.

In the present application, the term "A46R" generally refers to the TIR domain-containing viral protein of vaccinia virus. In this application, the term also covers homologues, derivatives, mutants and functionally active fragments thereof. In the present application, the left arm and the right arm of the A46R region are generally defined as upstream and downstream of the inserted gene of interest. For example, the left arm of the A46R region may be located upstream of the inserted gene of interest. For example, the right arm of the A46R region may be located downstream of the inserted gene of interest.

In this application, the term "growth factor" generally refers to a protein or polypeptide that regulates cell growth. In certain embodiments, the growth factor comprises an interleukin-like growth factor, eg, the growth factor comprises IL-2.

In this application, the term "IL-21", also called "interleukin-21", is a cell activating factor. The term also covers homologues, derivatives, mutants and functionally active fragments of IL-21. For example, the IL-21 can comprise human IL-21. For example, the cDNA and amino acid sequence of human IL-21 can be found, eg, in GenBank Accession Nos. BC066260.1 and AAH69124.1. For example, the IL-21 may comprise the amino acid sequence shown in SEQ ID NO:9.

In this application, the term "IL-2" is also referred to as "Interleukin-2", which is a cell growth factor. The term also covers homologues, derivatives, mutants and functionally active fragments of IL-2. For example, the IL-2 can comprise human IL-2. For example, the IL-2 may comprise the amino acid sequence shown in SEQ ID NO: 11.

In this application, the term "Lister strain" can be used interchangeably with "Lister strain", and usually refers to the preparation based on the VACV-List strain obtained from sheep in 1961 and passed on calves modified vaccinia virus strains. The Lister strain has been widely used in vaccine production (conventional vaccine production). The GenBank accession number of the genome information of the Lister strain is DQ121394.

In this application, the term "nucleic acid molecule" generally refers to a polymeric form of nucleotides, such as ribonucleotides, deoxyribonucleotides, and/or modified forms of any of the foregoing. The nucleic acid molecule may include RNA, cDNA, sense and antisense strands of genomic DNA, and synthetic forms and mixed polymers thereof. The nucleic acid molecule can comprise any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular and padlocked conformations. The nucleic acid molecule may comprise naturally occurring and/or non-naturally occurring nucleotides.

In this application, the term "pharmaceutical composition" generally refers to a combination of one or more active pharmaceutical ingredients and one or more pharmaceutically acceptable carriers. The pharmaceutical composition may exist in the final pharmaceutical dosage form, or may be an intermediate for preparation of the dosage form. The carrier may include stabilizers, diluents, dispersants, suspending agents, thickeners and/or excipients.

As used herein, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are inert to the cells or animals to which they are exposed at the dosages and concentrations employed. poisonous. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, Sugar alcohols, salt-forming counterions such as sodium, and/or nonionic surfactants.

In this application, the term "tumor" generally refers to a swelling or lesion formed by abnormal growth of cells, such as tumor cells. The tumor is used interchangeably with cancer in this application. The tumors can include solid tumors and non-solid tumors. The tumor may be a diffuse tumor, or may be a circulating tumor.

In this application, the term "treatment" generally refers to measures that can reduce or alleviate the progression, severity and/or duration of a proliferative disease or improve one or more symptoms of a proliferative disease.

In this application, the term "immune effector cells" generally refers to immune cells that participate in immune responses and perform effector functions. In this application, the immune effector cells may include but not limited to T cells, natural killer T cells, lymphocytes. The term also includes engineered immune cells, such as immune cells that have been genetically modified by adding foreign genetic material in the form of DNA or RNA to the total genetic material of the cell, such as immune cells that have been genetically altered by gene editing techniques cell.

Detailed description of the invention

In one aspect, the application provides a modified vaccinia virus comprising genes encoding one or more chemokines, and encoding one or more T cell growth factors and/or encoding one or more T cell growth factors Genes for cell activators.

In another aspect, the present application also provides a modified vaccinia virus comprising and/or expressing one or more exogenous chemokines, and comprising and/or expressing one or more exogenous T cell growth factors and and/or the genes of one or more exogenous T cell activators.

In the present application, the modified vaccinia virus may comprise a thymidine kinase (TK)-deficient vaccinia virus. In the present application, the TK-deficient vaccinia virus may comprise a vaccinia virus having a mutation in a gene encoding TK. In the present application, the TK-deficient vaccinia virus generally refers to a vaccinia virus whose TK gene contains at least one mutation compared to wild-type vaccinia virus. For example, the TK-deficient virus may contain a partial mutation in the gene encoding TK, or may contain a complete mutation in the gene encoding TK. For example, the mutations may involve additions, deletions and/or substitutions of genes. In the present application, the TK of the modified vaccinia virus may comprise the amino acid sequence shown in SEQ ID NO:13.

In the present application, the modified vaccinia virus may comprise a vaccinia virus in which all genes encoding TK are deleted.

In the present application, the modified vaccinia virus may comprise an A46R gene-deficient vaccinia virus. In the present application, the A46R gene-deficient vaccinia virus generally refers to a vaccinia virus whose A46R gene contains at least one mutation compared with wild-type vaccinia virus. For example, the modified vaccinia virus may contain a partial mutation of the A46R gene, or may contain a complete mutation of the A46R gene.

In the present application, the modified vaccinia virus may comprise a vaccinia virus in which all A46R genes are deleted. In the present application, the A46R of the modified vaccinia virus may comprise the amino acid sequence shown in SEQ ID NO:14.

In the present application, the modified vaccinia virus may contain both TK deficiency and A46R deficiency. In the present application, compared with the wild-type vaccinia virus, the modified vaccinia virus may have mutations in both the TK gene and the A46R gene. For example, the modified vaccinia virus may comprise a total deletion of the TK gene and a total deletion of the A46R gene.

In the present application, the chemokines may include exogenous chemokines. For example, the chemokine may comprise T-lymphocyte chemokine. For example, the modified vaccinia virus can comprise and/or express one or more exogenous chemokines. For example, the modified vaccinia virus can comprise and/or express two different exogenous chemokines. For example, the modified vacciniaviridae comprises and/or expresses three different exogenous chemokines. For example, the chemokine may comprise CXCL9, CXCL10 and/or CXCL11. In certain embodiments, the modified vaccinia virus comprises a CXCL9 gene. In certain embodiments, the modified vaccinia virus comprises a CXCL10 gene. In certain embodiments, the modified vaccinia virus comprises CXCL9 and CXCL10 genes. In certain embodiments, the chemokine may be of human origin.

In the present application, the CXCL9 may comprise the amino acid sequence shown in SEQ ID NO:5. In the present application, the CXCL9 may comprise the nucleotide sequence shown in SEQ ID NO:6. In the present application, the CXCL10 may comprise the amino acid sequence shown in SEQ ID NO:7. In the present application, the CXCL10 may comprise the nucleotide sequence shown in SEQ ID NO:8.

In the present application, the gene encoding a chemokine may be located at the position where the TK gene is located in the modified vaccinia virus. For example, the gene encoding a chemokine may be located within the range of the start gene and stop gene of the TK gene. For example, the TK gene of the modified vaccinia virus can be replaced by the gene encoding a chemokine. For example, the TK gene of the modified vaccinia virus can be replaced by the CXCL9 gene and the CXCL10 gene.

In the present application, the modified vaccinia virus may further comprise and/or express one or more cytokines. For example, the cytokines may comprise T cell growth factors. For example, the cytokine may comprise a T cell activator. For example, the cytokines may comprise IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-14, IFN- Gamma, TNF-β and/or GM-CSF. For example, the modified vaccinia virus may also comprise and/or express two different cytokines. For example, the modified vaccinia virus can contain and/or express three different cytokines. For example, the cytokines may comprise human cytokines.

In the present application, the T cell growth factor may comprise IL-2. In the present application, the IL-2 may comprise human IL-2. For example, the IL-2 may comprise the amino acid sequence shown in SEQ ID NO: 11. For example, the IL-2 may comprise the nucleotide sequence shown in SEQ ID NO:12. In the present application, the T cell activation gene may comprise IL-21. In the present application, the IL-21 may comprise human IL-21. For example, the IL-21 may comprise the amino acid sequence shown in SEQ ID NO:9. For example, the IL-21 may comprise the nucleotide sequence shown in SEQ ID NO:10.

In the present application, the modified vaccinia virus may comprise T cell growth factors and/or T cell activating factors. In the present application, the modified vaccinia virus may comprise T cell growth factors. In the present application, the modified vaccinia virus may comprise a T cell activating factor. In the present application, the modified vaccinia virus may comprise T cell growth factors and/or T cell activating factors.

In the present application, the modified vaccinia virus may comprise IL-2 and/or IL-21. In the present application, the modified vaccinia virus may comprise IL2. In the present application, the modified vaccinia virus may comprise IL-21. In the present application, the modified vaccinia virus may comprise IL-2 and IL-21.

In the present application, the gene encoding the T cell growth factor and/or the T cell activating factor may be located at the position of the A46R gene in the modified vaccinia virus. For example, the gene encoding T cell growth factor and/or the T cell activator may be located within the range of the start gene and stop gene encoding the A46R gene. For example, the A46R gene of the modified vaccinia virus can be replaced by the gene encoding T cell growth factor and/or the T cell activator. For example, the A46R gene of the modified vaccinia virus can be replaced by the IL-2 gene and the IL-21 gene.

In the present application, the modified vaccinia virus may comprise CXCL9 gene, CXCL10 gene, IL-2 gene and IL-21 gene.

In the present application, the CXCL9 gene, CXCL10 gene, IL-2 gene and/or IL-21 can be inserted into the genome of vaccinia virus through the connection of each promoter. The respective promoters may be the same or different. For example, the promoter may comprise pLEO, Psel, PE/L and/or H5.

In the present application, the modified vaccinia virus comprises TK gene deletion, A46R gene deletion, and comprises CXCL9 gene, CXCL10 gene, IL-2 gene and IL-21 gene.

In the present application, the gene encoding the chemokine can be inserted at the position where the TK gene is deleted in the modified vaccinia virus, and the gene encoding the T cell activating factor and/or The gene encoding T cell growth factor.

In the present application, the CXCL9 gene and CXCL10 gene can be inserted at the position where the TK gene is deleted in the modified vaccinia virus, and the IL-2 gene and IL-21 gene can be inserted at the position where the A46R gene is deleted.

In the present application, the modified vaccinia virus may be Lister.

On the other hand, the present application also provides a nucleic acid molecule comprising a modified vaccinia virus gene sequence.

In certain embodiments, the nucleic acid molecule encodes the genome of the modified vaccinia virus.

In another aspect, the present application provides a cell comprising the modified vaccinia virus described in the present application, and/or the nucleic acid molecule described in the present application.

In the present application, the cells may include host cells.

In the present application, the cells may include tumor cells.

In another aspect, the present application provides a pharmaceutical combination, which comprises the modified vaccinia virus, and immune effector cells.

In the present application, the immune effector cells may comprise modified immune effector cells.

In the present application, the immune effector cells may comprise T cells.

In the present application, the immune effector cells may comprise modified T cells.

In the present application, the immune effector cells may comprise human T cells.

In the present application, the modified vaccinia virus and the immune effector cells in the pharmaceutical combination can be placed in the same container. In the present application, the modified vaccinia virus and the immune effector cells in the pharmaceutical combination can be placed in different containers.

In this application, the modified oncolytic virus can be administered simultaneously with the immune effector cells. In this application, the modified oncolytic virus can be administered separately from the immune effector cells. For example, the modified oncolytic virus can be administered first, followed by the immune effector cells. For example, the immune effector cells can be administered first, followed by the modified oncolytic virus. For example, the components of the pharmaceutical combination can be separated by about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, About 2 hours, about 3 hours, or about half a day, about one day, about two days, about three days, about four days, about five days, about one week, about one and a half weeks, about two weeks, about three weeks, about Apply four weeks.

In another aspect, the present application provides a pharmaceutical composition, which comprises the modified vaccinia virus described in the present application, and optionally a pharmaceutically acceptable carrier. In this application, the pharmaceutical composition may also contain other active ingredients. For example, the pharmaceutical composition may also comprise immune cells.

In the present application, the pharmaceutical composition may include "therapeutically effective dose" or "prophylactically effective dose" of the modified vaccinia virus described in the present application. The "therapeutically effective dose" may be an amount effective, at dosages and for durations necessary, to achieve the desired therapeutic result. The therapeutically effective dose can be determined by those skilled in the art. For example, it may vary according to the state of the disease, age, sex, body weight of the subject, the ability of the modified vaccinia virus to elicit the response in the subject, and the like. The "prophylactically effective dose" may be an amount effective to achieve the desired prophylactic result at the necessary dosage and duration. For example, the prophylactically effective dose may be lower than the therapeutically effective dose.

In the present application, the pharmaceutical composition may be formulated in a form suitable for administration (eg, suitable for parenteral, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous, intratumoral and/or mucosal administration).

In the present application, the pharmaceutically acceptable carrier may include any or all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. The pharmaceutically acceptable carrier may include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. The pharmaceutically acceptable carrier may also include isotonic agents, wetting agents, emulsifying agents, preservatives and/or buffering agents.

In the present application, the pharmaceutical composition may be formulated for systemic administration, and/or, it may be formulated for intravenous administration.

In another aspect, the present application provides a modified vaccinia virus described in the present application, the nucleic acid molecule described in the present application, the cell described in the present application, the pharmaceutical composition described in the present application, and/or, the nucleic acid molecule described in the present application The use of the drug combination in the preparation of medicaments for preventing and/or treating diseases and/or conditions.

On the other hand, the application provides the modified vaccinia virus described in the application, the nucleic acid molecule described in the application, the cell described in the application, the pharmaceutical composition described in the application, and/or, the nucleic acid molecule described in the application A pharmaceutical combination for the prevention and/or treatment of diseases and/or conditions.

In another aspect, the present application provides a method for preventing and/or treating diseases and/or disorders, which comprises administering the modified vaccinia virus described in the present application, the nucleic acid molecule described in the present application to a subject in need , the cell described in the present application, the pharmaceutical composition described in the present application, and/or, the pharmaceutical combination described in the present application.

In the present application, the diseases and/or conditions may include tumors.

In this application, the tumor may include solid tumors and non-solid tumors.

In another aspect, the present application provides a method of increasing the efficiency of a modified vaccinia virus for entry of immune effector cells into a tumor, comprising: causing the modified vaccinia virus to include a gene encoding one or more chemokines and genes encoding one or more T cell growth factors and/or one or more T cell activating factors.

In the present application, the modified vaccinia virus significantly increases the efficiency of immune effector cell entry into tumors compared to unmodified vaccinia virus (eg, wild-type vaccinia virus). For example, an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least About 200%, at least about 300%, or more.

In the present application, compared with wild-type vaccinia virus, the modified vaccinia virus has significantly improved replication speed and/or ability to kill host cells. For example, an increase of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least About 200%, at least about 300%, or more.

In the present application, the modified vaccinia virus can effectively improve the efficiency of in vivo immune cells entering tumors. In the present application, the modified vaccinia virus can effectively improve the killing ability of tumor cells.

For example, compared to a vaccinia virus that only contains genes encoding chemokines or T cell growth factors or T cell activating factors, the vaccinia virus described herein also contains genes encoding one or more chemokines, and encodes a Genes of one or more T cell growth factors and/or genes encoding one or more T cell activating factors can effectively improve the efficiency of in vivo immune cells entering tumors.

For example, compared to a vaccinia virus that only contains genes encoding chemokines or T cell growth factors or T cell activating factors, the vaccinia virus described herein also contains genes encoding one or more chemokines, and encodes a The genes of one or more T cell growth factors and/or the genes encoding one or more T cell activating factors can effectively improve the killing ability of tumor cells.

In the present application, the modified vaccinia virus may include one or more mutations in the A46R gene and/or the TK gene.

In the present application, the A46R gene and/or TK gene of the modified vaccinia virus can be completely deleted.

In the present application, the modified vaccinia virus may comprise genes encoding CXCL9, CXCL10, IL-2 and/or IL-21, respectively.

In the present application, the modified vaccinia virus may comprise genes encoding CXCL9, CXCL10, IL-2 and IL-21, respectively.

In the present application, the modified vaccinia virus can insert the gene encoding CXCL9 and the gene encoding CXCL10 at the position where the TK gene is deleted, and insert the gene encoding IL-21 and the gene encoding IL-2 at the position where the A46R gene is deleted

In the present application, the modified vaccinia virus may comprise any strain of vaccinia virus known in the art. For example, the vaccinia virus strain may comprise a deletion of the TK gene. For example, the vaccinia virus strain may comprise a deletion of the A46R gene. In the present application, the modified vaccinia virus may be Listeria strain.

In the present application, the modified vaccinia virus may comprise Onco-T. In some embodiments, the Onco-T is the modified vaccinia virus prepared in Example 1 of the present application. In some embodiments, the Onco-T can effectively induce T cells to undergo chemotaxis in vivo animal experiments. In some embodiments, the Onco-T can effectively promote the proliferation of T cells in animal experiments in vivo. In certain embodiments, the combination of Onco-T and T cells is used to detect tumor killing ability.

In some embodiments, compared with the combined effects of TK and A46R-deleted vaccinia virus with only chemokines inserted and cell growth factors, the killing of Onco-T showed more chemotaxis and pro-proliferation effects on T cells. Good results.

In certain embodiments, simultaneous expression of chemokines and cell growth factors using vaccinia virus vectors with other mutations (e.g., other vaccinia virus strains; e.g., with mutations other than TK and A46R gene deletions) can also be beneficial. Effects (eg, induction of T cell chemotaxis, promotion of T cell proliferation, antitumor effects).

Not intending to be limited by any theory, the following examples are only for explaining various technical solutions of the invention of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Embodiment 1 prepares the modified vaccinia virus

Using Listeria vaccinia virus as a carrier, the wild-type vaccinia virus was purchased from ATCC (ATCC VR-1549), and the following transformation was carried out on the basis of the wild-type vaccinia virus: delete the A46R gene and TK gene of the vaccinia virus, and insert at the A46R gene Genes encoding IL-21 and IL-2, genes encoding CXCL9 and CXCL10 were inserted at the TK gene. The specific operation is as follows:

Construction of TK shuttle vector

The TK shuttle vector includes a TK left arm targeting the left side of the TK gene (L089), and a TK right arm targeting the right side of the TK gene (L091). H5 promoter (SEQ ID NO: 1) and red fluorescent protein RFP, T cell chemokine gene CXCL9 (amino acid sequence is shown in SEQ ID NO: 5, nucleotide sequence is shown in SEQ ID NO: 6) and CXCL10 (The amino acid sequence is shown in SEQ ID NO: 7, and the nucleotide sequence is shown in SEQ ID NO: 7), using the promoters pLEO (SEQ ID NO: 2) and Psel (SEQ ID NO: 3) respectively, the above All the sequences were spliced together and synthesized by Nanjing GenScript Biotechnology Co., Ltd., and cloned into the PUC57 vector. The schematic diagram of the TK shuttle carrier structure is shown in Figure 1.

Construction of A46R shuttle vector

The A46R shuttle vector includes an A46R left arm targeting the left side of the A46R gene (L163), and an A46R right arm targeting the right side of the A46R gene (L165). The Psel promoter (SEQ ID NO:3) drives the expression of green fluorescent protein GFP. PE/L promoter (SEQ ID NO:4) drives the expression of IL-21 (amino acid sequence as shown in SEQ ID NO:9, nucleotide sequence as shown in SEQ ID NO:10) gene, PE/L promoter Drive IL-2 (amino acid sequence as shown in SEQ ID NO: 11, nucleotide sequence as shown in SEQ ID NO: 12) gene expression. All the above sequences were spliced together and synthesized by Nanjing GenScript Biotechnology Co., Ltd., and cloned into the PUC57 vector. The schematic diagram of the structure of the A46R shuttle carrier is shown in Figure 2.

Cas9-mediated homologous recombination of vaccinia virus recombination

Seed 3 × ¹⁰⁵ CV-1 cells into one well of a six-well plate one day before transfection. The gRNA vector was used for the TKgRNA targeting the TK region, and the A46RgRNA targeting the A46R region was co-transfected with Cas9 into CV-1 cells in a six-well plate. The next day, the wells transfected with the gRNA vector and the Cas9 gene were infected with 0.01 PFU/cell of backbone virus. Two hours after virus infection, the shuttle vector for homologous recombination was transfected into the infected wells. Cells were harvested after 24 hours and frozen at -80°C for recombinant virus purification.

Purification of recombinant virus

Cell lysates collected from Cas9-mediated homologous recombination were thawed and 0.5 μl of this lysate was used to infect all 6 wells of a six-well plate containing CV1 cells grown to 80-90% confluency. Forty-eight hours after infection, carefully examine each well under a fluorescent microscope, looking for viruses that fluoresce red or green. After identifying positive infected plaques, mark their location on the lower surface of the plate with a marker pen. Viral plaques were then carefully picked with a 20 μl tip after aspirating the medium from the wells in the fume hood where the cells were grown. The tip was then submerged in a cryovial containing 200 μl of cell culture medium. After one freeze-thaw cycle, 5-20 [mu]l of the virus solution was added to each well of a new 6-well plate containing CV1 cells. This process is repeated until each virus spot is red or green fluorescent, that is, all virus spots are caused by the recombinant virus. Typically, 3 to 5 rounds of plaque purification are required to obtain pure recombinant virus. After confirming that the virus has been purified, the infected cells were scraped off and centrifuged to obtain cell pellets. A portion of the cells is then taken to extract viral DNA. The purity of the virus was confirmed by PCR amplification of the target gene from the extracted viral DNA.

The recombinant virus obtained deleted TK gene and carried T cell chemokine gene CXCL9 and 10 virus.

The wild-type Listerian vaccinia virus was used as the mother virus, and the recombinant vector and the mother virus were recombined using Cas9 and gRNA technology (the schematic diagram of TKcxcl virus recombination is shown in Figure 3). After recombination, the recombinant virus was screened under a fluorescent microscope until the purified virus was obtained, and the presence of TK gene deletion and insertion gene was identified by PCR method, and TKcxcl virus was obtained by identification (the schematic diagram of TKcxcl virus is shown in Figure 4).

The recombinant virus TKcxcl obtained above is a mother virus, and the recombinant virus is obtained by recombination to obtain a virus in which the A46R gene is deleted and the T cell activating factor IL-21 and T cell growth factor IL-2 genes are inserted (the schematic diagram of viral recombination is shown in Figure 5). The recombinant vector and parent virus were recombined using Cas9 and gRNA technology, and the schematic diagram of the recombinant virus is shown in Figure 6. After recombination, the recombinant virus was screened under a fluorescent microscope until the purified virus was obtained, and the presence of the deletion of the A46R gene and the insertion gene was identified by PCR. The identification results are shown in Figure 7, where + is a positive control, - is a negative control, and S It is a modified vaccinia virus, L09 is the gene region encoding List09 protein, and is used as amplified control gene, and TK and A46R are genes for identification and deletion. The results showed that the TK gene and A46R gene of vaccinia virus were deleted.

The results of the modified vaccinia virus expressing T cell chemokine genes CXCL9 and CXCL10, T cell activator IL-21 and T cell growth factor IL-2 identified by the ELISA method, the ELISA results are shown in Figure 8, the results show , the CXCL9, CXCL10, IL-21 and IL-2 genes were inserted into the vaccinia virus.

Amplification of the virus

After confirming that the recombinant virus is the target recombinant virus, 50 μl of the virus lysate was added to the T175 culture flask containing CV1 cells, and grown to 80-90% confluence in the cell culture medium containing about 30 ml. After 48 hours the cells and medium were scraped and saved.

Example 2 Detection of the influence of the modified vaccinia virus on T cell chemotaxis

To verify the effect of the modified vaccinia virus prepared by the present application on T cell chemotaxis. Onco-T is used to represent the modified vaccinia virus prepared by the present application, and the effect of the foreign protein expressed by the modified vaccinia virus prepared by the present application on T cell transmembrane chemotaxis is detected. The control group was the wild-type vaccinia virus Listeria strain. Transwell was used to detect the number of T cells transmembrane migration after 4 hours.

The results are shown in FIG. 9 , and the results show that testing with the cell culture fluid containing the foreign protein expressed by the modified vaccinia virus prepared in the present application can effectively induce T cells to undergo chemotaxis.

Example 3 Detection of the Effect of the Modified Vaccinia Virus on the Proliferation of T Cells

Use Onco-T to represent the modified vaccinia virus prepared by the present application, and verify the effect of the modified vaccinia virus prepared by the present application on the proliferation of T cells. The control group was the wild-type vaccinia virus Listeria strain. The effect on cell proliferation after adding vaccinia virus for 24 hours was detected by cell counting.

The results are shown in FIG. 10 , and the results show that testing with the cell culture medium containing the foreign protein expressed by the modified vaccinia virus prepared in the present application can effectively promote the proliferation of T cells.

Example 4 The ability of the modified vaccinia virus combined with T cells to kill tumors

A BALB/c nude mouse human rhabdomyosarcoma cell A673 subcutaneous tumor model was established to evaluate the tumor killing ability of vaccinia virus Onco-T combined with T cells.

1. Animal modeling

BALB/c nude mice qualified for quarantine, subcutaneously injected 0.1mL human rhabdomyosarcoma cells A673 (5×107 cells/mL) in the back of the mouse, observed the tumor growth every day after inoculation; after the tumor grew obviously, measured every 2 days Tumor volume once (recorded as M1 on the day of modeling); when the tumor volume grows to about 80-160mm3, random grouping was performed according to the tumor volume (recorded as D1 on the day of administration).

2. Grouping of animals

Animals that were screened and modeled were randomly divided into vehicle control group, positive control group, Onco-T group, and T cell therapy group respectively. T cells were injected 5 days after virus injection in each group. The effect of Onco-T on T cell migration and proliferation, tumor growth and animal survival was evaluated. The specific groups and administration information are as follows:

3. Detection of animal tumor treatment effect

Compared with the vehicle control group and the positive control group, the tumors of the animals in the Onco-T group were relatively smaller; in the combined T cell group, the tumors of the animals in the Onco-T and T cell combined group were relatively smaller, and were smaller than those in the Onco-T group alone. -T heals better. Tumor treatment effect: Onco-T and T cell combination group > HY01 and T cell combination group > vehicle control and T cell combination group.

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

A modified vaccinia virus comprising genes encoding one or more chemokines, and genes encoding one or more T cell growth factors and/or genes encoding one or more T cell activating factors.
The modified vaccinia virus of claim 1, wherein the modified vaccinia virus comprises a thymidine kinase (TK)-deficient vaccinia virus.
The modified vaccinia virus of claim 2, wherein the TK gene in the TK-deficient vaccinia virus comprises one or more mutations.
The modified vaccinia virus according to any one of claims 2-3, wherein all TK genes in the TK-deficient vaccinia virus are deleted.
The modified vaccinia virus according to any one of claims 1-4, wherein the modified vaccinia virus comprises a vaccinia virus in which the A46R gene comprises one or more mutations.
The modified vaccinia virus according to any one of claims 1-5, wherein the modified vaccinia virus comprises a vaccinia virus in which the entire A46R gene has been deleted.
The modified vaccinia virus according to any one of claims 1-6, which comprises a TK-deficient and A46R gene-deleted vaccinia virus.
The modified vaccinia virus according to any one of claims 1-7, which comprises a vaccinia virus in which all TK genes and A46R genes are deleted.
The modified vaccinia virus according to any one of claims 1-8, wherein the chemokines comprise CXCL9, CXCL10 and/or CXCL11.
The modified vaccinia virus according to claim 9, wherein said CXCL9 comprises the amino acid sequence shown in SEQ ID NO:5.
The modified vaccinia virus according to any one of claims 9-10, wherein said CXCL9 comprises the nucleotide sequence shown in SEQ ID NO:6.
The modified vaccinia virus according to any one of claims 9-11, wherein said CXCL10 comprises the amino acid sequence shown in SEQ ID NO:7.
The modified vaccinia virus according to any one of claims 9-12, wherein said CXCL10 comprises the nucleotide sequence shown in SEQ ID NO:8.
The modified vaccinia virus of any one of claims 1-13, wherein the T cell growth factor comprises IL-2.
The modified vaccinia virus according to claim 14, wherein said IL-2 comprises the amino acid sequence shown in SEQ ID NO: 11.
The modified vaccinia virus according to any one of claims 14-15, wherein said IL-2 comprises the nucleotide sequence shown in SEQ ID NO:12.
The modified vaccinia virus of any one of claims 1-16, wherein the T cell activator comprises IL-21.
The modified vaccinia virus according to claim 17, wherein said IL-21 comprises the amino acid sequence shown in SEQ ID NO:9.
The modified vaccinia virus according to any one of claims 17-18, wherein the IL-21 comprises the nucleotide sequence shown in SEQ ID NO:10.
The modified vaccinia virus according to any one of claims 1-19, comprising genes encoding CXCL9, CXCL10, IL-2 and IL-21, respectively.
The modified vaccinia virus according to any one of claims 1-20, wherein the gene encoding a chemokine is located at the position where the TK gene of the modified vaccinia virus is located.
The modified vaccinia virus of claim 21, wherein the TK gene of the modified vaccinia virus is replaced by the gene encoding a chemokine.
The modified vaccinia virus according to any one of claims 1-22, wherein the gene encoding a T cell growth factor and/or the gene encoding a T cell activator is located at the A46R gene of the modified vaccinia virus location.
The modified vaccinia virus according to claim 23, wherein the A46R gene of the modified vaccinia virus is replaced by the gene encoding T cell growth factor and/or the gene encoding T cell activating factor.
The modified vaccinia virus according to any one of claims 1-24, wherein the gene encoding a chemokine is inserted at the position where the TK gene is deleted, and the gene encoding the chemokine is inserted at the position where the A46R gene is deleted. Encoding T cell activating factor and/or the gene encoding T cell growth factor.
The modified vaccinia virus according to any one of claims 20-25, wherein the gene encoding CXCL9 and the gene encoding CXCL10 are inserted at the position where the TK gene is deleted, and the A46R gene is deleted Insert the gene encoding IL-21 and the gene encoding IL-2 at the position.
The modified vaccinia virus according to any one of claims 1-26, wherein the modified vaccinia virus is Lister.
A nucleic acid molecule encoding the modified vaccinia virus of any one of claims 1-27.
A cell comprising the modified vaccinia virus of any one of claims 1-27, and/or, the nucleic acid molecule of claim 28.
The cell of claim 29, wherein the cell comprises a host cell.
The cell according to any one of claims 29-30, wherein the cell comprises a tumor cell.
A pharmaceutical composition comprising the modified vaccinia virus of any one of claims 1-27, and optionally a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 32, which is formulated for systemic administration, and/or, which is formulated for intravenous administration.
a drug combination that contains:

1) The modified vaccinia virus of any one of claims 1-27, and

2) Immune effector cells.
The pharmaceutical combination according to claim 34, wherein the immune effector cells comprise modified immune effector cells.
The pharmaceutical combination according to any one of claims 34-35, wherein the immune effector cells comprise T cells.
The pharmaceutical combination according to any one of claims 34-36, wherein the immune effector cells comprise human T cells.
The modified vaccinia virus according to any one of claims 1-27, the nucleic acid molecule according to claim 28, the cell according to any one of claims 29-31, any one of claims 32-33 The pharmaceutical composition, and/or, the use of the pharmaceutical combination according to any one of claims 34-37 in the preparation of medicines for preventing and/or treating diseases and/or conditions.
The use according to claim 38, wherein the disease and/or condition comprises a tumor.
The use according to any one of claims 38-39, wherein the tumor comprises a solid tumor and/or a non-solid tumor.
A method of increasing the efficiency of a modified vaccinia virus for entry of immune effector cells into a tumor, comprising: providing the modified vaccinia virus with genes encoding one or more chemokines and encoding one or more T cell growth Factor genes and/or genes for one or more T cell activating factors.
The method of claim 41, wherein the modified vaccinia virus comprises one or more mutations in the A46R gene and/or the TK gene.
The method according to any one of claims 41-42, wherein the A46R gene and/or the TK gene of the modified vaccinia virus are all deleted.
The method according to any one of claims 41-43, wherein the modified vaccinia virus comprises genes encoding CXCL9, CXCL10, IL-2 and/or IL-21, respectively.
The method according to any one of claims 43-44, wherein the modified vaccinia virus comprises genes encoding CXCL9, CXCL10, IL-2 and IL-21, respectively.
The method according to any one of claims 41-45, wherein the modified vaccinia virus inserts the gene encoding CXCL9 and the gene encoding CXCL10 at the position where the TK gene is deleted, and inserts the gene encoding IL at the position where the A46R gene is deleted. -21 and the gene encoding IL-2.
The method according to any one of claims 41-46, wherein the modified vaccinia virus is a Listeria strain.