WO2020192684A1 - Agent thérapeutique contenant un adénovirus oncolytique recombinant isolé et des cellules immunitaires, et son utilisation - Google Patents

Agent thérapeutique contenant un adénovirus oncolytique recombinant isolé et des cellules immunitaires, et son utilisation Download PDF

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WO2020192684A1
WO2020192684A1 PCT/CN2020/081090 CN2020081090W WO2020192684A1 WO 2020192684 A1 WO2020192684 A1 WO 2020192684A1 CN 2020081090 W CN2020081090 W CN 2020081090W WO 2020192684 A1 WO2020192684 A1 WO 2020192684A1
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cancer
cells
tumor
cell
virus
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绳纪坡
侯亚非
翁立红
谭贤魁
董小明
王雪
胡放
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杭州康万达医药科技有限公司
合成免疫股份有限公司
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    • 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
    • A61K35/761—Adenovirus
    • 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
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464406—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/861—Adenoviral vectors
    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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
    • A61K2239/50—Colon

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, it relates to a therapeutic agent and application, a kit, and a method for treating tumors and/or cancers comprising isolated recombinant oncolytic adenovirus and immune cells.
  • Tumorigenesis is a multi-factor and multi-step process.
  • the main reason is that cells are stimulated by external carcinogens (including physical radiation, chemical substances, viruses, etc.), and cells are damaged, which leads to changes in DNA and other genetic materials inside the cells.
  • causes the abnormality and disorder of the signal transduction pathway inside the cell the cell manifests as crazy proliferation, resistance to apoptosis, stop differentiation and the ability to invade tissues and migration, and ultimately affect the function of important human organs and endanger life.
  • the main treatment methods for tumors include surgery, radiation therapy, chemotherapy, biological therapy and immunotherapy. Although these treatments can help tumor control to a certain extent, they still cannot solve the problem fundamentally.
  • Oncolytic virus therapy also belongs to the category of biological therapy.
  • the research on oncolytic virus can be traced back to the 1950s. It was discovered that a patient with cervical cancer was infected with rabies virus and the tumor subsided. Inspired by the phenomenon of spontaneous tumor remission in some tumor patients after infection, the first wave of oncolytic virus research began.
  • Oncolytic virus refers to a type of virus that can selectively replicate in target cells after infecting tumor cells, and ultimately lead to tumor cell lysis and death. This type of virus relies on its own specificity to replicate in tumor cells to lyse tumor cells. The virus released after cell lysis can further infect surrounding tumor cells, and at the same time has no damage to normal cells and tissues, or has little impact .
  • Oncolytic viruses are generally divided into two categories: one is wild-type viruses and naturally mutated attenuated virus strains. These viruses naturally have affinity for certain tumor cells, such as reovirus, Newcastle disease virus, and self-replicating small Viruses, etc., these viruses can multiply and lyse cells in certain tumor cells, and have natural specific oncolytic activity; the other type is viruses that can only replicate in tumor cells after modifying the viral genome.
  • adenovirus herpes simplex virus, influenza virus and human vaccinia virus through genetic engineering methods.
  • adenovirus is a relatively early oncolytic virus research. It is relatively clear that the mechanism of oncolysis is relatively early, and the type 5 adenovirus is more clearly studied.
  • Adenovirus has been used to treat malignant tumors of the head and neck shortly after its discovery.
  • the tumor shrinks to varying degrees after injection of adenovirus, but the tumor recurs easily after treatment, and the effect is difficult to last; until 1996, Bischoff et al. first reported the removal of some E1B recombinant Adenovirus Onyx-015 can selectively replicate in tumor cells with abnormal p53 and cause tumor killing. Only when oncolytic adenovirus research has received widespread attention and is developing rapidly, many new types of oncolytic adenovirus have emerged.
  • tumor immunotherapy is also a very important method in the fight against tumors. It mainly includes antibody therapy, T cell therapy and tumor vaccines.
  • Antibodies are called "drugs" for new target molecules for cancer, which can assist in activating effector cells by targeting immune cells around the tumor and promote more effective anti-tumor immunity; they can also kill tumor cells through complement-dependent cytotoxicity, or by induction Tumor cell apoptosis.
  • T cell therapy is a therapy in which tumor-specific autologous T cells (such as CAR-T) expanded in vitro are injected into the body through intravenous administration.
  • Tumor vaccine therapy is a method that modulates the body's immune system to produce specific antibodies and effector T cells, which is called active specific immunotherapy.
  • tumor immunotherapy has played a very active role in the treatment of tumors, but the biggest problem in the process of tumor immunotherapy is tumor escape.
  • the biggest problem in the process of tumor immunotherapy is tumor escape.
  • the immune escape mechanism of tumor There is a very complicated relationship between the immune escape mechanism of tumor and the body's immune response to tumor.
  • the early tumor-specific CD8 + T cells are activated and lose their killing function as the tumor grows to the later stage.
  • T cell activation requires the presentation of MHC-antigen peptides through APC to provide the first signal to antigen-specific T cells, and a series of co-stimulatory molecules to provide the second signal, so that T cells can reach the physiological activation threshold and produce normal Immune response.
  • the second signal provided by costimulatory molecules is absent, it will lead to T cell anergy or specific immune tolerance and even enter apoptosis. Therefore, the regulation of positive and negative co-stimulatory signals and the balance between the two play an important role in the whole process of the body's immune response.
  • T Chimeric Antigen Receptor-T Cell
  • Tumor immunotherapy has become one of the most promising fields, and it is expected to transform malignant tumors into controllable chronic diseases, and even cure certain advanced cancers.
  • an effective tumor immunotherapy strategy is adoptive transfer of T cells obtained in vitro that can effectively recognize tumor antigens.
  • T cells are derived from infiltrating T lymphocytes (TILs) of tumor tissues, or they can be peripheral blood T cells (TCR-T) that can effectively recognize tumor antigens after being genetically modified by antigen-specific T cell receptor (TCR).
  • TILs infiltrating T lymphocytes
  • TCR-T peripheral blood T cells
  • In vitro culture avoids the inhibitory microenvironment of tumor tissues and provides optimized culture conditions, so that a sufficient number of anti-tumor T cells can be obtained for adoptive transfusion therapy (see the literature "Adv Immunol.2016; 130:279-94").
  • Adoptive T cell therapy includes CAR-T therapy based on chimeric antigen receptor and TCR-T therapy based on T cell receptor.
  • CAR-T mainly targets tumor antigens expressed on the cell surface.
  • TCR-T mainly targets polypeptide antigens presented by human major histocompatibility antigen HLA molecules. Epitope polypeptides can be derived from intracellular proteins and cell membrane surface proteins. The types and numbers of target antigens are much greater than CAR-T recognizes. Tumor antigens (see the literature “Int Immunol. 2016 07; 28(7):349-53”).
  • immunotherapy based on TCR-T and CAR-T both exhibit various shortcomings, which affect the therapeutic effect of tumors.
  • the present invention provides therapeutic agents and applications, kits, and methods for treating tumors and/or cancers comprising isolated recombinant oncolytic adenovirus and immune cells.
  • the present invention provides:
  • a therapeutic agent for treating tumor and/or cancer comprising:
  • a first composition wherein the first composition comprises a first active ingredient in a first pharmaceutically acceptable carrier, and the first active ingredient comprises an isolated recombinant oncolytic adenovirus, wherein the recombinant oncolytic
  • the adenovirus is a selective replication-type oncolytic adenovirus, and the recombinant oncolytic adenovirus has integrated into the genome the coding sequence of an exogenous shRNA capable of inhibiting the expression of PDL1 in tumor cells;
  • a second composition wherein the second composition comprises a second active ingredient in a second pharmaceutically acceptable carrier, and the second active ingredient comprises a T cell receptor modified immune cell or a chimeric antigen receptor modification Of immune cells.
  • T cell receptor-modified immune cells include primitive T cells or their precursor cells, NKT cells, or T cell strains; and the chimeric antigen Receptor-modified immune cells include primitive T cells or their precursor cells, NKT cells, T cell strains or NK cells.
  • tumors and/or cancers include breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon Cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder Carcinoma, ureteral carcinoma, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervix Carcinoma, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testi
  • a kit of synergistic combination drugs for the treatment of tumors and/or cancers comprising:
  • a first container containing the first composition of the therapeutic agent according to any one of (1) to (12);
  • a second container containing the second composition in the therapeutic agent according to any one of (1) to (12), wherein the first container and the second container are independent; as well as
  • tumor and/or cancer include: breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon Cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder Carcinoma, ureteral carcinoma, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervix Carcinoma, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular cancer
  • a method of treating tumor and/or cancer including:
  • the second composition of the therapeutic agent according to any one of (1) to (12) is administered to the tumor and/or cancer patient.
  • tumors and/or cancers include breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon Cancer, rectal cancer, brain cancer, liver cancer, bone cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder Carcinoma, ureteral carcinoma, glioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervix Carcinoma, gallbladder cancer, eye cancer, Kaposi's sarcoma, prostate cancer, testicular
  • the present invention has the following advantages and positive effects:
  • the present invention proposes to delete the E1B19K gene, E1B55K gene, and the coding regions of all E3 genes in the oncolytic adenovirus, and at the same time make it carry the coding sequence of exogenous shRNA, so that the obtained recombinant oncolytic adenovirus can selectively be used in tumor cells. It replicates in tumor cells and expresses shRNA that can inhibit PDL1 expression in tumor cells.
  • the oncolytic adenovirus provided based on this concept has strong tumor-killing ability, and its replication ability in normal cells is much lower than its replication ability in tumor cells, so it has low toxicity to normal cells and improves safety And the shRNA expressed by the virus can significantly reduce the expression level of PDL1 protein in tumor cells, thereby reducing the immunosuppression of tumor cells on immune cells (including T lymphocytes and NK cells), thus enhancing the anti-tumor of immune cells Immune killing effect.
  • the present invention finds that integrating the shRNA coding sequence in an oncolytic adenovirus can produce a synergistic effect between the oncolytic killing effect of the oncolytic virus and the anti-tumor immune stimulation effect of immune cells.
  • the inventors of the present invention unexpectedly discovered that the above-mentioned oncolytic adenovirus, in addition to its oncolytic ability on tumor cells, can also enhance immune cells affected by PDL1/PD1 immune checkpoints (such as TCR-T, CAR -T or CAR-NK) has a killing effect on tumors (especially solid tumors), thus having a synergistic effect of 1+1>2.
  • PDL1/PD1 immune checkpoints such as TCR-T, CAR -T or CAR-NK
  • the present invention aims at the problems and difficulties faced by adoptive immune cell therapy and oncolytic virus treatment of tumors. Through theoretical research and experimental verification, the invention proposes the use of the oncolytic adenovirus constructed above and the use of immune checkpoints affected by PDL1/PD1.
  • Immune cells are designed for combined therapy.
  • the therapeutic agents and methods provided based on this concept can fully play the role of the recombinant oncolytic adenovirus of the present invention in selectively replicating in tumor cells and killing tumor cells, and further causing the subsequent immune response of the body, while also being able to fully Play T cell receptor modified immune cells and chimeric antigen receptor modified immune cells to kill tumor cells. This ultimately produces a further enhanced synergistic effect of killing tumors.
  • the combined use of oncolytic viruses and immune killer cells to kill tumors has enriched the treatment modes and methods of tumor treatment, and is very likely to become one of the breakthroughs in the treatment of tumors (especially solid tumors).
  • oncolytic virus refers to a virus capable of selectively replicating in tumor cells and lysing tumor cells.
  • terapéuticaally effective amount refers to the amount of the functional agent or pharmaceutical composition capable of exhibiting a detectable therapeutic effect or inhibitory effect, or an amount capable of exerting an anti-tumor effect. The effect can be detected by any test method known in the art.
  • administration refers to providing a compound, complex or composition (including viruses and cells) to a subject.
  • patient refers to a human or non-human organism. Therefore, the methods and compositions described herein are suitable for human diseases and veterinary diseases.
  • the patient has a tumor. In some cases, the patient has one or more types of cancer at the same time.
  • the term "synergistic effect” as used herein refers to the effect of two or more agents that is greater than the sum of the individual effects of each agent.
  • plaque forming unit refers to: the amount of virus that produces a plaque is called a plaque forming unit (pfu).
  • VP refers to the number of virus particles.
  • VP/kg refers to the number of virus particles per kilogram of patient weight.
  • TID50 refers to the median tissue culture infective dose, which means that half of the tissue culture is infected and cytopathic is caused.
  • MOI Multiplicity of infection
  • Figure 1 shows the gel electrophoresis diagram of PCR amplification of the E1A gene of type 5 adenovirus; lane M is the DNA molecular weight marker, and lane 1 is the PCR product using H101 genomic DNA as a template.
  • Figure 2 shows the PCR screening results of the positive clones of the pShuttle-E1A plasmid; Lane M is the DNA molecular weight marker, and lanes 1-3 are candidate clones.
  • Figure 3 shows the construction process of the pShuttle-E1A plasmid and the map of the constructed plasmid.
  • Figure 4 shows a schematic diagram (left picture) and gel electrophoresis diagram (right picture) of PCR amplification of the E1A expression frame from the pShuttle-E1A plasmid; where lane M is the DNA molecular weight marker, and lanes 1-2 are PCR products.
  • Figure 5 shows the results of PCR screening of pShuttle-MCS-E1A candidate plasmids
  • Lane M is a DNA molecular weight marker
  • lanes 1-13 are candidate plasmids
  • lane NC is a negative control for the PCR system (ie, PCR product with a water template)
  • Lane PC is a positive control for the PCR system (ie, the template is pShuttle-E1A plasmid DNA containing the target fragment).
  • Figure 6 shows the results of BglII digestion of pShuttle-MCS-E1A candidate plasmids; Lane M is the DNA molecular weight marker, samples 1-3 are candidate plasmids, two lanes for each sample, respectively: Lane N is not digested Lane B is the candidate plasmid after digestion with BglII.
  • Figure 7 shows the construction process of the pShuttle-MCS-E1A plasmid and the map of the constructed plasmid.
  • Figure 8 shows the inhibition of human PDL1 mRNA in U251 and H460 cells by three shPDL1 according to an embodiment of the present invention.
  • the axis of abscissa represents 4 sets of cell samples collected at 24h and 48h after treatment of U251 and H460 cells with 4 types of shRNA, and the ordinate represents the expression level of PDL1 mRNA in the cells after each shRNA treatment and the expression level of PDL1 in the cells after control shRNA treatment.
  • the ratio of mRNA expression levels The ratio of mRNA expression levels.
  • Figure 9 shows the inhibition of exogenous hPDL1 expression in 293T cells by three shPDL1 according to an embodiment of the present invention.
  • the left picture shows the results of Western Blot, which shows the expression changes of hPDL1 (with FLAG tag) in cell samples after different shPDL1 treatment of cells and the expression of the protein internal reference ⁇ -actin ( ⁇ -actin) in the cells;
  • the picture on the right shows the gray-scale scan value of hPDL1 band obtained by using the protein internal control ⁇ -actin as a standardization control according to the Western Blot results.
  • the abscissa represents the 293 cell sample group after different shPDL1 treatments, and the “control” refers to only pcDNA3 transfection .3-hPDL1-FLAG
  • the control group expressing hPDL1 (with FLAG tag), the ordinate is the grayscale scan value of the target protein standardized by ⁇ -actin.
  • Figure 10 shows the construction process of the pShuttle-U6-shPDL1-CMV-E1A plasmid and the map of the constructed plasmid.
  • Figure 11 shows the result of restriction digestion of pShuttle-U6-shPDL1-CMV-E1A plasmid; Lane M is the DNA molecular weight marker, Lane C is the control plasmid (pShuttle-MCS-E1A) after digestion with KpnI/HindIII, Lane 1 -7 is the candidate plasmid after KpnI/HindIII digestion.
  • Figure 12 shows a schematic diagram of the process of homologous recombination between pShuttle-U6-shPDL1-CMV-E1A plasmid and pAdEasy-1 in BJ5183 bacteria.
  • Fig. 13 shows a schematic diagram of homologous recombination between pShuttle-related plasmid and pAdEasy-1 during the construction of pAdEasy-U6-shPDL1-CMV-E1A plasmid.
  • Figure 14 shows the result of PacI digestion of the constructed positive pAdEasy-U6-shPDL1-CMV-E1A plasmid; Lane M is the DNA molecular weight marker, and lanes 1-8 are the PacI digestion products of different plasmids, specifically, lanes 1 is C-4.5K PacI digestion product, Lane 2 is PacI digestion product of 1-4.5K, Lane 3 is C-3K PacI digestion product, Lane 4 is 1-3K PacI digestion product, lane 5 is the PacI digestion product of 2-4.5K, lane 6 is the PacI digestion product of 3-4.5K, lane 7 is the PacI digestion product of 2-3K, and lane 8 is the PacI digestion product of 3-3K.
  • Figure 15 shows a schematic diagram of the process of pAdEasy-U6-shPDL1-CMV-E1A plasmid and pAdEasy-CMV-E1A control plasmid respectively completing virus packaging in AD293 cells.
  • Figure 16 shows a schematic diagram of the sample layout of a 12-well plate in an embodiment of the present invention.
  • NC refers to the blank control group without any virus treatment.
  • Figure 17 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 1.
  • OAd-shPDL1#3-4.5K (3-4.5K) Compared with the replication ability of OAd-shPDL1#3-4.5K (3-4.5K)) in different cells, OAd-C-4.5K (C-4.5K) is used as the system control virus.
  • the abscissa represents different oncolytic virus groups, and the ordinate represents the multiple of the copy number of the oncolytic adenovirus specific gene E1A after treatment with the GAPDH gene in the cell as a standardized control.
  • Figure 18 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 2 And OAd-shPDL1#3-4.5K (3-4.5K)) and system control virus OAd-C-4.5K (C-4.5K), control group H101 and control group paclitaxel (Paclitaxel) killing U251 cells.
  • the abscissa represents the amount of different virus infections used to treat the cells (unit is MOI), and the ordinate represents the inhibition rate (%) of cell growth after virus treatment of the cells.
  • the picture on the left is the result of the 48-hour experiment, and the picture on the right is the result of the 72-hour experiment. "***" means p ⁇ 0.001.
  • Figure 19 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 2 And OAd-shPDL1#3-4.5K (3-4.5K)) and the system control virus OAd-C-4.5K (C-4.5K), the control group H101 and the control group paclitaxel have the killing effect on A549 cells.
  • the abscissa represents the amount of different virus infections used to treat the cells (unit is MOI), and the ordinate represents the inhibition rate (%) of cell growth after virus treatment of the cells.
  • the picture on the left is the result of the 48-hour experiment, and the picture on the right is the result of the 72-hour experiment.
  • Figure 20 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 2 And OAd-shPDL1#3-4.5K(3-4.5K)) and system control virus OAd-C-4.5K(C-4.5K), control group H101 and control group paclitaxel have the killing effect on Hela cells.
  • the abscissa represents the amount of different virus infections used to treat the cells (unit is MOI), and the ordinate represents the inhibition rate (%) of cell growth after virus treatment of the cells.
  • the picture on the left is the result of the 48-hour experiment, and the picture on the right is the result of the 72-hour experiment.
  • Figure 21 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 2 Comparing the IC 50 (72h) dose of different cells with OAd-shPDL1#3-4.5K (3-4.5K)) and the system control virus OAd-C-4.5K (C-4.5K) and the control group H101.
  • the abscissa represents different types of tumor cell groups, and the ordinate represents the number of viruses (in MOI) that can kill 50% of the corresponding tumor cells when the virus is incubated for 72 hours.
  • Figure 22 shows the oncolytic adenovirus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 3 ) And OAd-shPDL1#3-4.5K (3-4.5K)) and the system control virus OAd-C-4.5K (C-4.5K) and the control group H101 are overexpressed in the A549/hPD-L1-FLAG cell line Inhibition of hPD-L1.
  • the above figure shows the Western blot results, which show the expression changes of hPDL1 (with FLAG tag) in cell samples after different virus treatment of cells and the expression of the protein internal reference ⁇ -actin in the cells.
  • the “control” refers to those without any virus treatment.
  • Blank control group the following figure shows the gray-scale scan value of hPDL1 band obtained by using the protein internal control ⁇ -actin as the standardization control according to the Western blot results.
  • the abscissa represents different groups, and the ordinate represents the gray scan value.
  • Figure 23 shows the oncolytic adenovirus OAd-shPDL1 (OAd-shPDL1#1-4.5K (1-4.5K), OAd-shPDL1#2-4.5K (2-4.5K) constructed by the present invention in Example 3 ) And OAd-shPDL1#3-4.5K(3-4.5K)) and the control group H101 inhibit the over-expression of hPD-L1 in Hela/hPD-L1-FLAG cell line.
  • the above figure shows the Western blot results, which show the expression changes of hPDL1 (with FLAG tag) in cell samples after different virus treatment of cells and the expression of the protein internal reference ⁇ -actin in the cells.
  • the “control” refers to those without any virus treatment.
  • Blank control group the following figure shows the gray-scale scan value of hPDL1 band obtained by using the protein internal control ⁇ -actin as the standardization control according to the Western blot results.
  • Figure 24 shows a schematic diagram of the p53 and Rb signaling pathways of cells.
  • Figure 25 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 4 And OAd-shPDL1#3-4.5K(3-4.5K)), the system control virus OAd-C-4.5K(C-4.5K), the control group H101 and the control group paclitaxel have the killing effect on HCT116 cells.
  • the abscissa represents the amount of different virus infections used to treat the cells (unit is MOI), and the ordinate represents the inhibition rate (%) of cell growth after virus treatment of the cells.
  • Figure A shows the results of the 48-hour experiment
  • Figure B shows the results of the 72-hour experiment.
  • Figure 26 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 4 And OAd-shPDL1#3-4.5K (3-4.5K)), the system control virus OAd-C-4.5K (C-4.5K), the control group H101 and the control group paclitaxel have the killing effect on PANC1 cells.
  • the abscissa represents the amount of different virus infections used to treat the cells (unit is MOI), and the ordinate represents the inhibition rate (%) of cell growth after virus treatment of the cells.
  • Figure A shows the results of the 48-hour experiment
  • Figure B shows the results of the 72-hour experiment.
  • Figure 27 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 4 And OAd-shPDL1#3-4.5K(3-4.5K)), system control virus OAd-C-4.5K(C-4.5K), control group H101 and control group paclitaxel have the killing effect on HT29 cells.
  • the abscissa represents the amount of different virus infections used to treat the cells (unit is MOI), and the ordinate represents the inhibition rate (%) of cell growth after virus treatment of the cells.
  • Figure A shows the results of the 48-hour experiment
  • Figure B shows the results of the 72-hour experiment.
  • Figure 28 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 4 And OAd-shPDL1#3-4.5K(3-4.5K)), system control virus OAd-C-4.5K(C-4.5K), control group H101 and control group paclitaxel have killing effect on H460 cells.
  • the abscissa represents the amount of different virus infections used to treat the cells (unit is MOI), and the ordinate represents the inhibition rate (%) of cell growth after virus treatment of the cells.
  • Figure A shows the results of the 48-hour experiment
  • Figure B shows the results of the 72-hour experiment.
  • Figure 29 shows the oncolytic virus OAd-shPDL1 (OAd-shPDL1#1-4.5K(1-4.5K), OAd-shPDL1#2-4.5K(2-4.5K) constructed by the present invention in Example 4 Comparing the IC 50 (72h) dose of different cells with OAd-shPDL1#3-4.5K (3-4.5K)) and the system control virus OAd-C-4.5K (C-4.5K) and the control group H101.
  • the abscissa represents different types of tumor cell groups, and the ordinate represents the number of viruses (in MOI) that can kill 50% of the corresponding tumor cells when the virus is incubated for 72 hours.
  • Figure 30 shows that the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 5 affect humans Inhibition of hPDL1 expressed in breast cancer cell MDA-MB-231.
  • Figure A shows the results of Western blot, showing the expression changes of hPDL1 in cell samples and the expression of the protein internal reference ⁇ -actin in cells after different virus treatment of cells.
  • Control refers to the blank control group without any virus treatment;
  • B is the gray-scale scan value of hPDL1 bands obtained by using the protein internal reference ⁇ -actin as a standardized control according to the Western blot results.
  • the abscissa indicates different groups, and the ordinate is the gray-scale scan value.
  • Figure 31 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 5.
  • FACS was used to detect the percentage change of hPDL1 expressing cells on the tumor cell membrane; where "control” refers to a blank control group without any virus treatment.
  • the abscissa represents different groups, and the ordinate represents the percentage (%) of cells expressing hPDL1.
  • Figure 32 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 5.
  • FACS was used to detect the percentage change of cells expressing hPDL1 on the cell membrane; where "control” refers to a blank control group without any virus treatment.
  • the abscissa represents different groups, and the ordinate represents the percentage (%) of cells expressing hPDL1.
  • Figure 33 shows that the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention were detected by Western blot in Example 5.
  • the expression of the internal control ⁇ -actin, "control" refers to the blank control group without any virus treatment.
  • Fig. 34 shows the gray-scale scan value of the hPDL1 band obtained with the protein internal control ⁇ -actin as a normalization control according to the Western blot result of Fig. 33.
  • the abscissa represents different groups, and the ordinate represents the gray scan value.
  • Figure 35 shows the phenotype of Her2/neu 369-377 polypeptide (Her2-E75) specific killer T cells induced from HLA-A2 + normal donor PBMC (specifically #1PBMC) in Example 8 of the present invention And function test results.
  • Figure 35A shows the results of flow cytometric analysis of PBMC cells after two rounds of Her2-E75 antigen polypeptide stimulation in vitro after being stained with CD8-APC antibody and Her2-E75 pentamer-PE. The right figure shows the cells stimulated by the polypeptide. CD8 + pentamer + killer T cell population was sorted by FACS to obtain T cell clones. The left picture shows control cells without peptide stimulation.
  • the abscissa represents the fluorescence intensity expressed by the CD8 molecule, and the ordinate represents the fluorescence intensity of the bound Her2-E75 pentamer.
  • Figure 35B shows the flow cytometric analysis of CD8 + E75-tetramer + killer T cell clones after CD8-APC and Her2-E75 tetramer-PE staining.
  • the right panel shows CD8 + Her2 tetramer + T cells
  • Her2 CTL 6A5 is a purified Her2-E75 polypeptide specific CTL cell clone.
  • the left picture shows the control CTL cells without peptide stimulation.
  • the abscissa represents the fluorescence intensity expressed by the CD8 molecule, and the ordinate represents the fluorescence intensity of the bound Her2-E75 tetramer.
  • Figure 35C shows the main functional fragments of the constructed lentiviral vector carrying the Her2 TCR-6A5-mC gene (ie "pCDH-EF1 ⁇ -Her2 TCR vector"). The fragments shown express the TCR gene driven by the EF-1 ⁇ promoter.
  • the constant region fragments of the ⁇ and ⁇ chains of each TCR are murine constant region fragments, and the ⁇ and ⁇ chains of the TCR are cleavably connected
  • the coding sequence of the polypeptide (furin-F2A) is linked.
  • Figure 36 shows the phenotype and functional test results of peripheral blood mononuclear cells (PBMC) transfected with the Her2 TCR-6A5-mC TCR gene.
  • Figure 36A shows the results of flow cytometric analysis after transfection of a lentiviral vector encoding Her2 TCR-6A5-mC with PBMC from two different donors, staining with Her2-E75 tetramer-PE and anti-CD8-APC antibody. First, the lymphocyte population is divided according to the cell morphology and size. The Her2-E75 tetramer + cell population is the cells expressing Her2 TCR-6A5-mC TCR.
  • PBMC peripheral blood mononuclear cells
  • the abscissa represents the fluorescence intensity expressed by the CD8 molecule, and the ordinate represents the fluorescence intensity of the bound Her2-E75 tetramer.
  • the percentage shown is the ratio of each positive cell population to the number of lymphocytes separated.
  • the left image relates to peripheral blood mononuclear cells (#1PBMC) provided by one donor, and the right image relates to PBMC (#2PBMC) provided by a different donor.
  • CD8 + Her2-E75 tetramer + cells are killer T cells expressing Her2 TCR-6A5-mC.
  • CD8 - Her2-E75 tetramer + cells may be CD4 + helper T cells expressing Her2 TCR-6A5-mC.
  • FIG 36B shows that T cells expressing Her2 TCR-6A5-mC can recognize the Her2-E75 polypeptide presented by T2 cells.
  • Two different donor PBMCs transfected with a lentiviral vector encoding Her2 TCR-6A5-mC were mixed and cultured with T2 cells presenting different concentration gradients of Her2-E75 polypeptide for 16 hours, and the cell supernatant was taken for IFN- ⁇ ELISA analysis.
  • the target cells in the control group are T2 cells (not shown in the figure) that present the EBV virus antigen polypeptide LMP2 426-434 that can bind to HLA-A2 molecules.
  • 0.1 ⁇ g/ml refers to the T2 cell group presenting 0.1 ⁇ g/ml Her2-E75 polypeptide
  • 0.01 ⁇ g/ml refers to the T2 cell group presenting 0.01 ⁇ g/ml Her2-E75 polypeptide
  • 0.001 “ ⁇ g/ml” refers to the T2 cell group presenting 0.001 ⁇ g/ml Her2-E75 polypeptide
  • 0.0001 ⁇ g/ml refers to the T2 cell group presenting 0.0001 ⁇ g/ml Her2-E75 polypeptide.
  • the ordinate represents the concentration of IFN- ⁇ secreted by T cells.
  • Figure 36C shows the results of the CD8 antibody blocking test of T cell function.
  • T2+Her2-E75 means the T2 cell group without anti-human CD8 antibody and presenting 0.1 ⁇ g/ml Her2-E75 polypeptide
  • T2+Her2-E75+anti-CD8 means adding anti-human CD8 antibody The T2 cell group presenting 0.1 ⁇ g/ml Her2-E75 polypeptide.
  • the abscissa represents different experimental groups, and the ordinate represents the concentration of IFN- ⁇ secreted by T cells. "Ns" means that there is no significant difference between the two experimental groups.
  • Each experimental group and control group in Figures 36B and 36C are duplicate holes, and the results are shown as mean ⁇ SEM.
  • Figure 37 shows the results of the functional test of the peripheral blood mononuclear cells (PBMC) transfected with the Her2 TCR-6A5-mC TCR gene to recognize tumor cell lines.
  • Figure 37A shows the expression of HLA-A2 and Her2/neu in different tumor cell lines.
  • the abscissa represents different human tumor cell lines. “Colo205” and “HCT116” are colon cancer cells; “MDA-MB-231” and “MCF-7” are breast cancer cells; “PANC-1” are pancreatic cancer cells; “U87MG” are glioma cells; “NCI-H446” is lung cancer cell.
  • the ordinate "MFI” represents the average fluorescence intensity of cells stained with anti-HLA-A2 fluorescent antibody or anti-Her2/neu fluorescent antibody.
  • FIG. 37B shows the lentiviral vector encoding Her2 TCR-6A5-mC TCR gene transfected with #2 PBMC, mixed with cells of different tumor cell lines for 24 hours, and then the cell supernatant is taken for IFN- ⁇ ELISA analysis results.
  • Each test group and control group are three holes, and the results are shown as mean ⁇ SME.
  • the abscissa shows different target cells, and the ordinate shows the concentration of IFN- ⁇ secreted by T cells.
  • the effective target ratio E:T is 5:1.
  • Figure 37C, D, E, F, G, H, I, J, and K show the killing activity of #2 PBMC on different tumor cell lines after transfection with a lentiviral vector encoding Her2 TCR-6A5-mC TCR gene .
  • the killing activity of Figure 37C-37G was obtained by counting viable cells, and the killing activity of Figure 37H-37K was determined by the MTT method, and the reaction time was 24 hours.
  • Figures 37C and 37H show the results against the tumor cell line MCF-7
  • Figure 37D shows the results against the tumor cell line HCT116
  • Figure 37E shows the results against the tumor cell line U87MG
  • Figure 37F shows the results against the tumor cell line
  • Figure 37G shows the results against the tumor cell line SKOV3
  • Figure 37I shows the results against the tumor cell line PANC-1
  • Figure 37J shows the results against the tumor cell line HEPG2
  • Figure 37K shows the results against the tumor cell line HEPG2.
  • Results of tumor cell line HT-29 Each test group and control group are all three wells, and the results are shown as the mean ⁇ SME.
  • the abscissa shows the different effective target ratio E:T.
  • the ordinate shows the T cell versus target cell The percentage value of the killing rate.
  • the dot-shaped graph shows that the effector cells are control peripheral blood mononuclear cells that have not been transfected with the Her2 TCR-6A5-mC TCR gene.
  • the upper triangle graph shows that the effector cells are Her2 TCR-6A5 -mC TCR gene-transfected peripheral blood mononuclear cells.
  • the other group was added with paclitaxel 10 ⁇ M as a positive control (shown as a single lower triangle point in Figure 37H-37K).
  • Figure 38 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 6.
  • Changes in tumor volume after HCT116 tumor-bearing NOD-SCID immunodeficient mice the gray triangle marked on the abscissa in the figure indicates the time point of the administration treatment, the abscissa indicates the time (days) after the administration treatment, and the ordinate indicates Tumor volume (mm 3 ).
  • Figure 39 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 6.
  • Figure 40 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 6.
  • Figure 41 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 6. After HCT116 tumor-bearing NOD-SCID immunodeficient mice, the pictures of tumors taken out from the mice after sacrifice.
  • Figure 42 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 6. Changes in tumor volume after HCT116 tumor-bearing BALB/C nude mice; the gray triangle marked on the abscissa in the figure indicates the time point of the administration treatment, the abscissa indicates the time (days) after the administration treatment, and the ordinate indicates the tumor Volume (mm 3 ).
  • Figure 43 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 6.
  • Figure 44 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 6.
  • Figure 45 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 6. After HCT116 tumor-bearing BALB/C nude mice, the pictures of tumors taken out from the mice after sacrifice.
  • Figure 46 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 7
  • Figure 47 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 7
  • Figure 48 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed in the present invention in Example 7.
  • Figure 49 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 7.
  • the photos of the tumors taken from the sacrificed mice Figure A
  • the statistical results of the tumors in each group after weighing Figure B
  • the abscissa represents the different groups
  • the ordinate represents the tumor weight (g).
  • Figure 50 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 7.
  • FACS was used to detect the statistical results of the changes in the number of NK cells in the tumor, blood and spleen of each group of mice after normalization.
  • Panel A shows the results in tumors
  • panel B shows the results in blood
  • panel C shows the results in the spleen.
  • the abscissa in each figure represents the different groups set in the experiment, and the ordinate represents the number of NK cells after normalization.
  • Figure 51 shows the treatment with the oncolytic adenovirus OAd-shPDL1#1-4.5K (1-4.5K) and the system control virus OAd-C-4.5K (C-4.5K) constructed by the present invention in Example 7
  • FACS was used to detect the statistical results of the changes in the number of T cells in the tumor, blood and spleen of each group of mice after normalization.
  • Panel A shows the results in tumors
  • panel B shows the results in blood
  • panel C shows the results in the spleen.
  • the abscissa in each figure represents the different groups set in the experiment, and the ordinate represents the number of T cells after normalization.
  • Fig. 52 is a schematic diagram showing an example of the chimeric antigen receptor of the present invention.
  • Figure 53 shows the experimental protocol of Example 12, and specifically shows the dosing protocol of this Example.
  • Fig. 54 shows the curve of the average tumor volume change of each group of animals in Example 12.
  • FIG. 55 shows the curve of tumor volume change of a single animal in each group of animals in Example 12.
  • FIG. 56 shows the curve of the relative tumor growth inhibition rate of each group of animals in Example 12.
  • Fig. 57 shows the average body weight change curve of each group of animals in Example 12.
  • Fig. 58 shows the comparison of tumors and weights among the animals in each group in Example 12.
  • Figure A is a photo of the tumor tissue peeled from the skin of the four groups of animals at the end of the experiment
  • Figure B is a histogram obtained by weighing the subcutaneous tumor tissue peeled from each group of animals.
  • the abscissa represents the different groups
  • the ordinate represents the tumor weight (g).
  • FIG. 59 shows the results of flow cytometry analysis of the number of T cells in the tumor tissues of each group of animals in Example 12.
  • Figure A shows the number of human CD3 + T cells per 100,000 cells in the tumor tissues of the four groups of animals
  • Figure B shows the number of human CD8 + T cells per 100,000 cells in the tumor tissues of the four groups of animals
  • Figure C shows the number of human CD4 + T cells per 100,000 cells in the tumor tissues of the four groups of animals.
  • the abscissa in each figure represents the different groups set in the experiment, and the ordinate represents the number of T cells after normalization.
  • FIG. 60 shows the results of flow cytometry analysis of the number of T cells in the blood of each group of animals in Example 12.
  • Figure A shows the number of human CD3 + T cells per 100,000 cells (except red blood cells) in the blood of the four groups of animals
  • Figure B shows the number of human CD3 + T cells per 100,000 cells (except red blood cells) in the blood of the four groups of animals
  • the number of human CD8 + T cells shows the number of human CD4 + T cells per 100,000 cells (excluding red blood cells) in the blood of the four groups of animals.
  • the abscissa in each figure represents the different groups set in the experiment, and the ordinate represents the number of T cells after normalization.
  • Fig. 61 shows the results of flow cytometry analysis of the number of T cells in the spleen of each group of animals in Example 12.
  • Figure A shows the number of human CD3 + T cells per 100,000 cells in the spleen tissues of the four groups of animals
  • Figure B shows the number of human CD8 + T cells per 100,000 cells in the spleen tissues of the four groups of animals
  • Figure C shows the number of human CD4 + T cells per 100,000 cells in the spleen tissues of the four groups of animals.
  • the abscissa in each figure represents the different groups set in the experiment, and the ordinate represents the number of T cells after normalization.
  • Figure 62 shows the percentage of Her2 + /PDL1 + positive cells in the tumor tissues of each group of animals in Example 12.
  • the abscissa in the figure represents the different groups set in the experiment, and the ordinate represents the percentage (%) of Her2 + /PDL1 + positive cells in the tumor tissue.
  • the human body is a complex system, which is composed of ten major systems such as breathing, circulation, and digestion. These systems coordinate and cooperate to enable various complex life activities in the human body to proceed normally.
  • the body's anti-tumor mechanisms include cellular immunity and humoral immunity. They are closely related, influence each other, and involve a variety of immune effector molecules and effector cells. It is generally believed that cellular immunity plays a leading role in the anti-tumor process, and humoral immunity plays a synergistic effect in some cases.
  • the present invention proposes to take advantage of the characteristics of oncolytic adenovirus to selectively replicate and kill tumor cells in tumor cells, and at the same time make it carry the coding sequence of exogenous shRNA that can inhibit the expression of PDL1 in tumor cells, so as to make recombinant oncolytic glands
  • the virus cooperates to exert selective oncolysis and enhance the body's anti-tumor immune effect.
  • the inventor of the present invention through experimental research and theoretical exploration found that the oncolytic adenovirus E1B19K gene, E1B55K gene, and all the coding regions of the E3 gene were deleted at the same time, and the exogenous shRNA was integrated in the genome.
  • the coding sequence can well realize the above-mentioned synergy.
  • DNA viruses such as adenovirus
  • adenovirus can change the cell cycle of host cells.
  • the main reason is that the virus produces some proteins that act on the cell cycle regulatory protein of the host cell, allowing quiescent cells to enter the cell cycle to facilitate viral DNA replication.
  • Different viruses have different mechanisms to influence the cell cycle.
  • Adenoviruses mainly interfere with the cell cycle of host cells through Rb and p53 cell signaling pathways ( Figure 24) (refer to the literature: "Chen Jianfa et al., Research Progress of Oncolytic Adenovirus, Cancer Prevention Research, 2004) , 31(4): 243-245.”).
  • the oncolytic function of oncolytic adenovirus is based on this principle by changing the expression of adenovirus to regulate host cell cyclin.
  • the adenovirus genome includes 4 early transcription units (E1, E2, E3 and E4) with regulatory functions and 1 late transcription unit.
  • E1 is divided into two parts, E1A and E1B.
  • E1A combines with Rb to release free E2F, and the cell enters S phase from G1 phase.
  • Adenovirus simultaneously encodes and produces E1B55k and E1B19k proteins to inhibit p53 and Bax, respectively, and make the host Cell division and proliferation are not inhibited by the p53 cell signaling pathway.
  • a large number of host cells enter the division phase from the resting phase, and adenoviruses can replicate and multiply in large numbers.
  • the adenovirus with the E1A gene removed infects host cells, it cannot encode and produce E1A protein to release free E2F, and cells in G1 phase cannot enter S phase. Similarly, even if the E1B gene-removed adenovirus can produce E1A protein to make the host cell enter the S phase from the G1 phase, the cells entering the division cycle will also undergo apoptosis or block division through the p53 signaling pathway. Therefore, the adenovirus with E1A or E1B gene removed cannot replicate and proliferate in host cells with normal Rb and p53 signal pathways, and only tumor cells with abnormal Rb or p53 signal pathways can proliferate.
  • E1B55K expression cannot inhibit the function of wild-type p53
  • the normal expression of E1B19K protein can still inhibit the function of Bax downstream of p53, thereby making oncolytic glands.
  • the virus can replicate in normal cells.
  • the oncolytic adenovirus described in the present invention in addition to deleting the E3 region, two genes E1B55K and E1B19K are also deleted at the same time. Therefore, this type of virus is more selective in tumor cells than the oncolytic adenovirus in the prior art. Better, the ability to replicate in normal cells is lower, and the safety to normal cells is better.
  • PD-L1 (also known as PDL1 or B7-H1) belongs to the B7 family and has IgV and IgC-like regions, transmembrane regions and cytoplasmic regions. This molecule has a broad tissue expression profile and is highly expressed on some tumor cell lines. Many studies have shown that it is related to the immune escape mechanism of tumors. The microenvironment of the tumor site can induce the expression of PD-L1 on tumor cells, and the expression is extensive. The expressed PD-L1 is conducive to the occurrence and growth of tumors.
  • the PD-L1 expressed by tumor cells and APCs in the tumor microenvironment interacts with the receptor PD1 on T cells through the PD-1/PD-L1 signaling pathway to inhibit the activation of tumor antigen-specific T cells and down-regulate T cell-mediated Tumor immune response.
  • blocking the PD-L1/PD-1 signaling pathway can promote the proliferation of tumor antigen-specific T cells, up-regulate the secretion of IFN- ⁇ infiltrating CD8 + T cells, and effectively inhibit tumor growth, indicating that PD-1/PD
  • the blocking of L1 signaling pathway plays an important role in tumor immune response for the purpose of inducing immune response.
  • choosing anti-PD-L1 monoclonal antibody combined with tumor vaccines for tumor immunotherapy can effectively enhance the immune activation of tumor vaccines and reduce the impact of tumor microenvironment on the efficacy.
  • the oncolytic adenovirus of the present invention has not only modified the oncolytic virus genome structure to make it have higher oncolytic killing ability, but also added a shRNA that can express shPDL1 (shRNA that inhibits PDL1 expression) Encoding frame, it is expected that shPDL1 can efficiently degrade the mRNA of PDL1 in cells to achieve gene silencing, thereby reducing the expression of PDL1 in tumor cells, reducing the transmission of PD1/PDL1 signaling pathways to immune cell suppression signals, and enhancing the killing effect of immune cells on tumors .
  • shPDL1 shRNA that inhibits PDL1 expression
  • the oncolytic virus of the present invention can be used as an oncolytic agent alone or as an effective vector for the coding frame of shPDL1, allowing shPDL1 to be expressed in large quantities along with virus replication, and at the same time exerting the dual functions of virus therapy and gene therapy.
  • the inventors of the present invention unexpectedly discovered that, in addition to its oncolytic ability on tumor cells, the oncolytic adenovirus can also enhance immunity affected by the PDL1/PD1 immune checkpoint.
  • Cells such as TCR-T, CAR-T or CAR-NK
  • the present invention aims at the problems and difficulties faced by adoptive immune cell therapy and oncolytic virus treatment of tumors.
  • the invention proposes the use of the oncolytic adenovirus constructed above and the use of immune checkpoints affected by PDL1/PD1.
  • Immune cells are designed for combined therapy.
  • the combined use of oncolytic viruses and immune killer cells to kill tumors has enriched the treatment modes and methods of tumor treatment, and is very likely to become one of the breakthroughs in the treatment of tumors (especially solid tumors).
  • the present invention provides a therapeutic agent for treating tumors and/or cancers, comprising:
  • a first composition wherein the first composition comprises a first active ingredient in a first pharmaceutically acceptable carrier, and the first active ingredient comprises an isolated recombinant oncolytic adenovirus, wherein the recombinant oncolytic
  • the adenovirus is a selective replication-type oncolytic adenovirus, and the recombinant oncolytic adenovirus has integrated into the genome the coding sequence of an exogenous shRNA capable of inhibiting the expression of PDL1 in tumor cells;
  • a second composition wherein the second composition comprises a second active ingredient in a second pharmaceutically acceptable carrier, and the second active ingredient comprises a T cell receptor modified immune cell or a chimeric antigen receptor modification Of immune cells.
  • the present invention adopts an isolated recombinant oncolytic adenovirus, wherein the recombinant oncolytic adenovirus is a selective replication type oncolytic adenovirus, and the recombinant oncolytic adenovirus has a genome capable of inhibiting the expression of PDL1 in tumor cells.
  • the coding sequence of the foreign shRNA is a selective replication type oncolytic adenovirus, and the recombinant oncolytic adenovirus has a genome capable of inhibiting the expression of PDL1 in tumor cells.
  • the coding sequence of the exogenous shRNA is shown in any one of SEQ ID NOs. 45, 48, and 51.
  • the E1B19K gene, E1B55K gene, and all E3 region genes are deleted from the genome of the recombinant oncolytic adenovirus.
  • the possible mechanisms of tumor cell lysis after oncolytic viruses enter tumor cells are as follows: (1) Direct cytotoxicity of viral proteins: For example, the death protein and late protein produced by adenovirus can effectively mediate tumor cell lysis. (2) Produce anti-tumor immune response: On the one hand, the virus can play a tumor-killing effect by enhancing the sensitivity of tumor cells to a variety of cytokines. For example, adenovirus can enhance the effect of replicating and expressing E1A protein in infected tumor cells.
  • Tumor-killing effect mediated by tumor necrosis factor when tumor cells are infected by viruses, the viral antigens on the surface of tumor cells form complexes with the major histocompatibility complex type I antigens, which are easily affected by cytotoxic T lymphocytes. Recognized by cells, thereby mediating specific attacks on virus-infected tumor cells.
  • the product of adenovirus E1A gene expression is a powerful chemosensitizer. The expression product of E1A gene in tumor cells can induce high-level expression of p53 protein, and In order to enhance the damage of chemotherapy and radiotherapy to DNA.
  • the genome of the recombinant oncolytic adenovirus contains the E1A gene coding sequence. It is also preferred that the E1A gene coding sequence is under the control of the CMV promoter, so as to increase the expression of E1A to enhance its oncolytic and killing effect on tumor cells.
  • the recombinant oncolytic adenovirus is obtained by genetically modifying type 5 adenovirus.
  • adenovirus type 5 is H101.
  • the oncolytic adenovirus genome integrates a coding frame including the U6 promoter and human PDL1 shRNA (shPDL1) after the ES sequence, as well as the CMV promoter, E1A coding region and part of the 3'end E1A expression cassette including UTR region and SV40polyA.
  • shPDL1 human PDL1 shRNA
  • the recombinant oncolytic adenovirus of the present invention affects a variety of human tumor cells (e.g., human glioma cell U251, human lung cancer cell A549, human cervical cancer cell Hela, human large cell lung cancer H460, human colorectal cancer cell HCT116, human pancreas Cancer cells PANC1, human colon cancer cells HT29, etc.) have strong killing ability.
  • human tumor cells e.g., human glioma cell U251, human lung cancer cell A549, human cervical cancer cell Hela, human large cell lung cancer H460, human colorectal cancer cell HCT116, human pancreas Cancer cells PANC1, human colon cancer cells HT29, etc.
  • the replication ability of the virus in human normal primary cells is far lower than its replication ability in human tumor cells (a difference of about 2 orders of magnitude).
  • the human shPDL1 expressed by the virus can significantly reduce the level of PDL1 protein highly expressed in human tumor cells.
  • the present invention also relates to a vector for preparing the recombinant oncolytic adenovirus of the present invention, wherein the vector contains an exogenous shRNA coding sequence under the control of a promoter, and the shRNA coding sequence is as SEQ ID NOs. 45, Either 48 and 51 are shown.
  • the vector adopts pShuttle as the basic skeleton, and the basic skeleton sequentially includes an operably linked promoter that controls the expression of the exogenous shRNA coding sequence, and the exogenous shRNA coding sequence.
  • the invention also relates to a host cell containing the vector of the invention.
  • the host cell stably expresses the vector.
  • the present invention also relates to an isolated shRNA, wherein the coding sequence of the shRNA is shown in any one of SEQ ID NOs. 45, 48 and 51, and the shRNA can inhibit the expression of PDL1 in tumor cells.
  • the present invention also relates to an isolated recombinant oncolytic adenovirus, wherein the recombinant oncolytic adenovirus is a selective replication type oncolytic adenovirus, and the genome of the recombinant oncolytic adenovirus has deleted E1B19K gene, E1B55K gene, and all E3 region gene; preferably, the recombinant oncolytic adenovirus contains the E1A gene coding sequence in the genome; further preferably, the E1A gene coding sequence is under the control of the CMV promoter.
  • the oncolytic adenovirus has strong tumor-killing ability, and its replication ability in normal cells is far lower than its replication ability in tumor cells, so it has low toxicity to normal cells and improves safety.
  • the immune cells modified by T cell receptors include primitive T cells or their precursor cells, NKT cells, or T cell strains.
  • the T cell receptor includes at least one of an ⁇ chain and a ⁇ chain, and both the ⁇ chain and the ⁇ chain include a variable region and a constant region, and the T cell receptor can specifically recognize tumor cells and/or cancer The epitope polypeptide on the cell surface.
  • the T cell receptor can specifically recognize the antigen Her2/neu expressed by tumor cells, and the amino acid sequence of the variable region of the ⁇ chain is at least the same as that shown in SEQ ID NO:1. 98%, preferably at least 98.5%, more preferably at least 99% identity, the amino acid sequence of the variable region of the ⁇ chain has at least 98%, preferably at least 98.5% of the amino acid sequence shown in SEQ ID NO: 2 , More preferably at least 99% consistency, as long as it does not significantly affect the effect of the present invention. It is also preferred that the amino acid sequence of the variable region of the ⁇ chain is shown in SEQ ID NO: 1, and the amino acid sequence of the variable region of the ⁇ chain is shown in SEQ ID NO: 2.
  • variable regions of the TCR ⁇ chain and ⁇ chain are used to bind the antigen polypeptide/major histocompatibility complex (MHC I), and respectively include three hypervariable regions or called complementarity determining regions (complementarity determining regions, CDRs), that is, CDR1, CDR2, CDR3. Among them, the CDR3 region is very important for the specific recognition of antigen peptides presented by MHC molecules.
  • the TCR ⁇ chain is a recombination of different V and J gene segments, and the ⁇ chain is a recombination of different V, D, and J gene segments.
  • the MHC class I molecules include human HLA.
  • the HLA includes: HLA-A, B, and C.
  • the T cell receptor can specifically recognize the epitope polypeptide of the antigen Her2/neu presented by the HLA-A2 molecule.
  • the amino acid sequence of the antigen Her2/neu is shown in SEQ ID NO:21.
  • the epitope polypeptide includes Her2/neu 369-377 as shown in SEQ ID NO: 3.
  • HLA-A2 alleles expressed by HLA-A2 positive cells include HLA-A*0201, 0202, 0203, 0204, 0205, 0206, and 0207.
  • the HLA-A2 molecule is HLA-A*0201.
  • the epitope polypeptide of the antigen Her2/neu is the Her2/neu 369-377 polypeptide (SEQ ID NO: 3). In other embodiments, the epitope polypeptide of the antigen Her2/neu has 4-9 consecutive identical amino acids (for example, 4, 5, 6, 7, 8 or 9) with the Her2/neu 369-377 polypeptide. Consecutive identical amino acids) epitope polypeptides, and these polypeptides are 8-11 amino acids in length. For example, in one embodiment, the epitope polypeptide of the antigen Her2/neu is the Her2/neu 373-382 polypeptide (SEQ ID NO: 22).
  • the maximum half-reactive polypeptide concentration of the Her2/neu 369-377 polypeptide recognized by the T cell receptor is between 1.0-10 ng/ml. In one embodiment of the present invention, the maximum half-reactive polypeptide concentration is about 1.6 ng/ml-2.9 ng/ml.
  • the term "maximum half-reactive polypeptide concentration" refers to the concentration of the polypeptide required to induce 50% of the maximum T cell response. According to reports, the maximum half-reactive polypeptide concentration of specific T cells against cytomegalovirus (CMV) antigen CMV pp65 (495-503) polypeptide is between 0.1-1ng/ml, and this TCR is considered to have a high level of CMV antigen polypeptide.
  • CMV cytomegalovirus
  • the T cell receptor has a medium to high affinity for the Her2/neu antigen, thereby avoiding possible off-target toxicity caused by high affinity (the maximum half-reactive polypeptide concentration is less than 0.1 ng/ml).
  • the recognition of the Her2/neu 369-377 polypeptide by the T cell receptor does not rely on the assistance of the CD8 molecule, and the expression of CD8 negative CD4 positive T cells can also specifically recognize Her2 presented by HLA-A2.
  • /neu 369-377 polypeptide secretes cytokines, thereby enhancing the function of killer T cells that express the T cell receptor.
  • the exogenous TCR ⁇ and ⁇ chains expressed by T cells may be mismatched with the ⁇ and ⁇ chains of the TCR itself, which will not only dilute the expression of the correctly paired foreign TCR, but also the antigen specificity of the mismatched TCR. It is clear that there is a potential risk of recognizing self-antigens, so it is preferable to modify the constant regions of the TCR ⁇ chain and ⁇ chain to reduce or avoid mismatches.
  • the constant region of the ⁇ chain and/or the constant region of the ⁇ chain are derived from humans; preferably, the present invention has found that the constant region of the ⁇ chain may be all or part of The ground is replaced by a homologous sequence derived from another species, and/or the constant region of the ⁇ chain may be replaced in whole or in part by a homologous sequence derived from another species. More preferably, the other species is mouse.
  • the replacement can increase the expression level of TCR in the cell, and can further improve the specificity of the cell modified by the TCR to the Her2/neu antigen.
  • the constant region of the ⁇ chain may be modified with one or more disulfide bonds, and/or the constant region of the ⁇ chain may be modified with one or more disulfide bonds, for example, 1 or 2 disulfide bonds.
  • two TCRs modified in different ways are prepared.
  • One way is to add a disulfide bond in the constant region of the TCR through point mutation.
  • the method is described in the document "Cancer Res. 2007 Apr 15; 67(8): 3898-903.", which is incorporated herein by reference in its entirety.
  • Her2 TCR-1B5-mC replaces the corresponding human TCR constant region sequence with the mouse TCR constant region sequence.
  • the method is described in the document "Eur.J.Immunol.2006 36:3052-3059", which is incorporated by reference in its entirety. This article.
  • amino acid sequence of the ⁇ chain is shown in SEQ ID NOs: 4, 5 or 6, and the amino acid sequence of the ⁇ chain is shown in SEQ ID NOs: 7, 8 or 9.
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 4, and the amino acid sequence of the ⁇ chain is shown in SEQ ID NO: 7.
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 5
  • the amino acid sequence of the ⁇ chain is shown in SEQ ID NO: 8.
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 6, and the amino acid sequence of the ⁇ chain is shown in SEQ ID NO: 9.
  • the ⁇ chain of the TCR has an amino acid sequence obtained by substituting, deleting, and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NOs: 4, 5 or 6.
  • the ⁇ chain has at least 90%, preferably at least 95%, and more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 4, 5 or 6.
  • the ⁇ chain of the TCR has an amino acid sequence obtained by substituting, deleting, and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NOs: 7, 8 or 9
  • the ⁇ chain has at least 90%, preferably at least 95%, and more preferably at least 99% identity with the amino acid sequence shown in SEQ ID NOs: 7, 8 or 9.
  • the alpha chain and/or beta chain of the TCR of the present invention can also be combined with other functional sequences at the end (such as the C-terminus), such as the functional region sequence of the costimulatory signal CD28, 4-1BB and/or CD3zeta.
  • the present invention also relates to an isolated nucleic acid encoding a T cell receptor, comprising the coding sequence of at least one of the ⁇ chain and the ⁇ chain of the T cell receptor, and the ⁇ chain coding sequence and the ⁇ chain coding sequence both comprise Variable region coding sequence and constant region coding sequence, the T cell receptor can specifically recognize the epitope polypeptide on the surface of tumor cells and/or cancer cells.
  • the T cell receptor can specifically recognize the antigen Her2/neu expressed by tumor cells, and the amino acid sequence encoded by the alpha chain variable region coding sequence has at least 98% of the amino acid sequence shown in SEQ ID NO:1. %, preferably at least 98.5%, more preferably at least 99% identity, the amino acid sequence encoded by the ⁇ -chain variable region coding sequence has at least 98%, preferably at least 98.5%, and the amino acid sequence shown in SEQ ID NO: 2, A uniformity of at least 99% is more preferable, as long as it does not significantly affect the effect of the present invention. It is also preferred that the alpha chain variable region coding sequence encodes the amino acid sequence shown in SEQ ID NO: 1, and the beta chain variable region coding sequence encodes the amino acid sequence shown in SEQ ID NO: 2.
  • the nucleic acid can be DNA or RNA.
  • the coding sequence of the ⁇ chain variable region is shown in SEQ ID NO: 10, and the coding sequence of the ⁇ chain variable region is shown in SEQ ID NO: 11.
  • the T cell receptor encoded by the nucleic acid can specifically recognize the epitope polypeptide of the antigen Her2/neu presented by the HLA-A2 molecule.
  • the epitope polypeptide of the antigen Her2/neu is the Her2/neu 369-377 polypeptide (SEQ ID NO: 3). In other embodiments, the epitope polypeptide of the antigen Her2/neu has 4-9 consecutive identical amino acids (for example, 4, 5, 6, 7, 8 or 9) with the Her2/neu 369-377 polypeptide. Consecutive identical amino acids) epitope polypeptides, and the length of these polypeptides is 8-10 amino acids. For example, in one embodiment, the epitope polypeptide of the antigen Her2/neu is the Her2/neu 373-382 polypeptide (SEQ ID NO: 22).
  • the maximum half-reactive polypeptide concentration of the Her2/neu 369-377 polypeptide recognized by the T cell receptor encoded by the nucleic acid is between 1.0-10ng/ml (for example, between 3.0-8.0ng/ml, 5.0- 7.0ng/ml). In one embodiment of the present invention, the maximum half-reactive polypeptide concentration is about 1.6-2.9 ng/ml. In this case, the T cell receptor has a medium to high affinity to the Her2/neu antigen, which can avoid possible off-target toxicity caused by high affinity (the maximum half-reactive polypeptide concentration is less than 0.1 ng/ml).
  • the constant region of the ⁇ chain and/or the constant region of the ⁇ chain is derived from humans; preferably, the coding sequence of the ⁇ chain constant region is derived in whole or in part from other The homologous sequence of the species is replaced, and/or the beta chain constant region coding sequence is replaced in whole or in part by the homologous sequence derived from other species. More preferably, the other species is mouse.
  • the replacement can increase the expression level of TCR in the cell, and can further improve the specificity of the cell modified by the TCR to the Her2/neu antigen.
  • the ⁇ -chain constant region coding sequence may include one or more disulfide bond coding sequences, and/or the ⁇ -chain constant region coding sequence may include one or more disulfide bond coding sequences.
  • the alpha chain coding sequence is shown in SEQ ID NOs: 12, 13, or 14, and the beta chain coding sequence is shown in SEQ ID NOs: 15, 16 or 17.
  • the sequence is the original human sequence; for the ⁇ chain with the coding sequence shown in SEQ ID NO: 16, its constant region is modified with a double Sulfur bond: For the ⁇ chain whose coding sequence is shown in SEQ ID NO: 17, the constant region is replaced with a murine constant region.
  • the coding sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 12, and the coding sequence of the ⁇ chain is shown in SEQ ID NO: 15.
  • the coding sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 13, and the coding sequence of the ⁇ chain is shown in SEQ ID NO: 16.
  • the coding sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 14, and the coding sequence of the ⁇ chain is shown in SEQ ID NO: 17.
  • the ⁇ -chain coding sequence and the ⁇ -chain coding sequence are connected by a cleavable linking polypeptide coding sequence, which can increase the expression of TCR in the cell.
  • cleavable linking polypeptide means that the polypeptide plays a linking role and can be cleaved by a specific enzyme, or the nucleic acid sequence encoding this polypeptide is translated by ribosome skipping, so that it is linked by it.
  • the polypeptides are separated from each other. Examples of cleavable linking polypeptides are known in the art, such as F2A polypeptides.
  • F2A polypeptide sequences include, but are not limited to, F2A polypeptides from Picornavirus and similar Class 2A sequences from other viruses.
  • the cleavable linking polypeptide also includes a standard four amino acid motif (canonical four amino acid motif) that can be cleaved by Furin enzyme, that is, the R-X-[KR]-R amino acid sequence.
  • the TCR encoded in this embodiment is a single-chain chimeric T cell receptor. After the single-chain chimeric T cell receptor is expressed, the cleavable linking polypeptide connecting the ⁇ chain and the ⁇ chain will be cleaved by a specific enzyme in the cell , Thereby forming an equal amount of free ⁇ chain and ⁇ chain.
  • the ⁇ chain and ⁇ chain that make up the single-chain chimeric TCR can also be as described above, and the constant region (and its corresponding coding sequence) can be replaced in whole or in part by homologous sequences derived from other species, and/or modified with (Code) One or more disulfide bonds.
  • sequence of the nucleic acid is shown in SEQ ID NOs: 18, 19, or 20.
  • the nucleotide sequence of the nucleic acid is optimized to increase gene expression, protein translation efficiency and protein expression, thereby enhancing the ability of TCR to recognize antigens.
  • Codon optimization includes, but is not limited to, modification of the translation initiation region, changes in mRNA structural fragments, and the use of different codons encoding the same amino acid.
  • mutations can be made to the sequence of the aforementioned TCR-encoding nucleic acid, including removal, insertion and/or substitution of one or more amino acid codons, so that the expressed TCR's function of recognizing the Her2/neu antigen remains unchanged or enhanced .
  • conservative amino acid substitutions are performed, including the substitution of one amino acid in the variable region of the above-mentioned TCR alpha chain and/or beta chain with another amino acid with similar structure and/or chemical properties.
  • similar amino acids refers to amino acid residues with similar properties such as polarity, electrical load, solubility, hydrophobicity, and hydrophilicity.
  • TCR maturation (TCR maturation) modification is performed, that is, it includes the amino acids in the complementarity determining region 2 (CDR2) and/or CDR3 region in the variable region of the above-mentioned TCR ⁇ chain and/or ⁇ chain. Removal, insertion, and/or replacement, thereby changing the affinity of TCR to bind Her2/neu antigen.
  • CDR2 complementarity determining region 2
  • the invention also relates to an isolated mRNA transcribed from the DNA according to the invention.
  • the present invention also relates to a recombinant expression vector, which contains the nucleic acid (such as DNA) according to the present invention operatively linked to a promoter, and/or its complementary sequence.
  • the DNA of the present invention is suitably effectively connected to a promoter, enhancer, terminator and/or polyA signal sequence.
  • the combination of the above-mentioned action elements of the recombinant expression vector of the present invention can promote DNA transcription and translation, and enhance the stability of mRNA.
  • the basic skeleton of a recombinant expression vector can be any known expression vector, including plasmids or viruses.
  • Viral vectors include, but are not limited to, for example, retroviral vectors (the virus prototype is Moloney murine leukemia virus (MMLV)) and lentivirus Vector (the virus prototype is human immunodeficiency type I virus (HIV)).
  • the recombinant vector expressing the TCR of the present invention can be obtained by conventional recombinant DNA technology in the art.
  • the expression of the ⁇ -chain and ⁇ -chain genes on the recombinant expression vector can be driven by two different promoters.
  • the promoters include various known types, such as strong expression, weak expression, and continuous expression. Expressed, inducible, tissue-specific, and differentiation-specific promoters.
  • the promoter can be of viral or non-viral origin (e.g. eukaryotic promoter), such as the CMV promoter, the promoter on the LTR of MSCV, the EF1- ⁇ promoter, and the PGK-1 promoter, SV40 promoter , Ubc promoter, CAG promoter, TRE promoter, CaMKIIa promoter, human ⁇ -actin promoter.
  • the driving directions of the two promoters can be the same or reverse.
  • the expression of the ⁇ chain and ⁇ chain genes on the recombinant expression vector can be driven by the same promoter, for example, in the case of encoding single-stranded chimeric T cell receptors, the nucleotide sequence of the ⁇ chain and The nucleotide sequence of the ⁇ chain is connected by the Furin-F2A polypeptide coding sequence.
  • the recombinant expression vector may contain coding sequences for other functional molecules in addition to the ⁇ -chain and ⁇ -chain genes.
  • One embodiment includes the expression of autofluorescent proteins (such as GFP or other fluorescent proteins) for in vivo tracking imaging.
  • Another embodiment includes the expression of an inducible suicide gene system, such as inducing the expression of herpes simplex virus-thymidine kinase (HSV-TK) protein, or inducing the expression of Caspase 9 (iCasp9) protein.
  • HSV-TK herpes simplex virus-thymidine kinase
  • iCasp9 Caspase 9
  • Embodiments include the expression of human chemokine receptor genes, such as CCR2. These chemokine receptors can bind to corresponding chemokine ligands that are highly expressed in tumor tissues, thereby increasing the presence of TCR gene-modified cells in tumors. Homing in the organization.
  • the present invention adopts a T cell receptor modified immune cell whose surface is modified by the T cell receptor of the present invention, wherein the cell includes primitive T cells or their precursor cells, NKT cells, or T cells Strain.
  • the "modification" in the "T cell receptor modification” refers to the expression of the T cell receptor of the present invention by gene transfection, that is, the T cell receptor is anchored in the transmembrane region.
  • the cell membrane of the modified cell has the function of recognizing the antigen polypeptide/MHC complex.
  • the present invention also relates to a method for preparing T cell receptor modified immune cells according to the present invention, which includes the following steps:
  • the cells described in step 1) can be derived from mammals, including humans, dogs, mice, rats, and transgenic animals.
  • the cells can be derived from autologous or foreign bodies.
  • Allogeneic cells include cells from identical twins, allogeneic stem cells, and genetically modified allogeneic T cells.
  • the cells in step 1) include primitive T cells or their precursor cells, NKT cells, or T cell strains.
  • the term "naive T cell” refers to mature T cells in peripheral blood that have not been activated by the corresponding antigen.
  • These cells can be isolated by methods known in the art.
  • T cells can be obtained from different tissues and organs, including peripheral blood, bone marrow, lymphoid tissue, spleen, cord blood, and tumor tissue.
  • T cells can be derived from hematopoietic stem cells (HSCs), including bone marrow, peripheral blood or cord blood, and obtained by isolation of stem cell marker molecules such as CD34.
  • HSCs hematopoietic stem cells
  • T cells can be derived from induced pluripotent stem cells (iPS cells), including the introduction of specific genes or specific gene products into somatic cells to transform the somatic cells into stem cells and then induce differentiation into T cells or their precursors in vitro cell.
  • T cells can be obtained by common methods such as density gradient centrifugation. Examples of density gradient centrifugation include Ficoll or Percoll density centrifugation.
  • density gradient centrifugation include Ficoll or Percoll density centrifugation.
  • One embodiment is to use apheresis or leukapheresis to obtain enriched T cell products from peripheral blood.
  • One embodiment is to label specific cell populations with antibodies and then separate them by magnetic beads (such as System (Miltenyi Biotec)), or flow cytometry to obtain enriched CD8 + or CD4 + T cells.
  • the T cell precursor cells are hematopoietic stem cells.
  • the TCR coding gene of the present invention can be directly introduced into hematopoietic stem cells, and then transferred into the body to further differentiate into mature T cells; or the coding gene can be introduced into T cells that are differentiated from hematopoietic stem cells under specific conditions in vitro.
  • the cells can be resuspended in a cryopreservation solution and placed in liquid nitrogen for storage.
  • cryopreservation solutions include but are not limited to a PBS solution containing 20% DMSO and 80% human serum albumin.
  • the cells were frozen at -80°C with a temperature decrease of 1°C per minute, and then stored in the gas phase of a liquid nitrogen tank.
  • Other cryopreservation methods are to put the cells in the freezing solution directly into -80°C or liquid nitrogen for freezing.
  • the nucleic acid in step 2) is the nucleic acid according to the present invention, including the DNA and RNA.
  • the transfection includes physical, biological and chemical methods.
  • the physical method is to introduce the TCR gene into the cell in the form of DNA or RNA through calcium phosphate precipitation, liposome, microinjection, electroporation, gene gun, etc.
  • electrotransfer instruments such as Amaxa Nucleofector-II (Amaxa Biosystems, Germany), ECM 830 (BTX) (Harvard Instruments, U.S.), Gene Pulser II (BioRad, U.S.), Multiporator (Eppendort, Germany) ).
  • the biological method is to introduce TCR genes into cells through DNA or RNA vectors.
  • Retroviral vectors are commonly used tools for transfection and insertion of foreign gene fragments into animal cells (including human cells).
  • Other viral vectors are derived from lentivirus, poxvirus, herpes simplex virus, adenovirus and adenovirus-related viruses, etc.
  • the chemical method is to introduce polynucleotides into cells, including colloidal dispersion systems, such as macromolecular complexes, nanocapsules, Microspheres, microbeads, micelles and liposomes.
  • various detection methods are used to analyze whether the target gene is introduced into the target cell.
  • the detection methods include common molecular biology methods ( For example, Southern blotting and Northern blotting, RT-PCR and PCR, etc.), or common biochemical methods (such as ELISA and Western blotting), and the methods mentioned in the present invention.
  • the transfection is performed by a retroviral vector or a lentiviral vector.
  • T cells can be expanded in vitro after being activated by the TCR/CD3 complex on the surface and auxiliary stimulatory molecules (such as CD28).
  • Stimuli that activate TCR, CD3 and CD28 can be adsorbed on the surface of the culture vessel, or co-cultured (such as magnetic beads), or can be directly added to the cell culture medium for co-cultivation.
  • Another embodiment is to co-culture T cells and trophoblast cells, which express auxiliary stimulatory molecules or corresponding ligands, including but not limited to HLA-A2, ⁇ 2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
  • auxiliary stimulatory molecules or corresponding ligands including but not limited to HLA-A2, ⁇ 2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
  • T cell culture is cultured and expanded under appropriate culture conditions.
  • the cells can be passaged when they reach a confluence of more than 70%, and the culture medium is usually replaced with fresh culture medium in 2 to 3 days.
  • the cells reach a certain number, use it directly, or freeze it as described above.
  • the duration of in vitro culture can be within 24 hours, or as long as 14 days or longer.
  • the frozen cells can be used in the next step after thawing.
  • the cells can be cultured in vitro for a few hours to 14 days, or any number of hours in between.
  • T cell culture conditions include the use of basic culture media, including but not limited to RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo.
  • conditions required for cell survival and proliferation include but are not limited to the use of serum (human or fetal bovine serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGF- ⁇ and TNF-a, other culture supplements (including amino acids, sodium pyruvate, vitamin C, 2-mercaptoethanol, growth hormone, growth factor) .
  • the cells can be placed in appropriate culture conditions, for example, the temperature can be 37°C, 32°C, 30°C, or room temperature, and the air condition can be, for example, air containing 5% CO 2 .
  • the chimeric antigen receptor modified immune cells include primitive T cells or their precursor cells, NKT cells, T cell strains or NK cells.
  • the chimeric antigen receptor includes an antigen-binding domain, a spacer, a transmembrane region, and an intracellular domain that are operably connected in series, and the antigen-binding domain can specifically recognize and bind to the labeled
  • the extracellular epitope region of the polypeptide, the spacer region is used to separate the antigen binding domain and the transmembrane region, and the intracellular domain is used for signal transmission.
  • the antigen-binding domain is preferably a single-chain antibody (ScFv) including a light chain and a heavy chain, and the light chain and the heavy chain can be connected to each other through a linker, as shown in FIG. 52.
  • ScFv single-chain antibody
  • the chimeric antigen receptor can modify immune cells through lentivirus infection and mRNA electrotransduction.
  • the first composition and the second composition are each independently present in the therapeutic agent without mixing with each other.
  • the first pharmaceutically acceptable carrier and the second pharmaceutically acceptable carrier are the same. In other embodiments, the first pharmaceutically acceptable carrier and the second pharmaceutically acceptable carrier are different.
  • the therapeutic agent can also be understood as a combination of drugs.
  • the first composition comprises a therapeutically effective amount of the recombinant oncolytic adenovirus (preferably, the first composition comprises 5 ⁇ 10 7 -5 ⁇ 10 12 vp / dose of recombinant soluble day Oncolytic adenovirus, more preferably comprising 5 ⁇ 10 7 to 1.5 ⁇ 10 12 VP/day of said recombinant oncolytic adenovirus, more preferably comprising 5 ⁇ 10 8 to 1 ⁇ 10 12 VP/day of said recombinant Oncogenic adenovirus, more preferably comprising the recombinant oncolytic adenovirus at a dose of 1 ⁇ 10 9 to 5 ⁇ 10 11 VP/day, still more preferably comprising the recombinant oncolytic adenovirus at a dose of 3 ⁇ 10 10 to 3 ⁇ 10 11 VP/day Oncolytic adenovirus).
  • the first composition comprises 5 ⁇ 10 7 -5 ⁇ 10 12 vp / dose of recombinant soluble day Onco
  • the active ingredient of the first pharmaceutical composition is the recombinant oncolytic adenovirus.
  • the recombinant oncolytic adenovirus can be administered by a commonly used administration method in the art, for example, it can be formulated to be administered by intratumoral injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.
  • the second composition contains a therapeutically effective amount of the immune cell (T cell receptor modified immune cell or chimeric antigen receptor modified immune cell) of the present invention.
  • the second composition contains the immune cells with a total dose range of 1 ⁇ 10 3 -1 ⁇ 10 9 cells/Kg body weight per treatment course.
  • the immune cells can be formulated to be administered via arteries, veins, subcutaneous, intradermal, intratumor, intralymphatic, intralymph node, subarachnoid, intramarrow, intramuscular, and intraperitoneal administration.
  • the therapeutic agent consists of the first composition and the second composition.
  • the therapeutic agent of the present invention may also contain suitable pharmaceutically acceptable auxiliary materials, including pharmaceutical or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives , Including sugars, lipids, peptides, amino acids, antioxidants, adjuvants, preservatives, etc.
  • suitable pharmaceutically acceptable auxiliary materials including pharmaceutical or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives , Including sugars, lipids, peptides, amino acids, antioxidants, adjuvants, preservatives, etc.
  • the present invention also provides the use of the therapeutic agent in the preparation of drugs for treating tumors and/or cancers.
  • the tumors and/or cancers include breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, Choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder cancer, ureteral cancer, glioma, neuroblastoma Tumors, meningiomas, spinal cord tumors, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma , Prostate cancer, testicular cancer, skin squamous cell carcinoma, meso
  • the present invention also provides a synergistic combination medicine kit for treating tumors and/or cancers, including:
  • a second container containing the second composition in the therapeutic agent according to the present invention wherein the first container and the second container are independent;
  • the invention also provides the use of the kit in the preparation of a medicine for treating or preventing tumors and/or cancers.
  • the tumors and/or cancers include breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, Choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder cancer, ureteral cancer, glioma, neuroblastoma Tumors, meningiomas, spinal cord tumors, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, Paget's disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi's sarcoma , Prostate cancer, testicular cancer, skin squamous cell carcinoma, meso
  • the present invention also provides a method for treating tumors and/or cancers, including:
  • the second composition of the therapeutic agent according to the present invention is administered to the tumor and/or cancer patient.
  • the first composition and the second composition in the therapeutic agent can be administered simultaneously (for example, as a mixture for simultaneous intratumor injection), separately but simultaneously (for example, administered by intratumoral and intravenous injection, respectively) or sequentially (for example, The first composition is applied first, and then the second composition is applied; or the second composition is applied first, and then the first composition is applied).
  • the method includes the following sequential steps:
  • the second composition of the therapeutic agent is administered to the tumor and/or cancer patient from 1-30 days after the first composition is first administered.
  • administering of the second composition of the therapeutic agent to the tumor and/or cancer patient on the 1-30 days after the first administration of the first composition means the first administration of the second composition and
  • the time interval for the first application of the first composition is 1-30 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days), or the first application of the second composition to the first
  • the time interval of composition administration is 1-30 days (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days).
  • the time interval between the first application of the second composition and the immediately preceding application of the first composition is 3-14 days (for example, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14 days).
  • the administration dose of the recombinant oncolytic adenovirus is 5 ⁇ 10 7 -5 ⁇ 10 12 vp/day, 1-2 times a day for 1-7 consecutive days, or the above range Any value between.
  • the dose of the immune cells is in the range of 1 ⁇ 10 3 for each course of treatment. -1 ⁇ 10 9 cells/Kg body weight. Preferably, it is administered 1-3 times a day, and continuously administered for 1-7 days.
  • the method for treating tumors and/or cancers further comprises administering to the patient other drugs for treating tumors and/or cancers, and/or drugs for regulating the patient’s immune system to enhance the The number and function of immune cells in the body.
  • the other drugs used to treat tumors and/or cancers include, but are not limited to: chemotherapy drugs, such as cyclophosphamide, fludarabine; radiotherapy drugs; immunosuppressants, such as cyclosporine, azathioprine, Methotrexate, mycophenolate (mycophenolate), FK50; antibodies, such as antibodies against CD3, IL-2, IL-6, IL-17, TNF.
  • the method for treating tumors and/or cancers further includes administering to the patient other drugs for the treatment of tumors and/or cancers, and/or drugs for regulating the immune system of the patient, for the When the immune cells produce severe toxic and side effects, the number and functions of immune cells modified by the T cell receptor carrying suicide genes in the body are eliminated.
  • the other drugs used to treat tumors and/or cancers include but are not limited to: chemically induced dimerization (CID) drugs, AP1903, phosphorylated ganciclovir (ganciclovir), anti-Cd20 antibodies, anti-CMYC antibodies, and anti-EGFR antibody.
  • the recombinant oncolytic adenovirus can be formulated to be administered by intratumoral injection, intraperitoneal administration, subarachnoid administration, or intravenous administration.
  • the immune cells can be formulated for administration via arteries, veins, subcutaneous, intradermal, intratumoral, intralymphatic, intralymph node, subarachnoid, intramarrow, intramuscular, and intraperitoneal administration.
  • the tumors and/or cancers include breast cancer, head and neck tumors, synovial cancer, kidney cancer, connective tissue cancer, melanoma, lung cancer, esophageal cancer, colon cancer, rectal cancer, brain cancer, liver cancer, bone cancer, Choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, anal cancer, cholangiocarcinoma, bladder cancer, ureteral cancer, glioma, neuroblastoma Tumors, meningiomas, spinal cord tumors, osteochondroma, chondrosarcoma, Ewing’s sarcoma, carcinoma of unknown primary site, carcinoid, fibrosarcoma, Paget’s disease, cervical cancer, gallbladder cancer, eye cancer, Kaposi’s sarcoma , Prostate cancer, testicular cancer, skin squamous cell carcinoma, meso
  • the percentage concentration (%) of each reagent refers to the volume percentage concentration (%(v/v)) of the reagent.
  • Cells AD293, MRC-5, Hela, A549, U251, HCT116, PANC1, HT29, H460, and MDA-MB-231 were purchased from ATCC; HUVEC was purchased from Ocells Biotechnology (Shanghai) Co., Ltd.
  • Oncolytic adenovirus H101 was purchased from Shanghai Sanwei Biotechnology Co., Ltd.
  • mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.
  • PBS formula 8mM Na 2 HPO 4 , 136mM NaCl, 2mM KH 2 PO 4 , 2.6mM KCl, pH 7.2-7.4.
  • the cell line used to prepare lentiviral particles is 293T cell (ATCC CRL-3216).
  • the presenting cell line used to present antigen polypeptides is T2 cells (174xCEM.T2, ATCC CRL-1992).
  • the tumor cell lines used to detect the function are human colorectal cancer colo205 cells (ATCC CCL-222), HT-29 cells (HTB-38) and HCT116 cells (ATCC CCL-247), human breast cancer MDA-MB-231 cells ( ATCC HTB-26) and MCF7 cells (ATCCHTB-22), human ovarian cancer SKOV3 cells (ATCC HTB-77), human pancreatic cancer PANC-1 cells (ATCCCRL-1469), human glioma U87MG cells (ATCC HTB) -14), human hepatocellular carcinoma HepG2 cells (ATCC HB-8065), human non-small cell lung cancer NCI-H460 cells (ATCC HTB177) and small cell lung cancer NCI-H446 cells (ATCC H
  • RPMI-1640 complete medium (Lonza, cat#12-115F).
  • RPMI-1640 complete medium is supplemented with 10% calf serum FBS (ATCC 30-2020), 2mmol/L L-glutamic acid , 100 ⁇ g/ml penicillin and 100 ⁇ g/ml streptomycin.
  • Peripheral blood The human peripheral blood products used in the test were from healthy donors.
  • the human peripheral blood products used in Examples 8-11 were from the Pacific Blood Center in San Francisco (#1PBMC and #2PBMC are Trima residues from the Apheresis method collection kit, respectively) Cell components #R32334 and #R33941).
  • the cell counting method used in the following example is as follows:
  • the MTT method used in Example 4 Add 10 ⁇ l of MTT solution (5mg/ml) to each well of cells, incubate for 4 hours in a 37°C incubator, aspirate the culture medium, add 150 ⁇ l of DMSO to each well, shake at low speed for 10 minutes on a shaker, The crystals are fully dissolved and mixed, and the absorbance (OD 490 ) at 490 nm is detected by a microplate reader.
  • Inhibition rate calculation formula: cell proliferation inhibition percentage (IR%) 1-(OD 490 test product-OD 490 blank) / (OD 490 negative control-OD 490 blank) ⁇ 100%.
  • Counting by trypan blue staining method wash the cells with PBS, digest them with trypsin, suspend the cells in PBS, add trypan blue dye solution at a final concentration of 0.04% (w/v), count under a microscope, dead cells will Stained in light blue, live cells resist staining. Take the number of live cells as the final data.
  • Culture plate source 6-well cell culture plate (culture volume per well is 2ml), 12-well cell culture plate (culture volume per well 1ml), 24-well cell culture plate (culture volume per well 500 ⁇ l), 96-well cell culture plates (100 ⁇ l per well) were obtained from Corning.
  • CTL Her2/neu 369-377 specific killer T cells
  • PBMC mononuclear cells
  • HLA-A2 positive PBMC cells are cultured in the culture wells of a 24-well culture plate, and the culture medium is the above-mentioned RPMI-1640 complete medium.
  • Her2/neu 369-377 polypeptide (Her2-E75, synthesized with Peptide2.0, 10 ⁇ g/ml dissolved in DMSO), the final concentration is 1 ⁇ g/ml. Placed in an incubator under 5% CO 2 and 37°C for 16-24 hours, add the following final concentrations of cytokines: human IL-2 (Peprtech, cat#200-02) 100IU/ml, human IL-7 (Peprotech company, cat#200-07) 5ng/ml, human IL-15 (Peprotech company, cat#200-15) 5ng/ml.
  • the phenotype of T cells expressing Her2/neu 369-377-specific TCR was analyzed by flow cytometry. Collect the tested cells in a 1.5ml tube (the number of cells is about 10e5), use 1ml DPBS solution (2.7mM KCl, 1.5mM KH 2 PO 4 , 136.9mM NaCl, 8.9mM Na 2 HPO 4 ⁇ 7H 2 O, pH 7.4) wash it again and reset it to 100 ⁇ l DPBS containing 1% calf serum, add 5 ⁇ l fluorescein APC-labeled anti-human CD8 antibody (Biolegend, cat#300912), and 10 ⁇ l fluorescein PE-labeled Her2- E75/HLA-A2 tetramer (Her2-E75 tetramer, MBL International Co, cat#T01014) or Her2-E75/HLA-A2 pentamer (Her2-E75 pentamer, Proimmune, cat#F
  • the flow cytometer was MACSQuant Analyzer 10 (Miltenyi Biotec), and Flowjo software (Flowjo) was used for the result analysis.
  • T cell clones are obtained by culturing single cells after separation using a flow cytometer (FACS sorter).
  • PBMC stimulated by Her2/neu369-377 polypeptide antigen were stained with APC-labeled anti-human CD8 antibody and PE-labeled Her2-E75/HLA-A2 pentamer, and then subjected to flow cytometry (Model: Sony cell sorter SH800) .
  • HLA-A2-positive PBMC treated with 25 ⁇ g/ml mitomycin C for 2 hours are added Cells, 10e5 cells per well, add 1 ⁇ g/ml Her2/neu 369-377 polypeptide to culture overnight, add RPMI-1640 containing IL-2 100IU/ml, IL-7 5ng/ml, IL-15 5ng/ml Culture medium.
  • the culture medium containing the cytokine was replaced every 3-4 days, and the growth of T cell clones was observed under a microscope. Collect the proliferating T cells, perform antigen re-stimulation as described above to obtain a sufficient number of cells, perform phenotypic or functional testing, and extract RNA for cloning of the TCR gene.
  • T cell function test To test the ability of T cells transfected with TCR gene to recognize epitope polypeptides, 10e5 T cells transfected with TCR gene and 10e5 T2 cells were added to each well of a 96-well plate, at 100 ⁇ l/ Each well of RPMI-1640 complete medium was mixed culture, and each test group was a duplicate well.
  • Her2/ The neu 369-377 polypeptide was placed in an incubator at 5% CO 2 and 37°C for overnight culture.
  • 10e5 T cells transfected with the TCR gene and 10e5 T2 cells were added to each well of the 96-well plate, and then added with a final concentration of 0.1 ⁇ g/ml
  • the epitope polypeptide to be tested is then placed in an incubator at 5% CO 2 and 37°C for overnight culture. Collect the supernatant after 24 hours, and use the human IFN- ⁇ ELISA Read-set-Go kit (eBioscience, cat#88-7316) or human IFN- ⁇ DuoSet ELISA kit (R&D Systems cat#DY285B) according to the manufacturer’s instructions. IFN- ⁇ in the supernatant was detected.
  • PBMC cells and tumor cells transfected with TCR gene as target cells were added to each well of a 96-well plate according to different target ratios, and cultured 24 After hours, the supernatant was collected to detect the secreted gamma interferon in the supernatant.
  • Each test group has multiple holes or three holes.
  • an anti-human CD8 antibody Biolegend, cat#300912
  • an anti-human CD8 antibody Biolegend, cat#300912
  • Trypsin digests the cells in logarithmic growth phase collect them by centrifugation after termination, blow evenly to prepare a single cell suspension; adjust the cell concentration to 0.1-10 ⁇ 104/ml with cell culture fluid (adjust the number of inoculated cells according to different cell growth conditions ), seeded on a 96-well cell culture plate, the culture system is 100 ⁇ l/well, placed in a 37°C, 5% CO 2 incubator overnight to make the cells completely adhere to the wall, reaching 70-80% the next day; counting Count the plates and use a countstar counter to verify the correctness of the count.
  • PCR was performed with 5'-CDS primer and TCR ⁇ chain 3'primer 5'-GCCTCTGGAATCCTTTCTCTTG-3' (SEQ ID NO: 24) and ⁇ chain 3'primer 5'-TCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 25)
  • the full sequence gene fragments of TCR ⁇ and ⁇ were added and cloned into pRACE vector (Takara Bio, USA, cat#634858). Transform the competent bacteria Stellar (Takara Bio, USA, cat#636763) and obtain the plasmids for sequencing.
  • the viral vector used to express TCR is a replication-deficient lentiviral vector, including: the GFP-expressing lentiviral vector pCDH-EF1 ⁇ -MCS-(PGK-GFP), which can be purchased from System Biosciences (Cat#CD811A-1); and the vector pCDH-EF1 ⁇ -MCS that does not express GFP, obtained by removing the PGK promoter and GFP gene from the pCDH-EF1 ⁇ -MCS-(PGK-GFP) vector by using conventional techniques in the art.
  • the GFP-expressing lentiviral vector pCDH-EF1 ⁇ -MCS-(PGK-GFP) which can be purchased from System Biosciences (Cat#CD811A-1)
  • the vector pCDH-EF1 ⁇ -MCS that does not express GFP obtained by removing the PGK promoter and GFP gene from the pCDH-EF1 ⁇ -MCS-(PGK-GFP) vector by using conventional techniques in the art.
  • TCR gene sequence synthesize the complete gene sequence of TCR ⁇ chain and ⁇ chain, as well as the cleavable F2A sequence and Furin digestion fragment between them, and link to the multiple cloning site downstream of the EF-1 ⁇ promoter of the vector .
  • the transcription sequence of inserting TCR is TCR ⁇ chain (without stop codon), Furin fragment, F2A fragment, TCR ⁇ chain (for the method, please refer to the document “Gene Ther. 2008 Nov; 15(21): 1411-1423”).
  • the vector expressing GFP is driven by the reverse PGK promoter. For vectors that do not express GFP, the PGK promoter and GFP fragment have been removed.
  • TCR lentiviral particles are obtained by transfecting 293T/293FT cells with Lipofectaine 2000 transfection reagent (invitrogen, #11668019). Prepare 293T/293FT cells and transfection procedures according to the manufacturer's instructions. Transfection was carried out in a 6-well culture plate. Firstly, Opti-MEM 1 medium (Thermo Fisher Company, cat#51985091) was used to prepare a liposome mixture solution of the transfected plasmid. According to the manufacturer's instructions, add 6 ⁇ l of lipofectaine 2000 reagent to 250 ⁇ l of culture medium.
  • TCR lentiviral vector plasmid 0.8 ⁇ g and pCDH system virus packaging plasmid 1.8 ⁇ g (SBI company, cat#LV500A-1), mixed and incubated for 25 minutes and then added to 293T/293FT cell culture wells. Incubate for 16 hours under 5% CO 2 and 37°C, change to FBS-free DMEM medium (Thermo Fisher, cat#11965092), continue culturing for 24 hours and 48 hours, collect the cell supernatant, and centrifuge at 2000g for 10 minutes. The virus supernatant obtained after filtration with a 0.4 ⁇ m filter membrane was concentrated using lentivirus concentrate (GeneCopoeiaTM#LPR-LCS-01) according to the manufacturer's instructions and used to infect cells.
  • lentivirus concentrate GeneCopoeiaTM#LPR-LCS-01
  • Recombinant TCR lentivirus transfected human T cells The frozen primary PBMC cells were thawed and cultured in RPMI-1640 complete culture medium for 24 hours. Dead cells were removed by Ficoll-Paque Premium density gradient centrifugation ( ⁇ 400g) for 30 minutes.
  • phenotype and functional tests can be performed after 72 hours. Transfection of T cell strains is also carried out in accordance with the above steps. If the viral vector is labeled with GFP, GFP-positive cells can generally be observed under a fluorescence microscope 48 hours after transfection.
  • the segment sequence includes the coding region of the E1A gene (excluding the E1A promoter sequence) and part of the 3'UTR region.
  • the PCR product obtained was digested with BglII and cloned into the vector pShuttle-CMV (purchased from Agilent) between the BglII and EcoRV sites in the multiple cloning site region (MCS) to obtain the intermediate vector pShuttle-E1A,
  • MCS multiple cloning site region
  • the obtained pShuttle-E1A positive clones were confirmed by PCR screening with P1 and P2.
  • the results are shown in Figure 2, and the construction process is shown in Figure 3.
  • the positive clones were sequenced, and the sequencing results were completely consistent with the corresponding sequences on the AD5 genomic DNA.
  • PCR primers P3 and P4 (P3: CGC GTCGAC TACTGTAATAGTAATCAATTACGG ( SEQ ID No.32) , and P4: GAC GTCGAC TAAGATACATTGATGAGTTTGGAC (SEQ ID No.33); NOTE: 5 'end of the two primers are added to the SalI restriction site Dot, underlined), the obtained pShuttle-E1A positive clone is used as a template for high-fidelity PCR amplification.
  • the PCR product contains the CMV promoter, E1A gene fragment and the E1A expression box including SV40polyA.
  • the size of the PCR product is 2017bp ( Figure 4).
  • the obtained PCR product of the E1A expression box was digested with SalI and cloned into the SalI site in the MCS region of the pShuttle vector (purchased from Agilent), and the positive clones inserted into the E1A expression box were screened by PCR using P3 and P4 primers ( Figure 5), and confirmed by digestion with BglII, the clone with the E1A expression box inserted in the forward direction will produce 7200bp and 1400bp fragments after BglII digestion, and the clone with the E1A expression box inserted in the reverse direction will produce 7970bp after BglII digestion And 630bp two fragments ( Figure 6), select the #2 plasmid in Figure 6 for subsequent experiments. Finally, the intermediate vector pShuttle-MCS-E1A was obtained, and the construction process is shown in Figure 7. The obtained pShuttle-MCS-E1A positive clone was sequenced, and the result was completely consistent with the expected
  • shPDL1-1 (or called shPDL1-1) targeting the 168-190, 430-452, and 589-611 regions of its coding region mRNA were designed shPDL1-#1), shPDL1-2 (or shPDL1-#2), and shPDL1-3 (or shPDL1-#3), respectively SEQ ID NO.45, SEQ ID NO.48, SEQ ID NO.51)
  • shPDL1-NC a negative control sequence shPDL1-NC that has nothing to do with human PDL1mRNA was also designed. The sequence is as follows:
  • Synthetic sense sequence (SEQ ID No. 43):
  • Synthetic antisense sequence (SEQ ID No. 44):
  • Synthetic sense sequence (SEQ ID No. 46):
  • Synthetic antisense sequence (SEQ ID No.47):
  • Synthetic sense sequence (SEQ ID No.49):
  • Synthetic antisense sequence (SEQ ID No. 50):
  • the inhibitory effect of shPDL1 on hPDL1 mRNA was detected in U251 and H460 cells.
  • U251 and H460 were inoculated with 2 ⁇ 10 5 cells per well 12 hours in advance, and U251 and H460 were transfected with 4 ⁇ l lipofectamin 2000:1.6 ⁇ g shRNA expression vector DNA in each well, respectively, at 24 hours
  • Two cell samples were collected at and 48 hours.
  • Total RNA was extracted and reverse transcription was performed.
  • the GAPDH gene mRNA level was used as a control, Real-time PCR was performed to detect the expression level of human PDL1 mRNA in the cells.
  • the 4 plasmids pSGU6/GFP/Neo-shPDL1-NC, pSGU6/GFP/Neo-shPDL1-1, pSGU6/GFP/Neo-shPDL1-2 and pSGU6/GFP/Neo-shPDL1-3 were respectively compared with pcDNA3.3-hPDL1 -3 ⁇ FLAG equimolar ratio (1:1) was instantly transfected into 293T cells, and the plasmid pcDNA3.3-hPDL1-3 ⁇ FLAG could express human PDL1 protein fused with 3 ⁇ FLAG tag.
  • Cell samples were collected after 48 hours and subjected to Western blot analysis after lysis. The results (see Figure 9) proved that shPDL1-#1 can significantly reduce the over-expressed hPDL1.
  • the construction process of the above-mentioned pcDNA3.3-hPDL1-3 ⁇ FLAG plasmid is as follows: First, two primers are designed according to the mRNA sequence of the human PDL1 gene in NCBI (P11: CGCGTCGACATGAGGATATTTGCTGTCTTTAT (SEQ ID No. 40), P12: CCGCTCGAGCGTCTCCTCCAAATGTGTATCAC (SEQ ID No.
  • BJ5183 (transformed into pAdEasy-1 plasmid) stored in our laboratory at -80°C was inoculated into LB/Amp medium and cultured overnight at 37°C and 200RPM for activation.
  • Plasmids that can produce 4.5Kb or 3Kb fragments after PacI digestion are all plasmids with correct homologous recombination.
  • the AD293 cells in good growth condition were seeded in a 6-well plate one day in advance, and the number of cells seeded should be 60-70% when the transfection experiment was carried out the next day.
  • the cells can be recovered by gently pipetting the cells and the culture supernatant, stored in a refrigerator at -80°C or directly for further expansion. The process is shown in Figure 15.
  • inoculate AD293 cells in a good growth state in a 6cm cell culture dish one day in advance, and the appropriate number of cells inoculated is 70-80% when the transfection experiment is performed the next day.
  • Add 800-1000 ⁇ l of the virus supernatant collected before to each 6cm cell culture dish mix it with the same cross and put it back into the cell culture medium to continue culturing. Usually after 48 hours, a large number of cells can be seen rounding and falling off. At this time, cells and culture supernatant can be collected.
  • the cell density should also be about 70%.
  • VP method there are VP method, GTU/BFU method, plaque method, TCID50 method and Hexon staining counting (kit) method for the determination of adenovirus titer.
  • the more accurate and high repetition rate methods are TCID50 method and Hexon staining counting method.
  • the Hexon staining and counting method was used to determine the number of active virus particles (unit: PFU/ml) in the obtained virus supernatant. The results are shown below.
  • the 3 pShuttle-U6-shPDL1-CMV-E1A plasmids and 1 control plasmid pShuttle-MCS-E1A and pAdEasy-1 plasmids described in the present invention produce 6 pAdEasy-U6-shPDL1-CMV-E1A plasmids and 2 PAdEasy-CMV-E1A (refer to Figure 13 that each plasmid can produce 2 correct ways of homologous recombination, and obtain two correct plasmids, which can produce 4.5K or 3K bands respectively after PacI digestion), Using the obtained 8 homologous recombination plasmids to package the virus in AD293, a total of 8 oncolytic viruses were obtained: OAd-C-4.5K, OAd-C-3K, OAd-shPDL1#1-4.5K, OAd-shPDL1#1 -3K, OAd
  • C-4.5K and C-3K are the same virus with the same sequence
  • 1-4.5K and 1-3K are the same virus with the same sequence
  • 2-4.5K and 2-3K are the same virus with the same sequence
  • 3 -4.5K and 3-3K are the same virus with the same sequence.
  • the titer of the virus has been obtained:
  • Example 1 Oncolytic adenovirus (OAd-shPDL1) replication ability in cells (tumor cells and normal cells)
  • Inoculate cells (HUVEC, MRC-5, Hela, A549 and U251) in a 12-well plate with 1.5 ⁇ 10 5 cells per well.
  • Medium (HUVEC cells use Allcells special medium, Cells and culture medium were purchased from Ausails Biotechnology (Shanghai) Co., Ltd.; MRC-5 cell culture uses MEM+10% FBS medium; Hela cell culture uses RPMI1640+10% FBS medium; A549 cell culture uses DMEM /F12+10% FBS medium; U251 cell culture uses MEM+10% FBS, all medium is purchased from Gibco company)
  • the volume is 1ml.
  • the medium was aspirated and the cells were rinsed with PBS once, and 500 ⁇ l of virus suspension (the virus was prepared in Preparation Example 4) was added in the manner shown in FIG. 16, and the multiplicity of infection (MOI) of the virus was 10.
  • MOI multiplicity of infection
  • the virus suspension was aspirated, and the cells were rinsed twice with PBS.
  • the cell sample was collected by trypsinization at 0 hr, and the second cell sample was collected after 48 hr.
  • the genomic DNA of the cell samples collected at 0 hr and 48 hr were extracted respectively, and used for the type 5 adenovirus E1A (P5: TCCGGTTTCTATGCCAAACCT (SEQ ID No.
  • the results in Figure 17 show that the four types of oncolytic adenoviruses constructed in the present invention (control viruses C-4.5K, OAd-shPDL1 viruses 1-4.5K, 2-4.5K and 3-4.5K) are in the tested cells
  • the copying ability of the system varies greatly.
  • the oncolytic virus of the present invention has a very strong replication ability in the detected tumor cells, including in the immortalized human embryonic lung fibroblast cell line, it also shows strong replication ability, but in human primary cells HUVEC
  • the replication ability is very low, and the replication ability in normal human cells is about 42-lower than that in tumor cell lines or cell lines with tumorigenesis tendency (for example: immortalized human embryonic lung fibroblast cell line MRC5) 444 times. Therefore, it can be considered that the oncolytic adenovirus of the present invention has a strong tumor cell bias in selective replication, has higher safety in future clinical oncolytic virus applications, and has greater virus usage. space.
  • Example 2 Oncolytic adenovirus (OAd-shPDL1) killing ability on tumor cells
  • the CCK8 experiment was used to test the killing ability of the oncolytic adenovirus (OAd-shPDL1) of the present invention.
  • Inoculate cells U251, Hela and A549 in a 96-well plate, inoculate 1.5 ⁇ 10 3 cells per well, and medium per well (U251 cell culture uses MEM+10% FBS; Hela cell culture uses RPMI1640+10% FBS medium; DMEM/F12+10% FBS medium is used for A549 cell culture; all mediums are purchased from Gibco company)
  • the volume is 100 ⁇ l.
  • the commercial oncolytic adenovirus H101 was used as a control. In the experiment, the same amount of virus was used to treat the same cells with the same MOI, and the light absorption value was measured at the same time point. In addition, 1 ⁇ M paclitaxel solution was used as a positive control for the system.
  • Oncolytic adenovirus (OAd-shPDL1) Dose three tumor cell killing effects and anti-half dose (IC 50) The results shown in FIG. 18-21.
  • the oncolytic adenovirus of the present invention (control virus C-4.5K, OAd-shPDL1 virus 1-4.5K, 2-4.5K and 3-4.5K) has a significant dose of killing U251, A549 and Hela
  • the virus of the present invention has a similar killing effect, and the killing effect on U251 cells is even better than that of H101.
  • Statistical analysis of the killing effect shows that there is a significant difference.
  • OAd-shPDL1 oncolytic adenovirus
  • two cell lines that can stably express hPDL1 with FLAG tags were specifically constructed in this example: A549/hPDL1-FLAG and Hela /hPDL1-FLAG.
  • the construction process is briefly described as follows: the pcDNA3.3-hPDL1-FLAG vector (also called pcDNA3.3-hPDL1-FLAG-IRES-hrGFP vector) obtained by the method shown in Preparation Example 2 was transfected into A549 and Hela using lipofectamin 2000 In the cells, because the vector carries the Neomycin gene, G418 was subsequently added for three rounds of screening, and finally A549/hPDL1-FLAG and Hela/hPDL1-FLAG cell lines that can stably express the FLAG-tagged hPDL1 and GFP proteins were obtained.
  • H101 and the four oncolytic adenoviruses of the present invention were used to treat the above
  • MOI multiplicity of infection
  • Cell samples were collected 48 hours after virus infection, and Western blot analysis was performed after lysis.
  • Anti-FLAG antibody was used to detect changes in the expression level of FLAG-tagged hPDL1. The results are shown in Figures 22 and 23.
  • Infect 293 cells with the adenovirus culture supernatant obtained in Preparation Example 4 in a 15CM petri dish according to the volume ratio of virus supernatant to cell culture solution (AD:DMEM 2:20), and continue in the incubator at 37°C and 5% CO 2 Cultivate for about 36 hours, observe under the microscope that about 50% of the cells have cytopathic effect (CPE) and after the cells are suspended, collect the cells and culture fluid in the culture dish by pipetting or cell scraper, etc., can be stored at 4°C for a short time, and for a long It needs to be stored at -20°C or -80°C; under normal circumstances, the amount of virus purified by ultracentrifugation is about 60-80 15CM culture dishes (including medium supernatant and infected 293 cells).
  • the collected oncolytic virus-containing 293 cells and the culture supernatant were centrifuged at 4°C, 3000RPM for 30 minutes, and the following experiments were divided into two parts: the supernatant and the precipitation.
  • All the supernatant can be mixed with PEG8000 solution (2.5M NaCl aqueous solution containing 20% PEG8000) at a ratio of 2:1, and precipitated on ice for 1 hour or overnight. After centrifugation at 12000RPM for 20 minutes, the supernatant was discarded and the virus pellet was retained.
  • the cell suspension is thawed in a 37°C water bath, shaken vigorously for 30 seconds, then the sample is returned to the -80°C refrigerator or placed in a dry ice/pure ethanol mixture to quickly freeze the cell suspension. Repeat the freezing and thawing process 3-5 times to completely destroy the cell membrane, release the virus from the cell, and obtain the virus liquid. If not immediately purified, it can be stored at -20°C. Before ultracentrifugation and purification, melt the virus solution obtained in this step in a 37°C water bath and centrifuge at room temperature for 10 minutes at a speed of 16000RPM. Collect the virus-containing supernatant. The virus supernatant can be stored on ice temporarily.
  • Cesium chloride density gradient centrifugation is still the most commonly used method for the separation and purification of various viruses. The main reason is that different types of viruses have different buoyant densities, so that they can be separated from other components in the cell lysate in the CsCl solution. After collecting the specific bands of the target virus, use a PD-10 desalting column to remove the cesium chloride, and finally obtain a purified virus. Using this method can obtain extremely high purity virus.
  • the specific purification procedure is as follows:
  • the centrifuge tube After centrifugation, the centrifuge tube is firmly fixed on the universal clamp on the iron stand in the biological safety cabinet. Carefully use a 5ml syringe with an 18G needle to pierce the centrifuge tube from the bottom ( ⁇ 1cm) of the intact virus particle band at the bottom, aspirate only the intact virus particle band, and keep it on ice for a short time.
  • the high-quality virus liquid purified by ultracentrifugation is divided into different clean centrifuge tubes in different volumes according to the concentration and experimental needs, and the date and virus name are marked and stored at -80°C for use.
  • Example 4 The killing ability of oncolytic adenovirus (OAd-shPDL1) on tumor cells (HCT116, PANC1, HT29 and H460)
  • the killing ability of the oncolytic adenovirus was tested by MTT experiment.
  • Inoculate human tumor cells HCT116, PANC1, HT29, and H460
  • the number of cells inoculated per well is 3 ⁇ 10 3
  • the medium per well HCT116 cell culture uses McCoy's 5A+10% FBS medium; PANC-1 cell culture uses DMEM+10% FBS medium; HT29 cell culture uses DMEM/F12+10% FBS medium; H460 cell culture uses RPMI1640+10% FBS, all medium is purchased from Gibco
  • the volume is 100 ⁇ l .
  • the viruses used are the control virus C-4.5K, OAd-shPDL1 virus 1-4.5K, 2-4.5K, and 3 prepared by the method described in Preparation Example 5.
  • the multiplicity of infection (MOI) is 1, 3, 10, 30, 100 and 300, respectively. There are 3 replicate holes for each multiplicity of infection.
  • the oncolytic adenovirus prepared by the present invention can kill HCT116, PANC1, HT29 and H460. Obvious dose dependence, and both have strong killing ability. Compared with the commercial oncolytic adenovirus H101, the virus of the present invention has a similar killing effect. It is expected that the oncolytic adenovirus of the present invention can be used in the treatment of the above-mentioned types of tumors in future clinical applications.
  • This example includes in vitro function tests performed in cells and in vivo function tests performed in a tumor-bearing mouse model.
  • the oncolytic adenovirus used in the experiment was the control virus C-4.5K and OAd-shPDL1 virus 1-4.5K (the virus used was prepared by the method described in Preparation Example 5).
  • the cell line used in this part of the experiment is the human breast cancer cell MDA-MB-231 with high expression of human PDL1.
  • MDA-MB-231 cells were seeded in 3 wells of a 6-well plate, and the number of seedings per well was 1 ⁇ 10 6 cells.
  • the cells were digested with trypsin and the cell samples were recovered. Rinse the recovered cell sample twice with clean PBS, and suspend the recovered cell pellet in RIPA buffer (50mM Tris-Cl (pH7.4), 150mM NaCl, 1% NP-40, 0.5% deoxygenated) with protease inhibitor Sodium cholate, 0.1% SDS and 1/20 Cocktail protease inhibitor), place on ice to lyse cells for 30 minutes, occasionally shake to mix the cell samples, centrifuge at 12000RPM for 5 minutes, recover the lysate supernatant and cell pellet and store them at -20 °C.
  • RIPA buffer 50mM Tris-Cl (pH7.4), 150mM NaCl, 1% NP-40, 0.5% deoxygenated
  • protease inhibitor Sodium cholate 0.1% SDS and 1/20 Cocktail protease inhibitor
  • the protein level of hPDL1 in the three recovered MDA-MB-231 cell lysate supernatants was detected by conventional Western blotting.
  • the primary antibody was Novusbio's rabbit-derived PDL1 antibody (Cat. No.: NBP2-15791). Each experiment was repeated three times.
  • the Western results and gray-scale scan value analysis are shown in Figure 30.
  • the experimental results showed that after treating MDA-MB-231 cells with OAd-shPDL1 oncolytic virus of the present invention, the percentage of cells with hPDL1 detected on the cell membrane decreased by about 5 percentage points compared with the blank control group without any treatment. However, the percentage of cells in the oncolytic virus control group (C-4.5K group) where hPDL1 can be detected increased by about 9% compared with the blank control group without any treatment. This is in line with the oncolytic virus in the relevant literature. Stimulating tumor cells can cause the expression level of hPDL1 on the tumor surface to increase, which is consistent (see (for example) the following scientific and technological literature: "Dmitriy Zamarin, et al.
  • OAd-shPDL1 oncolytic virus treatment of MDA-MB-231 cells can stimulate the oncolytic virus to tumor cells and reduce the percentage of cells with elevated hPDL1 expression to 95% of the percentage of unstimulated cells.
  • shPDL1 expressed in OAd-shPDL1 oncolytic virus indeed knocked down the expression level of hPDL1 in MDA-MB-231 tumor cells and reduced the number of cells expressing hPDL1 on the surface of MDA-MB-231 tumor cells.
  • mice 9 BALB/C nude mice were first subcutaneously inoculated with human colon cancer cell HCT116, and the number of cells inoculated in each nude mouse was 5 ⁇ 10 6 cells. After about 9 days, the cells were randomly divided into three groups after subcutaneous tumor formation in nude mice. The first group was treated as a blank control group without any treatment, and the second group was injected with oncolytic adenovirus into the subcutaneous tumor by intratumoral injection. Control virus C-4.5K, the virus injection volume per nude mouse is 1 ⁇ 10 9 PFU, and the injection volume is 100 ⁇ l. The third group also uses intratumoral injection to inject oncolytic adenovirus 1-4.5K into the subcutaneous tumor.
  • the virus injection volume of each nude mouse is 1 ⁇ 10 9 PFU, and the injection volume is 100 ⁇ l.
  • the injection was performed once a day for 3 consecutive days, and no treatment was done for 1 day on the 4th day.
  • the nude mice were sacrificed on the 5th day to recover the tumor tissue.
  • a part of all tissue samples were added to RIPA buffer containing protease inhibitors (the formula is the same as above) and then homogenized to extract tissue protein.
  • Western blotting was performed to detect the protein expression level of hPDL1 in tumor tissues after different treatments. The results are shown in the figure. 33 and Figure 34.
  • the strip scan of the Western result shown in Figure 33 is converted into gray value and normalized according to the gray value of the respective housekeeping gene ( ⁇ -actin) to obtain the scatter distribution map of Figure 34, according to the respective median value It can be seen that after treating HCT116 cells inoculated subcutaneously with BALB/C with oncolytic adenovirus control virus (C-4.5), the expression of hPDL1 in this group was increased by 20% compared with the expression of hPDL1 in the blank control group without any treatment.
  • Example 6 In vivo growth inhibition experiment of oncolytic adenovirus OAd-shPDL1 on human tumor cells inoculated subcutaneously in immunodeficient mice
  • NOD-SCID immunodeficiency mice were first subcutaneously inoculated (the number of cells inoculated subcutaneously in each mouse is 5 ⁇ 10 6 cells) human colon cancer cell HCT116 to prepare a tumor-bearing mouse animal model for the detection of oncolytic glands Virus OAd-shPDL1 (1-4.5K) inhibits the growth of HCT116, and oncolytic control virus C-4.5K is used as a negative control virus.
  • the control virus C-4.5K and OAd-shPDL1 virus 1-4.5K used in the experiment were prepared by the method described in Preparation Example 5.
  • mice with subcutaneously inoculated tumor volume up to the required were selected and divided into 4 groups according to a random grouping method, with 3 mice in each group.
  • the first group is the control group (blank control Control), each mouse is injected with 100 ⁇ l adenovirus preservation solution (ie, 10mM Tris solution (pH7.4) containing 1mM MgCl 2 and 10% glycerol);
  • the second group is Medium-dose oncolytic virus group (C-4.5K (middle)), each mouse was injected with 100 ⁇ l of C-4.5K virus suspension containing 1 ⁇ 10 8 PFU;
  • the third group was medium-dose oncolytic virus group (1-4.5K (middle)), each mouse is injected with 100 ⁇ l of 1-4.5K virus suspension containing 1 ⁇ 10 8 PFU each time;
  • the fourth group is the high-dose oncolytic virus group (1-4.5K (high) )), each mouse is injected with 100 ⁇ l of 1-4.5K
  • the oncolytic adenovirus OAd-shPDL1 (1-4.5K) treatment of HCT116 tumor cells inoculated subcutaneously in NOD-SCID immunodeficiency mice can inhibit the growth of tumor cells to a certain extent.
  • mice were subcutaneously inoculated (each mouse was inoculated with 5 ⁇ 10 6 cells) human colon cancer cell HCT116 to prepare an animal model of tumor-bearing mice to verify the oncolytic adenovirus OAd-shPDL1 (1-4.5 K)
  • the growth inhibitory effect on HCT116, the oncolytic control virus C-4.5K is used as the negative control virus.
  • Twenty-five tumor-bearing mice with subcutaneously inoculated tumor volume up to the requirement (tumor volume of 90-120 mm 3 ) were divided into 5 groups according to the random grouping method, with 5 mice in each group.
  • the first group is the control group (blank control (Control)), each mouse is injected with 100 ⁇ l adenovirus preservation solution; the second group is the medium dose oncolytic control virus group (C-4.5K (middle)), each mouse Mice were injected with 100 ⁇ l of C-4.5K virus suspension containing 1 ⁇ 10 8 PFU each time; the third group was the low-dose oncolytic virus group (1-4.5K (low)), and each mouse injected 100 ⁇ l each time containing 1 ⁇ 10 7 PFU of 1-4.5K virus suspension; the fourth group is the medium-dose oncolytic virus group (1-4.5K (middle)), each mouse is injected with 100 ⁇ l containing 1 ⁇ 10 8 PFU -4.5K virus suspension; the fifth group is the high-dose oncolytic virus group (1-4.5K (high)), and each mouse is injected with 100 ⁇ l of 1-4.5K virus suspension containing 1 ⁇ 10 9 PFU each time.
  • the control group blank control (Control)
  • C-4.5K (middle) medium dose on
  • the tumor cells in the high-dose group except one nude mouse attached to the muscles of the back spine making OAd-shPDL1 (1-4.5K) not very effective in its inhibitory effect.
  • the growth of tumor cells inoculated on the back of the remaining 4 nude mice was effectively inhibited.
  • the bodies of these 4 nude mice were rounder and stronger than the nude mice in other groups.
  • the tumor cells inoculated on the back of 2 nude mice passed OAd-shPDL1. (1-4.5K) After treatment, it is no longer visible to the naked eye (see Figure 44, 45).
  • nude mice in the OAd-shPDL1 (1-4.5K) medium dose group were inoculated subcutaneously
  • the growth of tumors also showed a more significant inhibitory effect (see Figures 42, 43, 45), which can be considered to be caused by the shPDL1 expressed in the shPDL1 expression box in the OAd-shPDL1 (1-4.5K) dose group
  • the reduction of HPDL1 expression in tumor cells plays a key role in inhibiting tumor growth.
  • the nude mice in the virus preservation solution group lost significant weight, and they mostly showed malignant wasting physique related to tumor growth.
  • Example 7 Verification of the growth inhibition of oncolytic adenovirus OAd-shPDL1 on human tumor cells HCT116 subcutaneously inoculated in BALB/C nude mice
  • this example expanded the sample size of the animal model to verify that OAd-shPDL1 (1-4.5K) ) Effective dose.
  • human colon cancer cells HCT116 were inoculated into BALB/C nude mice subcutaneously to prepare an animal model of tumor-bearing mice.
  • the cell inoculation amount for each nude mouse was 5 ⁇ 10 6 cells.
  • the oncolytic virus C-4.5K was also selected as the negative control virus.
  • mice with subcutaneously inoculated tumor volume up to the requirement were divided into 5 groups according to the random grouping method, with 7 mice in each group.
  • the first group is the control group (Control group).
  • Each mouse is injected with 100 ⁇ l of adenovirus preservation solution (that is, 10mM Tris solution (pH7.4) containing 1mM MgCl 2 and 10% glycerol); the second group is the solution Oncological control virus medium dose group (C-4.5K (1 ⁇ 10 8 )), each mouse is injected with 100 ⁇ l C-4.5K virus suspension containing 1 ⁇ 10 8 PFU; the third group is oncolytic control virus High-dose group (C-4.5K (1 ⁇ 10 9 )), each mouse was injected with 100 ⁇ l of C-4.5K virus suspension containing 1 ⁇ 10 9 PFU; the fourth group was oncolytic virus 1-4.5K In the medium-dose group (1-4.5K (1 ⁇ 10 8 )), each mouse was injected with 100 ⁇ l of 1-4.5K virus suspension containing 1 ⁇ 10 8 PFU each time; the fifth group was the oncolytic virus high-dose group ( 1-4.5K (1 ⁇ 10 9 )), each mouse is injected with 100 ⁇ l of 1-4.5K virus suspension containing 1 ⁇ 10 9 P
  • Preparation of blood cell suspension After removing the mouse eyeballs, blood is taken and added to an anticoagulation tube, shaken and mixed well, and placed on ice. Centrifuge at 500 ⁇ g for 5 minutes, discard the supernatant, and add the red blood cell lysate (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: #122-02), mix well to pellet the cells and react at room temperature for 10-15 minutes Centrifuge again at 500 ⁇ g for 5 minutes and discard the supernatant to retain the cell pellet. Resuspend the cells in a PBS solution containing 1% BSA and centrifuge at 500 ⁇ g for 5 minutes and discard the supernatant to retain the cell pellet.
  • red blood cell lysate purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number: #122-02
  • Preparation of spleen cell suspension dissecting the mouse and taking the mouse spleen, placing it in a 1.5ml centrifuge tube and temporarily storing it on ice. Add 5ml of PBS solution containing 1% BSA to a clean 6cm petri dish, wrap the spleen with nylon mesh, squeeze and grind the spleen in the petri dish to fully release the spleen cells. Centrifuge the cell suspension at 500 ⁇ g for 5 minutes and discard the supernatant. Add the red blood cell lysate to mix well to pellet the cells and react at room temperature for 10-15 minutes. Centrifuge again at 500 ⁇ g for 5 minutes. Discard the supernatant and retain the cell pellet.
  • Preparation of tumor tissue cell suspension dissecting the mouse to take the subcutaneous tumor and place it in a 1.5ml centrifuge tube and temporarily store it on ice. Cut each tumor tissue into a small piece of equal volume and put it in another set of centrifuge tubes. Add 500 ⁇ l collagenase. Use ophthalmic scissors to cut the tumor into small pieces and react at 37°C for 30 minutes. The cell suspension is 500 ⁇ g After centrifugation for 5 minutes, discard the supernatant and add the red blood cell lysate to fully mix the pelleted cells and react at room temperature for 10-15 minutes. Centrifuge again at 500 ⁇ g for 5 minutes and discard the supernatant to retain the cell pellet. Reuse the cell pellet containing 1% BSA.
  • the cells were resuspended in PBS solution and centrifuged at 500 ⁇ g for 5 minutes. The supernatant was discarded to retain the cell pellet.
  • the cell pellet was divided into two, one without any treatment as a control, and the other with anti-BALB/C mouse CD3 and CD49b flow cytometry antibody, after mixing, react at room temperature and avoid light for 30 minutes, then go to the machine for FACS.
  • C-4.5K and 1-4.5K oncolytic viruses fully exhibit the characteristics of oncolytic viruses.
  • 1-4.5K can express shPDL1, which is more effective than C-4.5K on HCT116 cells. Longer duration of growth inhibition.
  • shPDL1 expressed in the oncolytic virus 1-4.5K changes the expression level of hPDL1 in human tumor cells, which may weaken or relieve the immunosuppressive effect around the tumor and activate the surrounding immune cells. Therefore, Realize the combined killing effect on tumor.
  • the body weight of all five groups of mice did not change significantly, indicating that the two oncolytic adenoviruses were not significantly toxic to BALB/C nude mice (see Figure 48).
  • the NK cells in the spleen of animals in each group did not change significantly (see Figure 50).
  • the analysis of T cells found that the proportion of T cells in the blood of each group of animals did not change significantly.
  • the T cells in the spleen and tumor tissues of the 1-4.5K (1 ⁇ 10 8 ) experimental group were higher than those in the C-4.5K (1 ⁇ 10 8 ) experimental group. 108 ) T cells in the same tissue have been significantly increased (see Figure 51).
  • Another scaffold control virus (C-4.5K) at the same concentration (1 ⁇ 10 9 ) that does not contain the shPDL1 expression cassette showed subcutaneously inoculated BALB/C nude mice with HCT116 only in the early stage of administration, which showed the same concentration as OAd-shPDL1(1 -4.5K)
  • Oncolytic virus has similar anti-tumor effect, but it cannot continue to inhibit tumor growth like OAd-shPDL1 (1-4.5K) after stopping administration.
  • tumor cells will highly express ligands such as PDL1 or PDL2 on their own surface. The combination of the above ligands and PD-1 will cause the intracellular domain of PD-1.
  • B cells are present, they have a lack of function, and NK cells have extremely low functions; while BALB/C nude mice have The degree of immunodeficiency is low, and T cells are also missing in the body.
  • B cells are present but lacking in function, they retain intact NK cells, macrophages and antigen-presenting cells, mainly dendritic cells. It has been reported in the literature that NK, macrophages or dendritic cells in mice may play an important combined killing effect in the process of inhibiting tumor growth (see (for example) the following scientific literature: "Kevin C. Barry, et al.
  • HCT116 oncolytic adenovirus OAd-shPDL1 (1-4.5K) shPDL1 expressed in human tumor cells HCT116 caused the decrease in the expression of hPDL1 in tumor cells, which weakened and relieved the immune immunity around the tumor on BALB/C nude mice.
  • Cells NK, macrophages or dendritic cells
  • HCT116 inhibited the growth of HCT116.
  • Example 8 Inducing Her2/neu 369-377 polypeptide (Her2-E75 epitope polypeptide) to specifically kill T cells from the peripheral blood of HLA-A2-positive normal donors
  • the right panel of Figure 35A shows that 0.024% of lymphocytes are CD8-positive killer T cells that can bind to the Her2/neu 369-377/HLA-A2 pentamer (Her2-E75 pentamer).
  • the polypeptide-stimulated control cells did not show CD8-positive pentamer-positive cells.
  • the results show that in the natural T cell pool, the number of specific T cells that recognize the Her2/neu 369-377 antigen polypeptide is very small. Despite the small number, this group of T cells that can recognize the Her2/neu 369-377 polypeptide can still be clearly distinguished.
  • the positive cells contained high-affinity T cells and low-affinity T cells.
  • Example 9 Obtaining the full sequence of Her2/neu 369-377 polypeptide-specific TCR
  • RNA was purified directly from a certain number of Her2 CTL 6A5 cells obtained in Example 8, and the paired TCR ⁇ chain and ⁇ chain gene sequences were obtained through the 5'-RACE RT-PCR method (that is, the two chains can be shared It constitutes a functional TCR that recognizes the antigen polypeptide, and the encoded TCR is called "Her2 TCR-6A5".
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 4, the coding sequence is shown in SEQ ID NO: 12, and the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 7, and the coding sequence is shown in SEQ ID NO: 15.
  • TCR This TCR exists in the peripheral T cell pool of HLA-A2-positive normal people, and will not cross-react with normal cells that slightly express the Her2/neu protein to cause autoimmune reactions.
  • the TCR alpha chain and beta chain sequences were cloned into a replication-deficient lentiviral expression vector.
  • Figure 35C shows a schematic diagram of the constructed TCR lentiviral vector structure fragment. The constant regions of the TCR alpha chain and beta chain are replaced by human sequences with murine sequences, and are connected by a cleavable linking polypeptide. The expression of 6A5TCR ⁇ chain and ⁇ chain is driven by the EF-1 ⁇ promoter.
  • This promoter is a highly expressed promoter in eukaryotic cells, and will not be affected by methylation and other factors to cause loss of function, and is suitable for long-term expression of foreign genes in vivo.
  • the TCR alpha chain and beta chain are connected by the F2A polypeptide sequence, and the TCR alpha chain and beta chain genes can be transcribed at the same time, and translated through ribosome skipping, so that the TCR alpha chain and beta chain polypeptides are separated from each other. This ensures the consistency of the expression of the TCR alpha chain and the beta chain, thereby more efficiently composing TCR dimers.
  • TCR ⁇ chain and ⁇ chain SEQ ID NO: 20
  • corresponding TCR is Her2 TCR-6A5-mC
  • the amino acid sequence is connected to the above vector to obtain the Her2 TCR-6A5-mC recombinant lentiviral vector.
  • Her2 TCR-6A5-mC gene fragment was amplified by PCR and cloned into the downstream of the EF1-promoter of the above-mentioned lentiviral vector (ie pCDH-EF1 ⁇ -MCS): ⁇ of Her2 TCR-6A5-mC carrying mouse constant region sequence
  • the fragment is amplified by the 5'primer 5'-AGAGCTAGCGAATTCAACATGGGCTGCAGGCTGCTC-3' (SEQ ID NO: 26) and the 3'primer 5'-GGATCGCTTGGCACGTGAATTCTTTCTTTTGACCATAGCCAT-3' (SEQ ID NO: 27); it carries the mouse constant region sequence
  • the alpha gene of Her2 TCR-6A5-mC is amplified by 5'primer 5'-TCCAACCCTGGGCCCATGCTCCTGTTGCTCATACCAGTG-3' (SEQ ID NO: 28) and 3'primer 5'-GTTGATTGTCGACGCCCTCAACTGGACCACA
  • PCR uses the Q5 high-fidelity PCR kit (NEB, cat#M0543S), and the reaction conditions are: 98°C for 30 seconds, followed by 25 cycles: 98°C for 10 seconds, 65°C for 10 seconds, and 72°C for 3 minutes.
  • the obtained TCR fragment was cloned into the MCS region downstream of the EF1 ⁇ promoter of the pCDH-EF1 ⁇ -MCS vector.
  • the constructed recombinant TCR lentiviral expression vector was prepared according to the aforementioned method to obtain respective recombinant TCR lentiviral particles.
  • Example 10 Normal peripheral blood T cells were transfected with Her2 TCR-6A5-mC recombinant lentivirus to express a specific TCR that can recognize the Her2/neu 369-377 polypeptide.
  • TCR obtained in the present invention can be expressed in primary T cells and has the function of recognizing Her2/neu antigen polypeptides
  • recombinant lentiviral particles carrying Her2 TCR-6A5-mC gene (Her2 TCR-6A5-mC recombinant slow Viral vector) was transfected with CD3/CD28 antibody activated peripheral blood T cells from two different normal donors. After 14 days, the cells were collected for Her2-E75 tetramer staining. The specific method is as described above. The results are as follows:
  • Figure 36A shows that the two donor peripheral blood mononuclear cells (#1PBMC and #2PBMC, respectively) have lymphocytes that can bind to Her2-E75 tetramer, indicating that the Her2 TCR-6A5-mC expressed by these cells can be specific Recognizes the Her2/neu antigen polypeptide presented by HLA-A2.
  • Her2-E75 tetramer positive cells that is, Her2 TCR-6A5-mC
  • the positive rate of CD8 + T killer cells was similar to that of CD8 - lymphocytes.
  • CD8 - lymphocytes are likely to be CD4 + T helper cells.
  • the transfection efficiency of CD8 + and CD4 + T cells with the lentivirus is the same, it means that the exogenous Her2/neu 369-377 specific TCR on CD4 + cells can be Effectively binds Her2-E75 tetramer.
  • the transfected Her2 TCR-6A5-mC does not require the auxiliary function of the CD8 molecule and can effectively bind to the Her2/HLA-A2 complex, that is, Her2 TCR-6A5-mC recognizes Her2/neu presented by HLA-A2
  • the 369-377 epitope polypeptide is CD8 independent.
  • CD4 cells expressing Her2 TCR-6A5-mC TCR recognize Her2 antigen and secrete cytokines, which can not only help kill T cell function and survival time in the body, but also induce endogenous tumor antigens by regulating the tumor microenvironment Specific T cells, thereby enhancing anti-tumor immunity.
  • Figure 36B shows that PBMC expressing Her2 TCR-6A5-mC can be activated by the Her2/neu 369-377 antigen polypeptide presented by T2 cells to secrete IFN- ⁇ , indicating that the primary T expressing exogenous Her2 TCR-6A5-mC Cells can specifically recognize the Her2/neu 369-377 polypeptide presented by HLA-A2 molecules.
  • the ability to recognize antigen polypeptides is related to the amount of expression of exogenous TCR on T cells.
  • EC50 maximum half-maximum reaction
  • FIG 36C shows that T cells and T2 cells presented antigen polypeptides (T2+Her2-E75, namely Her2/neu 369-377 polypeptide) when anti-human CD8 antibody was added during co-cultivation, the function of T cells to secrete IFN- ⁇ was not significant inhibition.
  • T2+Her2-E75 namely Her2/neu 369-377 polypeptide
  • the function of the exogenous TCR to recognize the Her2/neu 369-377 antigen polypeptide does not require the assistance of the CD8 molecule, and also shows that the recognition function of the Her2 TCR-6A5-mC TCR of the present invention is non-CD8 function-dependent.
  • Example 11 The Her2/neu 369-377 polypeptide-specific TCR expressed by normal peripheral blood T cells transfected with Her2 TCR-6A5-mC recombinant lentivirus can recognize HLA-A2 + Her2/neu + tumor cells
  • Tumor cell lines include colorectal cancer Colo205 and HCT116, breast cancer MDB-MB-231 and MCF-7, pancreatic cancer PANC-1, glioma U87MG, and small cell lung cancer NCI-H446.
  • Tumor cells were stained with anti-HLA-A2 antibody (BD Bioscences, cat#561341) and anti-human CD340 (erbB2) antibody (Biolegend, cat#324406) for flow cytometric analysis.
  • FIG. 37A show that colo205, MDB-MB-231, MCF-7, HCT116, and PANC-1 are all HLA-A2 + Her/neu + ; U87MG is HLA-A2 + , Her2/neu - ; NCI-H446 is HLA -Both A2 and Her2/neu are negative. These tumor cell lines not only originate from different tissues, but also express different HLA-A2 and Her2/neu. Among them, U87MG and NCI-H446 cells can be used as negative controls for Her2 TCR-6A5-mC T cell function testing.
  • FIG. 37B shows that T cells expressing Her2 TCR-6A5-mC can be activated by HLA-A2 + Her2/neu + tumor cell lines and secrete IFN- ⁇ .
  • Tumor cell lines include colon cancer Colo205 and HCT116, breast cancer MDA -MB-231 and MCF-7, pancreatic cancer PANC-1.
  • the control group HLA-A2 + Her2/neu - glioma U87MG and HLA-A2 - Her2/neu - lung cancer NCI-H446 cannot activate the T cells transfected with Her2 TCR-6A5-mC, indicating that Her2 TCR -6A5-mC TCR can specifically recognize the Her2/neu antigen presented by HLA-A2 on the surface of tumor cells.
  • Control T cells from the same donor PBMC and cultured in parallel but not transfected with Her2 TCR-6A5-mC could not be activated by the listed tumor cell lines, indicating that the response to tumor cells is not non-specific.
  • the results show that the ability of Her2TCR-6A5-mC T cells to recognize the Her2/neu antigen presented by HLA-A2 is not closely related to the expression of HLA-A2 and Her2/neu molecules on the surface of tumor cells. Different tumor cells may have different inhibitory effects on T cells.
  • the expression level on the cell surface does not necessarily reflect the total expression level of Her2/neu. Some tumor cells express Her2/neu mainly in the cell cytoplasm. Therefore, these antigens are more easily presented by HLA-A2 (see the literature "J Immunol 2006; 177:5088-5097").
  • FIG. 37C-K shows that, compared with control T cells without TCR transfection, T cells expressing Her2 TCR-6A5-mC TCR can specifically recognize and kill the HLA-A2 + Her2/neu + tumor cell line MCF-7 , HCT116, PANC-1 and HEPG-2.
  • the killing ability has a dose-effect relationship with the number of Her2 TCR-6A5-mCT cells.
  • the control group HLA-A2 + Her2/neu - glioma U87MG, HLA-A2-Her2/neu+ SKOV3 and HT-29, and HLA-A2 - Her2/neu - lung cancer NCI-H446 can not be Her2 TCR.
  • -6A5-mC T cell specific killing The results also showed that when Her2 TCR-6A5-mC T cells increased to a certain number, they showed significant specific recognition and killing functions for HLA-A2 + Her2/neu + tumor cells, when the effective target ratio was less than 10 :1, the specific killing function is not obvious, which may be related to the number of Her2/neu epitope peptides presented by HLA-A2 on the surface of tumor cells.
  • one strategy is to increase the number of tumor target cells expressing HLA-A2 and Her2/neu.
  • Example 12 Combined killing effect of oncolytic adenovirus OAd-shPDL1 and human T cells expressing Her2 TCR-6A5-mC on human colorectal cancer cells HCT116 subcutaneously inoculated in NCG severely immunodeficient mice
  • human colorectal cancer cells HCT116 were inoculated subcutaneously on the dorsal side of the right forelimb of NCG severe immunodeficiency mice to prepare a tumor-bearing animal model that simulates the human environment for the detection of oncolytic adenovirus OAd-shPDL1 (prepared with the method described in Preparation Example 5.
  • the combined killing effect of human T cells expressing Her2 TCR-6A5-mC (prepared by referring to the method in Example 10) on human colorectal cancer cells HCT116 subcutaneously inoculated with NCG in severely immunodeficient mice.
  • the amount of cell inoculation for each animal is 5 ⁇ 10 6 cells.
  • mice with the required subcutaneous tumor volume (tumor volume of 90-120mm 3 ) were selected and divided into 4 groups according to the block randomization method , 3 in each group, and the grouping day is set to day 0.
  • the first group is a blank control group.
  • Each mouse in the group is injected with 100 ⁇ l of Ad preservation solution (ie, 10mM Tris solution (pH7.4) containing 1mM MgCl 2 and 10% glycerol) intratumorally on day 0 and day 4, respectively.
  • Ad preservation solution ie, 10mM Tris solution (pH7.4) containing 1mM MgCl 2 and 10% glycerol
  • the second group is 1-4.5K group, each mouse in the group was injected with 100 ⁇ l oncolytic adenovirus intratumorally on the 0th and 4th day OAd-shPDL1 (1-4.5K), the virus injection volume per animal is 5 ⁇ 10 8 PFU, 200 ⁇ l of normal saline is injected into the tail vein on the 2nd and 6th days;
  • the third group is 1-4.5K+Mock -T group (where "Mock-T" is the control T cell), each mouse in the group was injected with 100 ⁇ l of oncolytic adenovirus OAd-shPDL1(1-4.5K) into the tumor on day 0 and day 4, respectively.
  • the virus injection volume per animal is 5 ⁇ 10 8 PFU, and 200 ⁇ l of ordinary T cell suspension without any modification (suspension medium is normal saline) is injected into the tail vein on the 2nd and 6th day, and the number of cells is 2 ⁇ 10 7 ;
  • the fourth group is the 1-4.5K+E75-TCRT group (where "E75-TCRT” is the human T cell expressing Her2 TCR-6A5-mC), each mouse in the group is on day 0
  • 100 ⁇ l of oncolytic adenovirus OAd-shPDL1 (1-4.5K) was injected intratumorally, the virus injection amount per animal was 5 ⁇ 10 8 PFU, and 200 ⁇ l was injected into the tail vein on the 2nd and 6th days.
  • Her2 TCR-6A5-mC human T cells the number of cells is 2 ⁇ 10 7 cells
  • the two injections of human T cells expressing Her2 TCR-6A5-mC in this group were cultured to the 9th day E75-TCR positive rate 25% of cells cultured to the 13th day with a positive rate of 20%.
  • Each group of animals were injected subcutaneously with 100,000 IUIL2 next to the tumor on day 2, day 3, day 6 and day 7 to stimulate T Cell proliferation and activation enable human T cells to survive longer in NCG mice.
  • the detailed dosing schedule for this experiment is shown in Figure 53.
  • the flow cytometric analysis results of the number of T cells in the blood are shown in Figure 60, and the flow cytometric analysis results of the number of T cells in the spleen of each group of animals are shown in Figure 61.
  • the flow cytometric analysis of the number of Her2 + /PDL1 + double positive cells in the tumors of each group of animals The results are shown in Figure 62.
  • the combined killing effect of the oncolytic adenovirus OAd-shPDL1 (1-4.5K) and human T cells expressing Her2 TCR-6A5-mC (group 4) on HCT116 cells inoculated subcutaneously by NCG is far Stronger than oncolytic adenovirus OAd-shPDL1 (1-4.5K) alone administration group (group 2) and oncolytic adenovirus OAd-shPDL1 (1-4.5K) and control T cell (Mock-T) combined group ( The third group), considering that ordinary T cells (i.e.
  • human T cells expressing Her2 TCR-6A5-mC can kill HCT116 cells.
  • the oncolytic adenovirus OAd-shPDL1 (1-4.5K) may also enhance other immune killer T cells (TCR-T and CAR-T) affected by the PDL1/PD1 immune checkpoint on tumors (especially Is the killing effect of solid tumors.
  • TCR-T and CAR-T immune killer T cells
  • the combined use of oncolytic viruses and immune killer cells to kill tumors has enriched the treatment modes and methods of tumor treatment, and is very likely to become one of the breakthroughs in the treatment of tumors (especially solid tumors).

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Abstract

L'invention concerne un agent thérapeutique contenant un adénovirus oncolytique recombinant isolé et des cellules immunitaires, et son utilisation. L'agent thérapeutique contient : une première composition contenant une première substance active située dans un premier support pharmaceutiquement acceptable, ladite première substance active contenant un adénovirus oncolytique recombinant à réplication conditionnelle isolé dans lequel une séquence codante d'ARNsh exogène capable d'inhiber l'expression de PDL1 dans des cellules tumorales a été intégrée; et une seconde composition contenant une seconde substance active située dans un second support pharmaceutiquement acceptable, la seconde substance active contenant des cellules immunitaires modifiées par le récepteur des lymphocytes T ou des cellules immunitaires modifiées par un récepteur d'antigène chimère. Le présent agent thérapeutique peut être utilisé dans le traitement de tumeurs et/ou du cancer.
PCT/CN2020/081090 2019-03-27 2020-03-25 Agent thérapeutique contenant un adénovirus oncolytique recombinant isolé et des cellules immunitaires, et son utilisation WO2020192684A1 (fr)

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CN112481299A (zh) * 2020-11-20 2021-03-12 郑州大学 用于调节PD-1/PD-L1通路的RNAi表达质粒
CN113244411B (zh) * 2021-06-25 2021-09-17 诺赛联合(北京)生物医学科技有限公司 一种基因修饰的溶瘤病毒诱导ctl细胞方法及其在肿瘤治疗的用途
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