WO2020173123A1 - 兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 - Google Patents

兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 Download PDF

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WO2020173123A1
WO2020173123A1 PCT/CN2019/115627 CN2019115627W WO2020173123A1 WO 2020173123 A1 WO2020173123 A1 WO 2020173123A1 CN 2019115627 W CN2019115627 W CN 2019115627W WO 2020173123 A1 WO2020173123 A1 WO 2020173123A1
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immune
tumor
spd1cd137l
blocking
cells
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魏继武
张永辉
张海林
吴俊华
董杰
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南京大学
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Definitions

  • the present invention relates to the field of tumor immunotherapy, in particular to a replicating oncolytic adenovirus capable of activating immune costimulation signal pathways and blocking immune checkpoints and applications thereof.
  • Cancer has become the first disease that endangers human life and health. There are about 4 million new cancer patients in my country every year, and nearly 3 million people die from cancer every year. In addition to conventional surgery, radiotherapy and chemotherapy, new and effective treatment methods are urgently needed. Therefore, the research and development of anticancer drugs has always been a hot spot in pharmaceutical research.
  • the immune system has the ability to recognize and eliminate dissidents. During the development of tumors, it not only suppresses the innate immune response of the immune system through a variety of ways, but also evades the body's normal immunity through many methods such as "hiding identity” and initiating negative immune regulation. The monitoring of the system will not be cleared by the body. Tumor cells hijack the PD-1/PD-L1 pathway by themselves or by promoting other cells to express PD-L1, which is a negative immunoregulatory molecule, thereby inhibiting the anti-tumor activity of effector lymphocytes and entering a disabled state to avoid immune surveillance and Purpose of immune clearance. Therefore, blocking the immune checkpoint PD-1/PD-L1 pathway has received extensive attention. Blocking immune checkpoints has been proven to be one of the effective anti-tumor methods. So far, multiple immune checkpoint blockers have been approved as drugs for clinical tumor treatment.
  • CD137 is an extremely important immune co-stimulatory molecule of lymphocytes and participates in the activation process of T lymphocytes. Without the activation signal of this molecule, T cells that recognize tumors still cannot effectively produce anti-tumor responses. However, due to the body's normal balance regulation mechanism, activated lymphocytes often up-regulate the expression of immunonegative regulatory molecules, including PD1, CTLA4, TIGIT, etc., to avoid excessive activation of the immune system. Therefore, we hypothesize that the combination of blocking immune checkpoint PD-1/PD-L1 and activating immune costimulatory molecule CD137 can more effectively exert the anti-tumor effect of immune cells.
  • TILs tumor infiltrating lymphocytes
  • IFN ⁇ / ⁇ type I interferon pathway
  • recombinant oncolytic viruses can be injected locally into tumors to highly express target genes (proteins) that have immune activation effects in the infected tumor microenvironment.
  • target genes proteins
  • the purpose of the present invention is to provide a replicating oncolytic adenovirus that can provide a second signal for T cell activation and block the T cell immune checkpoint suppression signal and its application.
  • the present invention adopts the following technical solutions: a soluble fusion protein of the present invention that both activates the immune costimulatory signal pathway, blocks immune checkpoints, and bridges tumor cells and immune cells, and the soluble fusion protein
  • the two ends of the protein are respectively PD1 which binds to PD-L1 (tumor cells) and CD137L which binds to CD137 (immune cells).
  • PD1 and CD137L are connected by a linker sequence.
  • the soluble fusion protein is sPD1CD137L
  • the protein sequence and amino acid sequence of sPD1CD137L are shown in SEQ ID NO: 1 and SEQ ID NO: 6 of the sequence table, respectively.
  • the application of the soluble fusion protein capable of activating the immune costimulation signal pathway and blocking the immune checkpoint of the present invention in the preparation of activating anti-tumor immune drugs.
  • the tumor is liver cancer, ascites cancer, melanoma or breast cancer.
  • the replicating oncolytic adenovirus capable of activating the immune costimulatory signal pathway and blocking immune checkpoints of the present invention is characterized in that: the replicating oncolytic adenovirus replicates in tumor cells, and expresses and secretes A soluble fusion protein.
  • the two ends of the soluble fusion protein are respectively PD1 binding PD-L1 and CD137L binding CD137, and PD1 and CD137L are connected by a linker sequence.
  • the soluble fusion protein is sPD1CD137L, and the amino acid sequence of the protein sequence of sPD1CD137L is shown in SEQ ID NO:1 in the sequence table.
  • the replicating oncolytic adenovirus capable of activating the immune costimulatory signal pathway and blocking immune checkpoints according to the present invention is characterized in that the replicating oncolytic adenovirus can be oncolytic.
  • the tumor is liver cancer, ascites cancer, melanoma or breast cancer.
  • the method for constructing replicating oncolytic adenovirus AD5 sPD1CD137L which has the function of activating the immune costimulatory signal pathway and blocking immune checkpoints of the present invention, includes the following steps: (1) AD5 sPD1CD137L full-length plasmid construction: the constructed Shuttle vector AD5-pShuttle-sPD1-CD137L was linearized with PmeI and then transferred to competent pAdEasy-BJ5183, and screened using LB plates containing 50ug/ml kanamycin, and positive clones were selected for culture identification, and the correct clone plasmid was identified Re-transform DH5a competence for secondary screening and identification. After the identification is correct, carry out plasmid extraction to obtain the full-length AD5-sPD1-CD137L plasmid;
  • AD5 sPD1CD137L full-length plasmid was linearized with PacI, after purification, 1ug/well was transfected into 293T cells in a 6-well plate, cultured in 5% CO 2 , 37°C, after 2 days, the cells were digested and transferred to 10cm Change the plate for 2-3 days until 80% of the cells show disease. Use 10ml medium to blow down the cells and collect them into a 15ml centrifuge tube. Repeat the freeze-thaw cycle twice, centrifuge at 3000rpm/min for 15min, collect the virus supernatant and store at -80°C As a virus seed
  • Virus amplification Take 50ul of virus seed solution and add 60% 293T cells to a 10cm plate, culture in 5% CO 2 at 37°C, and the cell density is above 90%. Passage 1 to 3 until 80% of the cells show disease. There are about 10 plate cells, the virus is collected according to the above method, and the virus is purified by cesium chloride density gradient centrifugation; the TCID50 method is used for titer determination.
  • the present invention provides a method for designing and constructing a replicating oncolytic adenovirus AD5 sPD1CD137L that can activate immune costimulatory signal pathways and block immune checkpoints, and successfully obtained a new strain with both activating immune
  • the replicating oncolytic adenovirus AD5 sPD1CD137L that stimulates signal pathways and blocks immune checkpoints.
  • This virus can selectively replicate in tumor cells and tumor sites, has tumor targeting, can effectively oncolysis, and induce immunogenicity Cell death.
  • the present invention has the following advantages: (1) At the same time, the virus can highly express the soluble fusion protein sPD1CD137L, which can be secreted to the outside of the cell to block immune checkpoints in the tumor microenvironment. Activate the immune costimulatory signal pathway, and at the same time can bridge the tumor cells and immune cells to enhance the anti-tumor immunity.
  • the replicating oncolytic adenovirus AD5 sPD1CD137L of the present invention which has both activating immune costimulatory signal pathways and blocking immune checkpoints, has a significant activating anti-tumor immunity, and can significantly stimulate the high expression of IFN- ⁇ in tumors and It has no obvious systemic toxicity, significantly inhibits tumor growth, prolongs survival, and has a significant anti-tumor effect.
  • a virus which integrates multiple unique anti-tumor mechanisms at the same time, has unexpected anti-tumor effects. Can be used to prepare anti-tumor drugs.
  • the present invention designs a method for constructing a new type of replicating oncolytic adenovirus AD5 sPD1CD137L that has both activating immune costimulatory signal pathways and blocking immune checkpoints, and obtained a new type of both activating immune costimulatory signals
  • the replication-type oncolytic adenovirus AD5sPD1CD137L which can access pathways and block immune checkpoints, can replicate specifically in tumor cells and tumor sites, and can highly express the soluble fusion protein sPD1CD137L.
  • the N-terminus of the soluble fusion protein sPD1CD137L is the extracellular region of PD1, which can specifically bind to PD-L1, thereby blocking the binding of PD-L1 to the specific receptor PD1 on effector lymphocytes, and finally blocking PD1/PD -L1 negative regulatory pathway;
  • the C-terminal of the fusion protein is the extracellular region of CD137L, which can specifically bind to its receptor CD137, activate the downstream immune activation pathway of CD137, and play an immune activation role.
  • the fusion protein sPD1CD137L can be secreted to the outside of cells in large quantities, and can simultaneously play the biological and immunological functions of blocking immune checkpoints and immune co-stimulation in the tumor microenvironment, and significantly activating immunity. At the same time, it can also rise between tumor cells and immune cells. To bridge, enhance anti-tumor immunity.
  • the present invention provides a method for designing and constructing a replicating oncolytic adenovirus AD5 sPD1CD137L that both activates the immune costimulatory signal pathway and blocks immune checkpoints, and successfully obtained a strain that also activates immune costimulatory signals.
  • the replication-type oncolytic adenovirus AD5 sPD1CD137L which can access pathways and block immune checkpoints, can replicate in tumor cells and tumor sites, and can highly express the soluble fusion protein sPD1CD137L, which can be secreted outside the cell and exerted locally on the tumor Regulate the biological and immunological functions of immune checkpoints and immune costimulation.
  • the N-terminal of the soluble fusion protein sPD1CD137L is the extracellular region of PD1, which can specifically bind to PD-L1, thereby blocking the binding of PD-L1 to the specific receptor PD1 on the effector lymphocytes, and finally blocking The negative regulatory pathway of PD1/PD-L1; the C-terminus of the fusion protein is the extracellular region of CD137L, which can specifically bind to its receptor CD137, activate the downstream immune activation pathway of CD137, play an immune activation role, and also It can play a bridge between tumor cells and immune cells to enhance anti-tumor immunity.
  • the replicating oncolytic adenovirus AD5 sPD1CD137L of the present invention which is capable of activating the immune costimulatory signal pathway and blocking immune checkpoints, has a significant activating anti-tumor immunity and can stimulate high expression of IFN- ⁇ .
  • the replicating oncolytic adenovirus AD5 sPD1CD137L which both activates the immune costimulatory signal pathway and blocks immune checkpoints, has significant anti-tumor effects and can be used to prepare anti-tumor drugs.
  • Figure 1 shows the results of the present invention showing that we have successfully constructed a replicating oncolytic adenovirus AD5 sPD1CD137L that both activates the immune costimulatory signal pathway and blocks immune checkpoints.
  • the corresponding position of the control group in the figure has no bands and is blank.
  • the dark black band corresponding to the replicating oncolytic adenovirus AD5 sPD1CD137L, which activates the immune costimulatory signaling pathway and blocks immune checkpoints, proves that it can activate the immune costimulatory signaling pathway and block immune checkpoints.
  • the tumor adenovirus AD5 sPD1CD137L can be expressed in tumor cells and secreted outside the cell, and judging from the protein size, it is the target protein we intend to express: soluble SPD1CD137L.
  • Figure 2 shows the results of the present invention showing that the replicating oncolytic adenovirus AD5 sPD1CD137L constructed by us that activates the immune costimulatory signal pathway and blocks immune checkpoints has the same replication and oncolytic abilities in tumor cells as the control virus .
  • Figure 3 is the result of the tumor volume measurement data of the present invention showing that the replicating oncolytic adenovirus AD5 sPD1CD137L, which has both activating the immune costimulatory signal pathway and blocking immune checkpoints, has a significant anti-tumor effect in vivo (B16/F10 solid tumor Model), can inhibit tumor growth.
  • Figure 4 shows the results of the present invention showing that the replicating oncolytic adenovirus AD5 sPD1CD137L can inhibit tumor growth in the mouse 4T1 breast cancer subcutaneous tumor model, and significantly prolong the survival time of mice.
  • Figure 5 shows the results of the present invention showing that the replicating oncolytic adenovirus AD5 sPD1CD137L can significantly inhibit tumor growth in the mouse Hepa1-6 hepatocarcinoma subcutaneous tumor model. About 60% of the mouse tumors are completely cured, and the mice achieve long-term survival.
  • Figure 6 is the survival result data of the present invention showing that the recombinant oncolytic adenovirus AD5 sPD1CD137L has an anti-tumor effect in vivo (H22 liver cancer ascites tumor model), which can significantly prolong the survival period of tumor-bearing mice, 70% of the mice are completely cured and long-term survive.
  • Figure 7 shows the detection of the number and expression level of IFN- ⁇ -secreting cells by the ELISpot and ELISA methods of the present invention.
  • the data results show that the replication-type oncolytic gland is capable of activating the immune costimulatory signal pathway and blocking the immune checkpoint.
  • the virus AD5 sPD1CD137L significantly enhances immune activation and improves anti-tumor effect (H22 liver cancer ascites tumor model), stimulates high expression of IFN- ⁇ , and maintains the activity of T cells.
  • Figure 8 shows that the present invention confirms that the replication-type oncolytic adenovirus AD5 sPD1CD137L promotes anti-tumor immune response dependent on CD8+ T cells, not NK cells. And CD8+T is the main source of IFN- ⁇ in ascites.
  • Figure 9 shows that the fusion protein sPD1CD137L of the present invention can significantly inhibit tumor growth in the Hepa1-6 hepatocarcinoma subcutaneous tumor model in mice.
  • Figure 10 shows that the replication-type oncolytic adenovirus AD5 sPD1CD137L of the present invention releases tumor-associated antigens by lysing tumor cells; at the same time, it recruits immune cells and provides signals to maintain immune cell activation, ultimately generating an effective anti-tumor immune response.
  • the primers are all synthesized by GenScript.
  • the DMEM high glucose medium, double antibodies, and serum required for tumor cell culture were all purchased from Invitrogen (Shanghai).
  • Quantitative RT-PCR reagent Faststart Universal SYBR Green Master (Roche, 04913914001).
  • protease inhibitor (Roche, 11873580001), cell lysate (Biyuntian: P0013), PVDF membrane (Roche, 03010040001), WB Immobilon ECL luminescent solution (Millipore, WBKLS0500), primary antibody diluent ( Biyuntian, P0023A), HRP-labeled secondary antibody (Multisciences, GAR007 and GAM007, 1:5000 dilution), and the rest of the required reagents are all domestically pure, purchased from the School of Chemistry and Chemical Engineering, Nanjing University. Trypan Blue (Biyuntian, C0011), Opti-MEM was purchased from Invitrogen (Shanghai).
  • Western Blot antibody anti-His (GenScript, MB001, 1:5000 dilution).
  • Mouse PD1 and CD137L are membrane proteins, and their structures are: N-terminal signal peptide-extracellular domain-transmembrane domain-intracellular domain C-terminal.
  • the functional unit that PD1 and CD137L bind to PD-L1 and CD137, respectively, is the extracellular region.
  • sPD1CD137L only fusion expression of the extracellular region of PD1 and CD137L, using the linker GGGSGGGSGGGS in the middle to connect, retaining the N-terminal signal peptide of PD1 Area (see Figure 1);
  • Gene cloning of the soluble protein sPD1CD137L design and synthesize primers PD1-F, PD1-R, CD137L-F, CD137L-R, respectively, using PD1-F and PD1-R primers.
  • the primers PD1-R and CD137L-F are about 16bp, respectively, which are exactly the same as the 5'and 3'of the linker sequence.
  • EXO-PD1, linker, and EXO-CD137L fragments were spliced according to the design to complete the sPD1CD137L gene cloning.
  • the protein sequences of sPD1CD137L, EXO-PD1, linker, EXO-CD137L and signal peptide are shown in the sequence listing SEQ ID NO: 1-SEQ ID NO: 5; EXO-PD1, EXO-CD137L, linker, sPD1CD137L and signal peptide
  • the DNA sequences are shown in the sequence list SEQ ID NO: 6-SEQ ID NO: 10 respectively.
  • Gene template construction related primers are shown in Table 1:
  • the sPD1 fragment was ligated with AD5-pShuttle (pZD55) using Infusion technology. Specific steps: First, use restriction enzyme BglII to linearize AD5-pShuttle (pZD55), and then ligate the purified fragments according to the 2:1 ratio of sPD1-CD137L: AD5-pShuttle using Infusion kit (clontech lab. Inc.) After transformation and amplification, the adenovirus shuttle plasmid AD5-pShuttle-sPD1-CD137L carrying the sPD1-CD137L gene was obtained.
  • AD5 sPD1CD137L full-length plasmid construction A. AD5 sPD1CD137L full-length plasmid construction:
  • the constructed shuttle vector AD5-pShuttle-sPD1-CD137L was linearized with PmeI and transferred to competent pAdEasy-BJ5183, and screened using LB plates containing 50ug/ml kanamycin, and positive clones were picked for culture identification and identification
  • the correct cloned plasmid was re-transformed into DH5a competence for secondary screening and identification. After the identification was correct, the plasmid was extracted to obtain the full-length AD5-sPD1-CD137L plasmid.
  • the AD5 sPD1CD137L full-length plasmid was linearized with Pad. After purification, 1ug/well was transfected into 293T cells in a 6-well plate, cultured in 5% CO2, 37°C, after 2 days, the cells were digested and transferred to a 10cm plate, and the medium was changed in 2-3 days. When 80% of the cells appear diseased, use 10ml of medium to blow down the cells and collect them into a 15ml centrifuge tube. Repeated freezing and thawing twice, centrifuging at 3000rpm/min for 15min, collecting the virus supernatant and storing at -80°C as the virus seed.
  • the virus was collected by the above method, and the virus was purified by cesium chloride density gradient centrifugation; the titer was determined by the TCID50 method.
  • AD5 sPD1CD137L and AD5 con viruses infect tumor cells at the same MOI.
  • the cells were harvested 72 hours later, and the same amount of virus suspension was obtained after repeated freezing and thawing and centrifugation. 293T cells were used for virus titer determination; virus replication ability changes were analyzed.
  • the AD5 sPD1CD137L and AD5 con viruses were used to infect tumor cells at MOIs ranging from 1 to 100 viruses. After 48 hours, MTT was used to detect cell activity and evaluate the tumor-killing effect of AD5 sPD1CD137L.
  • mice choose 6-8 weeks old C57BL/6 mice to establish a subcutaneous tumor model in the right axillary, inoculate 5 ⁇ 10 5 B16/F10 cells on one side of each mouse, and measure the tumor size to 200mm 3 after 4-6 days.
  • the mice were randomly divided into 3 groups, namely: no treatment group, control AD5 virus treatment group, AD5 sPD1CD137L virus treatment group; a. According to the group, use the corresponding intratumoral injection of virus, each injected virus amount 5 ⁇ 10 8 pfu, The tumor volume and body weight were tracked and measured until the tumor volume was greater than 2500 mm 3 to determine the death of the mouse, and the survival period of the mouse was recorded.
  • the virus was injected intratumorally, each with a virus amount of 5 ⁇ 10 8 pfu, injected twice, and elispot detected immune activation.
  • mice choose 6-8 weeks old C57BL/6 mice to establish an ascites tumor model in the abdominal cavity, and each mouse is inoculated with 1 ⁇ 10 7 H22 cells in the abdominal cavity. The mice have ascites after 7-8 days.
  • the rats were randomly divided into 3 groups, namely: no treatment group, control AD5 virus treatment group, AD5 sPD1CD137L virus treatment group; a. According to the group, use the corresponding virus intraperitoneal injection, each injected virus volume 5 ⁇ 10 8 pfu, and dynamically monitor the body weight. Until the death of the mouse, the survival period of the mouse was recorded.
  • b Inject the virus into the intraperitoneal cavity according to the group, each with 5 ⁇ 10 8 pfu of virus, and two injections. ELISpot detects immune activation.
  • 293T cells are planted in 96-well plates with approximately 1 ⁇ 103 cells per well, and the titer is determined after the cells adhere to the wall.
  • Virus gradient dilution prepare EP tubes, add 1170 ⁇ l DMEM containing fetal bovine serum to each EP tube; add 130 ⁇ l virus solution to the first EP tube, mix well, and mark as 10-1; from the first EP Pipette 50 ⁇ l from the tube into the second EP tube, mix well, mark as 10-2; and so on, until the dilution reaches the desired gradient.
  • the 96-well plate was placed under a microscope to observe GFP, and the number of wells with GFP in each gradient was recorded for the calculation of virus titer.
  • V the initial volume of cell culture medium per well (ml/well)
  • the 10 ⁇ l system composition of real-time quantitative PCR 2.6 ⁇ l PCR water, 0.2 ⁇ l upstream and downstream primers, 2 ⁇ l template and 5 ⁇ l SYBR Green fluorescent dye. After the samples are mixed, they are amplified on the ABI 384 PCR machine.
  • Glue preparation and electrophoresis SDS-PAGE separation gel and concentrated gel of different concentrations are prepared according to different requirements. According to the calculation results of protein quantification, the loading amount of each sample is adjusted to 30 ⁇ g. Electrophoresis conditions: concentrated gel 80V 30min, separation gel 120V, about 80min, provided that the bands are separated and will not run out.
  • Transfer membrane Prepare filter paper and PVDF membrane, first soak the PVDF membrane with methanol, and then soak the filter paper in the transfer buffer solution for use. Carefully remove the glue from the glass plate, soak it in the transfer buffer solution, and place it in the order of the negative electrode-filter paper-PVDF membrane-glue-filter paper-positive electrode sandwich to drive out bubbles, according to the required strip size, constant flow 110mA transfer film 60-70min.
  • Exposure Wash the strips with washing buffer for 10 minutes each time, a total of three times; Expose the strips with a chemiluminescent liquid on a WB exposure instrument, and obtain strip images.
  • Figure 1 is the construction of the recombinant oncolytic adenovirus expressing soluble sPD1CD137L of the present invention
  • A The gene structure principle diagram of the recombinant oncolytic adenovirus AD5con and AD5sPD1CD137L.
  • B B16/F10 mouse melanoma cells were infected with AD5 con and AD5 sPD1CD137L respectively, and the multiplicity of infection (MOI) was 10, 48 hours later, the supernatant of the infected cells was collected, and the western blot method was used to detect the fusion protein sPD1CD137L Expression and secretion. Data represents three independent replicate experiments. GFP, green fluorescent protein; E1A, virus early region 1 replication element (early region 1); sPD1CD137L, free fusion protein PD1CD137L.
  • Figure 2 shows the replication and oncolysis of the recombinant oncolytic adenovirus AD5 sPD1CD137L of the present invention
  • Figure 3 shows the anti-tumor effect of the recombinant oncolytic adenovirus AD5 sPD1CD137L in vivo (B16/F10 melanoma solid tumor model)
  • A Evaluating the anti-tumor effect of AD5 sPD1CD137L in the B16/F10 subcutaneous tumor model, experimental scheme diagram as the picture shows.
  • B C57BL/6 was subcutaneously inoculated with 5 ⁇ 10 6 B16/F10 mouse melanoma cells on the right side, and 5 ⁇ 10 8 pfu AD5 con and AD5 sPD1CD137L were injected intratumorally to monitor tumor size in real time. The data represents three independent repeated trials. Pfu, plaque forming unit; Mock, normal saline treatment as a negative control; no statistical difference in ns; **p ⁇ 0.01.
  • Figure 4 shows the anti-tumor effect of the recombinant oncolytic adenovirus AD5 sPD1CD137L in vivo (4T1 breast cancer solid tumor model)
  • A The anti-tumor effect of AD5 sPD1CD137L was evaluated in the 4T1 subcutaneous tumor model of breast cancer. Shown.
  • B Balb/c's right side was subcutaneously inoculated with 5 ⁇ 10 4 4T1 mouse breast cancer cells, 5 ⁇ 10 8 pfu AD5 con and AD5 sPD1CD137L were injected intratumorally, and tumor size was monitored in real time.
  • C The mouse is considered dead as the tumor volume is greater than 2 cm 3 and the survival curve is calculated.
  • the data represents three independent repeated trials. Pfu, plaque forming unit; Saline, saline treatment as a negative control; no statistical difference in ns; **p ⁇ 0.01.
  • Figure 5 shows the in vivo anti-tumor effect of the recombinant oncolytic adenovirus AD5 sPD1CD137L of the present invention (Hepa1-6 solid liver cancer model)
  • A The anti-tumor effect of AD5 sPD1CD137L was evaluated in the Hepa1-6 subcutaneous tumor model.
  • the experimental plan is as follows As shown in the figure.
  • B C57BL/6 mice were subcutaneously inoculated with 5 ⁇ 10 6 Hepa1-6 hepatocellular carcinoma cells on the right side, and 5 ⁇ 10 8 pfu AD5 con and AD5 sPD1CD137L were injected intratumorally. The tumor size was monitored in real time.
  • Figure 6 shows the anti-tumor effect in vivo of the recombinant oncolytic adenovirus AD5 sPD1CD137L of the present invention (H22 liver cancer ascites tumor model)
  • A The anti-tumor effect of AD5 sPD1CD137L was evaluated in the H22 liver cancer ascites tumor model. The experimental scheme is shown in the figure.
  • B C57BL/6 was intraperitoneally inoculated with 5 ⁇ 10 6 H22 mouse liver cancer ascites tumor cells. After the mice developed ascites, 5 ⁇ 10 8 pfu AD5 con and AD5 sPD1CD137L were injected intraperitoneally to monitor the survival time of the mice in real time.
  • mice were intraperitoneally inoculated again with 5 ⁇ 10 6 H22 mouse liver cancer ascites tumor cells, and mice without any treatment were inoculated with the same number of H22 cells as a control to monitor the survival of the mice .
  • Data represents three independent replicate experiments. Pfu, plaque forming unit; Mock, saline treatment as a negative control; Mice that have not been inoculated with tumor before; ns have no statistical difference; ***p ⁇ 0.001.
  • Fig. 7 shows that the recombinant oncolytic adenovirus AD5 sPD1CD137L enhances immune activation and evaluates the immune activation effect of AD5 sPD1CD137L in the H22 liver cancer ascites tumor model.
  • the experimental scheme is shown in Fig. 6A.
  • B ELISpot detects the level of activated immune cells.
  • Figure 8 shows that the recombinant oncolytic adenovirus AD5 sPD1CD137L of the present invention relies on CD8+ T cells to clear H22 liver cancer cells, not NK cells.
  • A The mechanism of the anti-tumor immune response induced by AD5 sPD1CD137L was studied through the H22 liver cancer ascites tumor model. The experimental plan is shown in the figure.
  • B C57BL/6 was intraperitoneally inoculated with 5 ⁇ 10 6 H22 mouse liver cancer ascites tumor cells, and anti-CD8a or Anti-NK1.1 antibodies were injected on the 10th and 18th days, respectively. Five days after the antibody injection, the clearance of lymphocyte subsets in the peripheral blood of mice was detected by flow cytometry.
  • Figure 9 shows the anti-tumor effect of the fusion protein sPD1CD137L in vivo (Hepa1-6 solid tumor model of liver cancer)
  • A The anti-tumor effect of the fusion protein sPD1CD137L was evaluated in the Hepa1-6 subcutaneous tumor model of liver cancer. Show.
  • B C57BL/6 mice were subcutaneously inoculated with 5 ⁇ 10 6 Hepa1-6 tumor cells on the right side. After the tumor appeared, 300 ⁇ l of supernatant containing the fusion protein sPD1CD137L was injected intraperitoneally to monitor the tumor size in real time. There is no statistical difference in ns; **p ⁇ 0.01.
  • Figure 10 shows the working principle of the recombinant oncolytic adenovirus AD5 sPD1CD137L of the present invention.
  • Recombinant oncolytic adenovirus AD5 sPD1CD137L infects tumor cells, on the one hand it promotes the lysis of tumor cells, causes tumor cell immunogenic cell death, activates and recruits immune cells; on the other hand, the infected tumor cells secrete the fusion protein sPD1CD137L and enter the tumor microenvironment .
  • the protein sPD1CD137L 1) The PD1 end can specifically bind to tumor cells and other PD-L1 molecules expressing PD-L1 immunosuppressive cells, and prevent anti-tumor effector T cells from depletion due to PD-L1/PD1 signals; 2) The CD137L end can specifically bind to the T cell surface costimulatory molecule receptor CD137, induce continuous T cell activation, and recruit more lymphocytes to infiltrate the tumor to produce a more effective anti-tumor immune response; 3) immune activation due to tumor microenvironment
  • the up-regulated PD-L1 enables sPD1CD137L to be trapped in the tumor tissue and not easily spread into the blood. Therefore, the off-target effect is reduced; 4)
  • the fusion protein is the parent molecule of tumor cells and effector T cells, so it can act as a bridge to promote the contact between tumor cells and T cells, and increase the killing of tumor cells by T cells.
  • PD1 cell death receptor
  • PD-L cell death receptor ligand
  • Adenovirus adenovirus
  • Ad5-PC adenovirus expressing PD1CD137L fusion protein
  • Perforin perforin
  • FAS suicide-related factor
  • FASL FAS body.
  • the present invention provides a method for designing and constructing a replicating oncolytic adenovirus AD5 sPD1CD137L that can activate the immune costimulatory signal pathway and block immune checkpoints, and successfully obtained a new strain with both activation Immune costimulatory signaling pathway and replicating oncolytic adenovirus AD5 sPD1CD137L that blocks immune checkpoints.
  • This virus can selectively replicate in tumor cells and tumor sites, has tumor targeting, can effectively oncolysis, and induce immunity Primary sex cell death.
  • the virus can highly express the soluble fusion protein sPD1CD137L, which can be secreted to the outside of the cell to block immune checkpoints, activate immune costimulatory signaling pathways in the tumor microenvironment, and then activate the biological function of immunity.
  • the replicating oncolytic adenovirus AD5 sPD1CD137L of the present invention which has both activating immune costimulatory signal pathways and blocking immune checkpoints, has significant activating anti-tumor immunity, and can significantly stimulate the high expression of IFN- ⁇ in tumors without obvious Systemic toxicity, significantly inhibits tumor growth, prolongs survival, and has significant anti-tumor effects.
  • a virus which integrates multiple unique anti-tumor mechanisms at the same time, has unexpected anti-tumor effects. Can be used to prepare anti-tumor drugs.

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Abstract

本发明属于肿瘤免疫治疗领域，具体提供了一种新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L及其在抗肿瘤药物中的应用。本发明公开了AD5 sPD1CD137L的设计和构建方法，成功获得了复制型溶瘤腺病毒AD5 sPD1CD137L，该病毒可以在肿瘤细胞内特异性复制，并且能够高表达分泌型融合蛋白sPD1CD137L，该融合蛋白分子能够分泌到胞外，发挥免疫共刺激和阻断免疫检查点的双重功能。实验表明，本发明的新型复制型溶瘤腺病毒具有显著的免疫共刺激和阻断免疫检查点的作用，显著激活抗肿瘤免疫反应，具有显著的抗肿瘤的活性，有极大的开发抗肿瘤药物的前景和价值。

Description

兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用 技术领域
本发明涉及肿瘤免疫治疗领域，具体涉及兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用。
背景技术
癌症已经成为危害人类生命健康的疾病之首，在我国每年新增的癌症患者约400万人，每年有近300万人死于癌症。除了常规的手术、放疗和化疗，急需新而有效的治疗手段，因此抗癌药物的研发一直是药学研究的热点。
随着不断出现的令人振奋的临床研究结果，抗肿瘤免疫治疗给肿瘤患者带来了希望。免疫系统具有识别并清除异己的能力，肿瘤在发生发展过程中不仅通过多种途径抑制免疫系统的先天性免疫反应，也通过“隐藏身份”和启动免疫负性调控等诸多方法来逃避机体正常免疫系统的监视，从而不被机体清除。肿瘤细胞通过自身或者促进其他细胞表达免疫负性调控分子PD-L1等，来劫持PD-1/PD-L1通路，从而抑制效应淋巴细胞的抗肿瘤活性，进入失能状态，达到逃避免疫监视和免疫清除的目的。因此，阻断免疫检查点PD-1/PD-L1这个通路受到了广泛的关注。阻断免疫检查点已经被证明为有效的抗肿瘤方法之一，至今已有多个免疫检查点阻断剂被批准为用于临床肿瘤治疗的药物。
随着临床研究的进展，阻断免疫检查点的抗肿瘤治疗也遇到了一些亟需解决的问题。首先，阻断免疫检查点的免疫治疗对肿瘤类型和肿瘤患者的普适性(有效率)有待提高。尤其是对一些“冷肿瘤”，即实体肿瘤内只有少量或缺乏淋巴细胞的浸润，抑制免疫检查点的治疗效果甚微。其次，在一些肿瘤患者中， 系统性阻断免疫检查点存在“脱靶”而“误伤”正常组织的情况，由此带来了自身性免疫损伤如心肌炎等副作用。
CD137是淋巴细胞的一个极其重要的免疫共刺激分子，参与T淋巴细胞的活化过程。如果缺乏该分子的激活信号，识别肿瘤的T细胞仍不能有效产生抗肿瘤反应。然而，由于机体正常的平衡调控机制，活化的淋巴细胞往往上调免疫负性调控分子的表达，包括PD1、CTLA4、TIGIT等，以避免免疫系统的过度活化。因此，我们假设将阻断免疫检查点PD-1/PD-L1与活化免疫共刺激分子CD137联合起来，能够更有效地发挥免疫细胞的抗肿瘤作用。
进一步的临床试验数据表明，肿瘤浸润淋巴细胞(TILs)的多少、肿瘤局部的免疫活化状态等，是靶向免疫检查点治疗能否显效的重要预测指标。CD8+T细胞介导肿瘤清除过程中，I型干扰素(IFNα/β)通路的活化是靶向免疫检查点治疗的重要事件。因此，如何在肿瘤局部有效诱导I型干扰素介导的免疫活化、增强肿瘤微环境免疫细胞的浸润，可以使肿瘤对靶向免疫检查点的治疗更加敏感，这也许是解决免疫检查点治疗普适性(有效率)不高的有效手段之一。
病毒作为外来侵入颗粒，能够有效激活机体的天然免疫和适应性免疫。随着溶瘤病毒T-Vec在2015年底被FDA批准上市，溶瘤病毒介导的抗肿瘤免疫治疗受到越来越多的关注。我们设想，溶瘤病毒免疫疗法是否能使肿瘤从“冷”变“热”，对靶向免疫检查点和免疫共刺激的治疗更加敏感，从而解决免疫检查点治疗普适性(有效率)不高这一问题。
另外，得益于溶瘤病毒在肿瘤细胞具有选择性复制的优势，重组溶瘤病毒经肿瘤局部注射后，能够在感染的肿瘤微环境内，高表达具有免疫活化作用的目的基因(蛋白)，使得免疫活化能够最大限度地被局限在肿瘤微环境，从而避免阻断免疫检查点“脱靶”或者活化CD137实现免疫共刺激而造成系统性“误伤”(免疫检查点治疗遇到的第2个问题)。
目前，缺乏一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒及其应用。
发明内容
本发明的目的在于提供一种既能提供T细胞活化第二信号又能阻断T细胞免疫检查点抑制信号的复制型溶瘤腺病毒及其应用。
为达到上述目的，本发明采用了下列技术方案：本发明的一种兼具激活免疫共刺激信号通路、阻断免疫检查点和桥联肿瘤细胞与免疫细胞的可溶性融合蛋白，所述的可溶性融合蛋白的两端分别为结合PD-L1(肿瘤细胞)的PD1和结合CD137(免疫细胞)的CD137L，PD1和CD137L之间通过linker序列连接。
进一步地，所述的可溶性融合蛋白为sPD1CD137L，sPD1CD137L的蛋白序列和氨基酸序列分别如序列表SEQ ID NO:1和SEQ ID NO:6所示。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备活化抗肿瘤免疫药物中的应用。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备刺激IFN-γ表达药物中的应用。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备抗肿瘤药物中的应用。
进一步地，所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
本发明的一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒，其特征在于：所述的复制型溶瘤腺病毒在肿瘤细胞内复制，并且表达和分泌可溶性融合蛋白，所述的可溶性融合蛋白的两端分别为结合PD-L1的PD1和结合CD137的CD137L，PD1和CD137L之间通过linker序列连接。
进一步地，所述的可溶性融合蛋白为sPD1CD137L，sPD1CD137L的蛋白序列的氨基酸序列如序列表SEQ ID NO:1所示。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒，其特征在于：所述的复制型溶瘤腺病毒能够溶瘤。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备活化抗肿瘤免疫药物中的应用。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备刺激IFN-γ表达药物中的应用。
本发明所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用。
进一步地，所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
本发明的一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的构建方法，包括如下步骤：(1)AD5 sPD1CD137L全长质粒构建：将构建好的穿梭载体AD5-pShuttle-sPD1-CD137L用PmeI线性化后转入感受态pAdEasy-BJ5183中，使用含50ug/ml卡那霉素LB平板的进行筛选，挑取阳性克隆培养鉴定，鉴定正确的克隆质粒重新转化DH5a感受态进行二次筛选鉴定，鉴定正确后进行质粒大提获得AD5-sPD1-CD137L全长质粒；
(2)AD5 sPD1CD137L病毒拯救：AD5 sPD1CD137L全长质粒使用PacI线性化，纯化后6孔板中1ug/well转染293T细胞，5％CO 2、37℃培养，2天后将细胞消化后转入10cm平皿，2-3天换液，至80％细胞出现病变，使用10ml培养基将细胞吹下收集至15ml离心管，反复冻融2次，3000rpm/min离心15min，收集病毒上清-80℃保存做为毒种；
(3)病毒扩增：取病毒种液50ul加入60％293T细胞10cm平皿中，5％CO 237℃培养，细胞密度至90％以上，按照1传3比例传代，直至80％细胞出现病变，大约有10个平皿细胞，按上述方法收病毒，使用氯化铯密度梯度离心纯化病毒；使用TCID50方法进行滴度测定。
有益效果：本发明提供了一种可以兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的设计和构建方法，成功获得了一株新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L，该病毒可以选择性地在肿瘤细胞内和肿瘤部位复制、具有肿瘤靶向性，能够有效溶瘤，并诱导免疫原性细胞死亡。
与现有技术相比，本发明具有如下优点：(1)与此同时，该病毒能够高表达 可溶性融合蛋白sPD1CD137L，该蛋白能够分泌到细胞外，在肿瘤微环境中发挥阻断免疫检查点、激活免疫共刺激信号通路，同时还能在肿瘤细胞和免疫细胞间起到桥联，增强抗肿瘤免疫作用。
(2)本发明的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的活化抗肿瘤免疫作用，能够显著刺激IFN-γ在肿瘤局部高表达且没有明显的全身毒性，显著抑制肿瘤生长、延长生存期，具有显著的抗肿瘤作用。一个病毒，同时整合多种独特的抗肿瘤机制于一身，具有预料不到的抗肿瘤效果。可以用来制备抗肿瘤药物。
(3)本发明设计了一个构建一种新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的方法，获得了一株新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5sPD1CD137L，该病毒可以在肿瘤细胞内和肿瘤部位特异性复制，并且能够高表达可溶性融合蛋白sPD1CD137L。可溶性融合蛋白sPD1CD137L的N端是PD1的胞外区，可以特异性地结合PD-L1，从而封闭了PD-L1与效应淋巴细胞上的特异性受体PD1点结合，最终能够阻断PD1/PD-L1的负性调控通路；该融合蛋白的C端是CD137L的胞外区，能够特异性地结合其受体CD137，激活CD137的下游免疫活化通路，起到免疫活化作用。融合蛋白sPD1CD137L能够被大量分泌到细胞外，在肿瘤微环境内同时发挥阻断免疫检查点和免疫共刺激、显著活化免疫的生物学与免疫学功能，同时还能在肿瘤细胞和免疫细胞间起到桥联，增强抗肿瘤免疫作用。
(4)药理学实验表明，本发明的新型复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的活化抗肿瘤免疫作用、刺激刺激IFN-γ高表达，有显著的抗肿瘤的活性，具有非常高的开发抗肿瘤药物的前景和价值。
(5)本发明提供了一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的设计和构建方法，成功获得了一株兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5  sPD1CD137L，该病毒可以在肿瘤细胞内和肿瘤部位复制，并且能够高表达可溶性融合蛋白sPD1CD137L，该蛋白能够分泌到细胞外，在肿瘤局部发挥调控免疫检查点和免疫共刺激的生物学和免疫学功能。
(6)可溶性融合蛋白sPD1CD137L的N端是PD1的胞外区，可以特异性地结合PD-L1，从而封闭了PD-L1与效应淋巴细胞上的特异性受体PD1点结合，最终能够阻断PD1/PD-L1的负性调控通路；该融合蛋白的C端是CD137L的胞外区，能够特异性地结合其受体CD137，激活CD137的下游免疫活化通路，起到免疫活化作用，同时还能在肿瘤细胞和免疫细胞间起到桥联，增强抗肿瘤免疫作用。
(7)进一步的本发明的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的活化抗肿瘤免疫作用，能够刺激IFN-γ高表达。在肿瘤动物模型中，兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的抗肿瘤作用，可以用来制备抗肿瘤药物。
附图说明
图1为本发明的结果显示我们已经成功构建兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L，图中对照组对应位置没有条带、是空白的，而兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L对应的条带深黑，证明兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L能够在肿瘤细胞表达并且分泌到细胞外，而且从蛋白大小判断，是我们拟表达的目标蛋白：可溶性的SPD1CD137L。
图2为本发明的结果显示我们所构建的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L在肿瘤细胞内具有与对照病毒相同的复制和溶瘤能力。
图3为本发明的肿瘤体积测量数据的结果显示兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L在体内具有显著的抗肿瘤作用(B16/F10实体瘤模型)，能够抑制肿瘤的生长。
图4为本发明结果显示复制型溶瘤腺病毒AD5 sPD1CD137L在小鼠4T1乳腺癌皮下瘤模型中能够抑制肿瘤的生长，显著延长小鼠的生存时间。
图5为本发明结果显示复制型溶瘤腺病毒AD5 sPD1CD137L在小鼠Hepa1-6肝癌细胞皮下瘤模型中能够显著抑制肿瘤的生长，约有60％小鼠肿瘤完全治愈，小鼠达到长期存活。
图6为本发明的生存结果数据显示重组溶瘤腺病毒AD5 sPD1CD137L在体内具有抗肿瘤作用(H22肝癌腹水瘤模型)，能显著延长荷瘤小鼠的生存期，70％小鼠完全治愈，长期生存。
图7为本发明通过酶联免疫斑点法ELISpot和ELISA法检测分泌IFN-γ的细胞数和表达水平，数据结果显示兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L显著增强免疫活化而提高抗肿瘤作用(H22肝癌腹水瘤模型)，刺激IFN-γ的高表达，并维持T细胞的活性。
图8为本发明证实复制型溶瘤腺病毒AD5 sPD1CD137L促进抗肿瘤免疫应答依赖于CD8+T细胞，而不依赖于NK细胞。而且CD8+T是腹水中IFN-γ的主要来源。
图9为本发明融合蛋白sPD1CD137L小鼠Hepa1-6肝癌细胞皮下瘤模型中能够显著抑制肿瘤的生长。
图10为本发明复制型溶瘤腺病毒AD5 sPD1CD137L通过裂解肿瘤细胞，使肿瘤相关抗原被释放；同时招募免疫细胞，并提供维持免疫细胞活化的信号，最终产生有效抗肿瘤免疫应答。
具体实施方式
下面结合具体实施例对本发明进行进一步的解释和说明，但应理解，所给 出的实施例只作为举例说明，不以任何方式对本发明构成任何限制。
实施例1
新型复制型溶瘤腺病毒AD5 sPD1CD137L的构建、制备、抗肿瘤免疫活化评估和抗肿瘤作用评价
1实验材料和方法
1.1实验材料和仪器
1.1.1实验细胞系
人胚肾细胞株293T、人肝癌细胞株LM3、小鼠黑色素瘤细胞株B16/F10小鼠肝癌细胞株H22和Hepa1-6、小鼠乳腺癌细胞株4T1，使用含10％胎牛血清，100U/I青霉素和1mg/ml链霉素的高糖DMEM培养基培养于37℃、5％CO 2的培养箱中。
1.1.2实验仪器
生物安全柜(
Figure PCTCN2019115627-appb-000001
IIIadvance，Class II Biological Safety Cabinet，The Baker Company)，二氧化碳培养箱(FORMA SERIES II WATER JACKET CO 2incubator，Thermo)，低温离心机(HERAEUS MEGAFUGE 1.0R，Thermo)，垂直电泳槽(BIO-RAD)，电泳仪(BIO-RAD)，半干转-转膜仪(BIO-RAD)，免疫印迹曝光系统(Alpha Innotech)，PCR仪(PCR Thermal Cycler Dice，TaKaRa)，实时定量PCR仪及分析软件(ABI384，Sequence Detection Software，Version1.3.1)，酶标仪(VERSA max microplate reader)，整套移液器(eppendorf和RAININ)，细胞计数仪(Countstar Automated cell counter，Inno-Alliance Biotech Inc.，Wilmington，USA)，流式细胞仪(FACSCalibur，Becton，Dickinson and Company，USA)，FlowJo software(Version 7.6.5，Tree Star Inc，Ashland，Oregon)，微孔板振荡器(QiLinBeiEr)，核酸纯度浓度检测仪(Biophotometer plus，eppendorf)，数显恒温水浴锅(国华电器)。
1.1.3主要实验试剂及耗材
引物均由金斯瑞公司合成。肿瘤细胞培养所需的DMEM高糖培养基，双抗， 血清均购自Invitrogen(上海)公司。定量RT-PCR试剂Faststart Universal SYBR Green Master(Roche，04913914001)。Western Blot所需试剂耗材：蛋白酶抑制剂(Roche，11873580001)，细胞裂解液(碧云天：P0013)，PVDF膜(Roche，03010040001)，WB Immobilon ECL发光液(Millipore，WBKLS0500)，一抗稀释液(碧云天，P0023A)，HRP标记的二抗(Multisciences，GAR007and GAM007，1:5000稀释)，其余所需试剂均为国产分析纯，购自南京大学化学化工学院。台盼蓝(碧云天，C0011)，Opti-MEM购自Invitrogen(上海)公司。Western Blot抗体:anti-His(金斯瑞，MB001，1:5000稀释)。
1.1.4实验方法
AD5 sPD1CD137L病毒构建
可溶性蛋白sPD1CD137L的基因克隆以及携载sPD1CD137L基因的腺病毒穿梭质粒的构建
小鼠PD1和CD137L都属于膜蛋白，其结构依次是:N端信号肽-胞外区-跨膜区-胞内区C端。PD1与CD137L分别与PD-L1和CD137结合的功能单位是胞外区，sPD1CD137L仅将PD1和CD137L的胞外区进行融合表达，中间使用连接肽(Linker)GGGSGGGSGGGS连接，保留PD1的N端信号肽区域(见图1)；
可溶性蛋白sPD1CD137L的基因克隆：分别设计合成引物PD1-F、PD1-R、CD137L-F、CD137L-R，使用PD1-F和PD1-R引物。以小鼠脾脏cDNA为模板扩增得到片段EXO-PD1；使用CD137L-F和CD137L-R引物，以小鼠肝癌细胞Hep1-6的cDNA为模板扩增得到片段EXO-CD137L；体外合成Linker DNA；引物PD1-R与CD137L-F分别有16bp左右与linker序列5’和3’完全一致。使用PCR技术以PD1-F和CD137L-R为引物，将EXO-PD1、linker、EXO-CD137L片段按设计拼接，完成sPD1CD137L基因克隆。sPD1CD137L、EXO-PD1、linker、EXO-CD137L和信号肽的蛋白序列分别如序列表SEQ ID NO:1-SEQ ID NO:5所示；EXO-PD1、EXO-CD137L、linker、sPD1CD137L和信号肽的DNA序列分 别如序列表SEQ ID NO:6-SEQ ID NO:10所示。基因模板构建相关引物如表1所示：
表1
Figure PCTCN2019115627-appb-000002
携载可溶性蛋白基因的腺病毒穿梭质粒AD5-pShuttle-sPD1CD137L载体的构建：
使用Infusion技术将sPD1片段与AD5-pShuttle(pZD55)连接。具体步骤：首先使用限制性内切酶BglII对AD5-pShuttle(pZD55)线性化，纯化后片段按照sPD1-CD137L：AD5-pShuttle的2:1比例使用Infusion试剂盒(clontech lab.Inc.)进行连接，后经转化扩增验证获得携载sPD1-CD137L基因的腺病毒穿梭质粒AD5-pShuttle-sPD1-CD137L。
1.1.5 AD5 sPD1CD137L病毒构建(质粒构建、病毒拯救与扩增)
A.AD5 sPD1CD137L全长质粒构建：
将构建好的穿梭载体AD5-pShuttle-sPD1-CD137L用PmeI线性化后转入感受态pAdEasy-BJ5183中，使用含50ug/ml卡那霉素LB平板的进行筛选，挑取阳性克隆培养鉴定，鉴定正确的克隆质粒重新转化DH5a感受态进行二次筛选鉴定，鉴定正确后进行质粒大提获得AD5-sPD1-CD137L全长质粒。
B.AD5 sPD1CD137L病毒拯救：
AD5 sPD1CD137L全长质粒使用PacI线性化，纯化后6孔板中1ug/well转染293T细胞，5％CO2、37℃培养，2天后将细胞消化后转入10cm平皿，2-3天换液，至80％细胞出现病变，使用10ml培养基将细胞吹下收集至15ml离心管，反复冻融2次，3000rpm/min离心15min，收集病毒上清-80℃保存做为毒种。
C.病毒扩增：
取病毒种液50ul加入60％293T细胞10cm平皿中，5％CO2 37℃培养，细胞密度至90％以上，按照1传3比例传代，直至80％细胞出现病变，大约有10个平皿细胞，按上述方法收病毒，使用氯化铯密度梯度离心纯化病毒；使用TCID50方法进行滴度测定。
AD5 sPD1CD137L病毒功能评价
A.sPD1CD137L的表达和分泌功能：
AD5 sPD1CD137L病毒感染肿瘤细胞72小时后，收细胞和上清，使用western Blot检测sPD1CD137L的表达和分泌功能。
B.病毒复制能力：
AD5 sPD1CD137L和AD5 con病毒相同MOI感染肿瘤细胞，72小时后收细胞，反复冻融离心后得到等量病毒悬液，使用293T细胞进行病毒滴度测定；分析病毒复制能力变化。
C.溶瘤功能：
分别使用AD5 sPD1CD137L和AD5 con病毒按照MOI 1到100病毒量感染肿瘤细胞，48小时后使用MTT检测细胞活性，评价AD5 sPD1CD137L的杀瘤作用。
1.1.6体内研究AD5 sPD1CD137L抗肿瘤效应与机制
A.选用6-8周龄C57BL/6小鼠在右侧腋窝建立皮下瘤模型，每只小鼠一侧接种B16/F10细胞5×10 5个细胞，4-6天后测量肿瘤大小至200mm 3，将小鼠随机分成3组，分别是：无处理组、对照AD5病毒治疗组、AD5 sPD1CD137L病毒治疗组；a.按照分组使用相应病毒瘤内注射，每只注射病毒量5×10 8pfu，跟 踪测量肿瘤体积，体重，至肿瘤体积大于2500mm 3判定小鼠死亡，记录小鼠生存期。b.按照分组瘤内注射病毒，每只注射病毒量5×10 8pfu，注射两次，elispot检测免疫活化。
B.选用6-8周龄C57BL/6小鼠在腹腔建立腹水瘤模型，每只小鼠腹腔接种H22细胞1×10 7个细胞，第7-8天左右看到小鼠有腹水，将小鼠随机分成3组，分别是：无处理组、对照AD5病毒治疗组、AD5 sPD1CD137L病毒治疗组；a.按照分组使用相应病毒腹腔注射，每只注射病毒量5×10 8pfu，动态监测体重，直至小鼠死亡，记录小鼠生存期。b.按照分组腹腔注射病毒，每只注射病毒量5×10 8pfu，注射两次，ELISpot检测免疫活化。
1.1.7 AD5 sPD1CD137L病毒的滴度测定
1.293T细胞种于96孔板，每孔约1×103个细胞，待细胞贴壁后进行滴度测定。
2.病毒梯度的稀释：准备EP管，每个EP管加入1170μl含胎牛血清的DMEM；往第一个EP管中加入130μl病毒溶液，混匀，标记为10-1；从第一个EP管中吸取50μl于第二个EP管中，混匀，标记为10-2；依次类推，直至稀释到所需梯度为止。
3.每孔加入100μl相应梯度的病毒稀释液，每个梯度重复10个孔，37℃培养过夜。
4.5天后，将96孔板放于显微镜下观察GFP，记下每个梯度有GFP的孔数，用于病毒滴度的计算。
5.病毒滴度TCID50的计算公式：
Log10(TCID 50)＝L+d(s-0.5)+log10  (1/v)
L＝Log10 最高稀释度(如最高稀释度为10倍稀释，L＝1)
V＝最初每孔细胞培养液的体积(ml/well)
d＝Log10 稀释度(如为10倍稀释，d＝1)
s＝各个梯度GFP比率之和
1.2.3实时定量PCR
实时定量PCR的10μl体系组成：2.6μl PCR water，上下游引物各0.2μl，2μl的模板和5μl的SYBR Green荧光染料。样品混合后，于ABI 384PCR仪上进行扩增。
1.2.4细胞总蛋白的提取及浓度测定
1)以六孔板为例，去掉细胞培养上清，用PBS洗涤2遍，去掉PBS，每孔加入200μl的胰酶，消化吹打细胞，并将细胞收入至EP管中，1500rpm离心5min。
2)去掉上清，加入PBS重悬细胞，1500rpm离心5min。
3)去掉PBS，每孔根据细胞量加入相应的含蛋白酶抑制剂的细胞裂解液，涡旋30s，置于冰上10min，重复操作三次。4℃，12000g离心15min。收集上清于另一干净的EP管中。
4)蛋白浓度的测定：根据BCA蛋白浓度测定盒说明书进行检测。取2μl蛋白样品于96孔板中，加入18μl的PBS稀释样品，最后在加入200μl的测定工作液(工作液由试剂A:试剂B＝50:1)，放置于60℃的烘箱中，30min后，用酶标仪在562nm测定吸光度，根据标准曲线，计算出蛋白样品的浓度。
5)每管加入1/4蛋白裂解液体积的5×loading buffer，混匀后，100℃金属浴5min，冷却后，-20℃保存备用。
1.2.5 Western blot实验
1)配胶和电泳：按照不同要求配制不同浓度的SDS-PAGE分离胶和浓缩胶根据蛋白定量的计算结果，每个样品上样量调为30μg。电泳条件：浓缩胶80V30min，分离胶120V，约80min，前提是将条带分开且不会跑出去。
2)转膜：准备滤纸和PVDF膜，先用甲醇浸泡PVDF膜，再和滤纸一同浸泡在转膜缓冲液中备用。从玻璃板中小心将胶取下，浸泡在转膜缓冲液中，按照负极-滤纸-PVDF膜-胶-滤纸-正极的三明治顺序放置，赶走气泡，根据所需条带大小不同，恒流110mA转膜60-70min。
3)封闭：转膜结束后，立即取出PVDF膜，放入5％脱脂奶粉中室温封闭1h。
4)一抗孵育：4℃孵育一抗过夜。
5)二抗孵育：用washing buffer洗涤条带，每次10min，共三次；再用相应的HPR标记的二抗室温孵育1h。
6)曝光：用washing buffer洗涤条带，每次10min，共三次；用化学发光液在WB曝光仪上曝光，并获取条带图像。
1.2.6台盼兰计数
以六孔板为例，去除细胞上清，用PBS洗涤2遍，去掉PBS，每孔加入200μl胰酶消化，轻轻吹打细胞并收集进入干净的EP管中，1500rpm，离心5min。去掉上清，加入PBS重悬细胞，1500rpm，离心5min。去掉PBS，根据细胞数量加入一定量的PBS重悬细胞，从中取出10μl细胞重悬液，加入10μl 0.2％台盼蓝溶液混合，取混合液20μl于细胞计数板中，用细胞计数仪计数。
1.2.7流式细胞仪检测细胞表面分子
1)取实体瘤细胞(腹水)105 cells，加PBS清洗一遍。
2)去掉PBS，每管加入100μl含相应量的流式抗体的PBS，重悬细胞，置于冰上30min，避光。期间拿出样品，轻轻吹打，防止其因沉淀而影响抗体结合效果。
3)30min后，每管加入1ml PBS混合细胞，1500rpm，离心5min。去掉上清，再加入PBS重悬细胞，1500rpm，离心5min。去掉PBS，每管加入300μl PBS重悬，避光。
4)将准备好的样品，用流式细胞仪检测。用FlowJo软件分析实验结果。
1.2.8 Mouse IFN-γELISpot检测
1)ELISpot板子在使用前每孔加入200μl含10％血清的培养基孵育30min以上，放入细胞培养箱中。
2)去掉培养基，每孔加入200μl含细胞的培养体系。细胞体系组成：100μl的肿瘤细胞和100μl的脾脏细胞。混合均匀之后，加入孔板中，放入细胞培养箱中。且在实验结束取出之前，不要随意挪动板子。12h后，取出板子检测。
3)去掉培养基，每孔加入200μl的PBS清洗，需要清洗五遍以上。
4)去掉PBS，每孔加入100μl含一抗的稀释液。一抗稀释液：含0.5％FBS的PBS；一抗稀释比例1:1000。室温放置2h。
5)去掉一抗稀释液，用PBS洗五遍以上；每孔加入100μl二抗稀释液。二抗稀释液：含0.5％FBS的PBS；二抗稀释比例1:1000。室温放置1h。
6)去掉二抗稀释液，用PBS洗五遍以上。每孔加入200μl显色剂显色。待有蓝色斑点出现，且又不显色过头的情况下，甩掉显色液，用自来水洗多遍。
7)将自来水甩掉，室温晾干。注意：不要在室温晾的过久，且晾干过程保持避光。最后用避光保存于封口袋中。扫描读板。
1.2.9肿瘤组织及脾细胞的mouse IFN-γ的ELISpot检测
1)肿瘤组织单细胞悬液制备及mouse IFN-γ的ELISpot检测：处死小鼠，取下一小块的肿瘤组织，用PBS清洗，再放入培养皿中，加入1ml的胶原酶溶液，用剪刀剪碎，将肿瘤组织浑浊液吸入干净的离心管中，再加入1ml胶原酶溶液，放入37℃培养箱2小时，使得肿瘤组织得以消化完全。期间，每隔15min要取出，用枪头吹打混匀。2h后，取出装有肿瘤组织浑浊液的离心管，确认肿瘤组织消化完全。将浑浊液离心，取沉淀即肿瘤组织细胞。用含血清的DMEM重悬，细胞计数，将细胞浓度调为2×10 6个/ml，取100μl细胞混合液做鼠IFN-γ的ELISpot检测，方法同1.2.8。
2)脾单细胞悬液的制备及鼠IFN-γ的ELISpot检测：处死小鼠，取出脾脏，用PBS清洗，剪下一小块脾脏组织，放于70μl的细胞够滤网中，用5ml的注射器研磨，边研磨边加入适量的PBS冲洗。用Ficoll法去掉红细胞，离心重悬获得脾脏单细胞混合液，细胞计数，将细胞浓度调为2×10 6个/ml，取100μl与肿瘤细胞混合做鼠IFN-γ的ELISpot检测，方法同1.2.8。
2.实验结果与结论
图1为本发明的表达可溶性sPD1CD137L的重组溶瘤腺病毒的构建(A)重组溶瘤腺病毒AD5 con和AD5 sPD1CD137L的基因结构原理图。(B)B16/F10 小鼠黑色素瘤细胞分别感染AD5 con和AD5 sPD1CD137L，感染复数(MOI)为10，48h后，被感染细胞的上清被收集起来，通过western blot的方法检测融合蛋白sPD1CD137L的表达与分泌。数据代表三次独立性重复实验。GFP，绿色荧光蛋白；E1A，病毒早期区域1复制元件(early region 1)；sPD1CD137L，游离融合蛋白PD1CD137L。
图2为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的复制与溶瘤(A)B16/F10小鼠黑色素瘤细胞株、H22小鼠肝癌腹水瘤细胞株、Hepa1-6小鼠肝癌细胞株、LM3人肝癌细胞株分别感染AD5 con和AD5 sPD1CD137L，MOI＝10，分别在12，24，36，48，60和72h收取细胞，提取病毒基因组DNA，通过Q-PCR检测AD5的拷贝数。(B)B16/F10小鼠黑色素瘤细胞株、H22小鼠肝癌腹水瘤细胞株、Hepa1-6小鼠肝癌细胞株、LM3人肝癌细胞株分别感染AD5 con和AD5 sPD1CD137后，CCK8检测细胞活率。数据代表三次独立性重复实验.
图3为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的体内抗肿瘤作用(B16/F10黑色素瘤实体瘤模型)(A)在B16/F10皮下瘤模型中评估AD5 sPD1CD137L的抗肿瘤效果，实验方案图如图所示。(B)C57BL/6右侧皮下接种5×10 6B16/F10小鼠黑色素瘤细胞，瘤内注射5×10 8pfu AD5 con和AD5 sPD1CD137L，实时监测肿瘤大小。数据代表三次独立性重复试验。Pfu，空斑形成单位；Mock，生理盐水处理作为阴性对照；n.s.无统计学差异；**p<0.01。
图4为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的体内抗肿瘤作用(4T1乳腺癌实体瘤模型)(A)在乳腺癌4T1皮下瘤模型中评估AD5 sPD1CD137L的抗肿瘤效果，实验方案图如图所示。(B)Balb/c右侧皮下接种5×10 4 4T1小鼠乳腺癌细胞，瘤内注射5×10 8pfu AD5 con和AD5 sPD1CD137L，实时监测肿瘤大小。(C)以肿瘤体积大于2cm 3认为小鼠死亡，统计生存曲线。数据代表三次独立性重复试验。Pfu，空斑形成单位；Saline，生理盐水处理作为阴性对照；n.s.无统计学差异；**p<0.01。
图5为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的体内抗肿瘤作用 (Hepa1-6肝癌实体瘤模型)(A)在Hepa1-6皮下瘤模型中评估AD5 sPD1CD137L的抗肿瘤效果，实验方案图如图所示。(B)C57BL/6小鼠右侧皮下接种5×10 6Hepa1-6肝癌细胞，瘤内注射5×10 8pfu AD5 con和AD5 sPD1CD137L，肿瘤大小被实时监测。(C)以肿瘤体积大于2cm 3认为小鼠死亡，统计生存曲线。数据代表三次独立性重复试验。Pfu，空斑形成单位；Saline，生理盐水处理作为阴性对照；n.s.，无统计学差异；**，p<0.01。
图6为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的体内抗肿瘤作用(H22肝癌腹水瘤模型)(A)在H22肝癌腹水瘤模型中评估AD5 sPD1CD137L的抗肿瘤效果，实验方案图如图所示。(B)C57BL/6腹腔接种5×10 6H22小鼠肝癌腹水瘤细胞，小鼠出现腹水后，腹腔注射5×10 8pfu AD5 con和AD5 sPD1CD137L，实时监测小鼠生存时间。(C)被治愈的小鼠在90天后，腹腔再次接种5×10 6H22小鼠肝癌腹水瘤细胞，以未经任何处理的小鼠腹腔接种同样数量的H22细胞作为对照，监测小鼠生存情况。数据代表三次独立性重复实验。Pfu，空斑形成单位；Mock，生理盐水处理作为阴性对照；
Figure PCTCN2019115627-appb-000003
之前未接种过肿瘤的小鼠；n.s.无统计学差异；***p<0.001。
图7为本发明的重组溶瘤腺病毒AD5 sPD1CD137L增强免疫活化通过H22肝癌腹水瘤模型中评估AD5 sPD1CD137L的免疫活化作用，实验方案图如图Fig.6A所示。(A)C57BL/6腹腔接种5×10 6H22小鼠肝癌腹水瘤细胞，小鼠出现腹水后经腹腔注射5×10 8pfu AD5 con和AD5 sPD1CD137L，第14天用ELISA方法检测腹水中游离sPD1CD137L水平。(B)ELISpot检测活化的免疫细胞水平。(C)ELISA检测腹水中的IFN-γ水平。数据代表三次独立性重复实验。Mock，生理盐水处理作为阴性对照；*p<0.05。
图8为本发明的重组溶瘤腺病毒AD5 sPD1CD137L清除H22肝癌细胞依赖于CD8+T细胞，而不依赖于NK细胞。(A)通过H22肝癌腹水瘤模型研究AD5 sPD1CD137L诱导的抗肿瘤免疫应答的机制，实验方案图如图所示。(B)C57BL/6腹腔接种5×10 6H22小鼠肝癌腹水瘤细胞，在第10和18天分别注射anti-CD8a 或Anti-NK1.1抗体。注射抗体5天后流式检测小鼠外周血中淋巴细胞亚群清除情况。(C)小鼠出现腹水后，经腹腔注射上述中和抗体清除CD8+T细胞或NK细胞后，再腹腔注射5×10 8pfu AD5 sPD1CD137L，监测小鼠生存率。(D)抗体封闭后检测腹水中IFN-γ的水平。数据代表三次独立性重复实验。Pfu，空斑形成单位；Saline，生理盐水处理组作为阴性对照；#无统计学差异；*p<0.05；***p<0.001。
图9为本发明的融合蛋白sPD1CD137L的体内抗肿瘤作用(Hepa1-6肝癌实体瘤模型)(A)在肝癌Hepa1-6皮下瘤模型中评估融合蛋白sPD1CD137L的抗肿瘤效果，实验方案图如图所示。(B)C57BL/6小鼠右侧皮下接种5×10 6Hepa1-6肿瘤细胞，待肿瘤出现后，腹腔注射300μl含融合蛋白sPD1CD137L的上清液，实时监测肿瘤大小。n.s.无统计学差异；**p<0.01。
图10为本发明的重组溶瘤腺病毒AD5 sPD1CD137L的工作原理。重组溶瘤腺病毒AD5 sPD1CD137L感染肿瘤细胞，一方面促进肿瘤细胞的裂解，引起肿瘤细胞免疫原性细胞死亡，活化并招募免疫细胞；另一方面感染的肿瘤细胞分泌融合蛋白sPD1CD137L，进入肿瘤微环境。该蛋白sPD1CD137L：1)PD1端能够特异性结合肿瘤细胞及其它表达PD-L1免疫抑制细胞的PD-L1分子，阻止抗肿瘤效应T细胞因PD-L1/PD1信号导致细胞失能耗竭；2)CD137L端能够特异性结合T细胞表面共刺激分子受体CD137，诱导持续的T细胞活化，并招募更多淋巴细胞浸润到肿瘤局部，产生更有效抗肿瘤免疫应答；3)因肿瘤微环境免疫活化而上调的PD-L1，使sPD1CD137L能够被捕获在肿瘤组织局部，不易扩散到血液中。因而降低了脱靶效应；4)融合蛋白是肿瘤细胞和效应T细胞双亲分子，因此可以作为桥梁，促进肿瘤细胞与T细胞的接触，增加T细胞对肿瘤细胞的杀伤。
PD1，细胞死亡受体；PD-L1，细胞死亡受体配体；Adenovirus，腺病毒；Ad5-PC，表达PD1CD137L融合蛋白的腺病毒；Perforin，穿孔素；FAS，自杀相关因子；FASL，FAS配体。
由以上结果可知，本发明提供了一种可以兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的设计和构建方法，成功获得了一株新型兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L，该病毒可以选择性地在肿瘤细胞内和肿瘤部位复制、具有肿瘤靶向性，能够有效溶瘤，并诱导免疫原性细胞死亡。与此同时，该病毒能够高表达可溶性融合蛋白sPD1CD137L，该蛋白能够分泌到细胞外，在肿瘤微环境中发挥阻断免疫检查点、激活免疫共刺激信号通路，进而活化免疫的生物学功能。本发明的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L具有显著的活化抗肿瘤免疫作用，能够显著刺激IFN-γ在肿瘤局部高表达且没有明显的全身毒性，显著抑制肿瘤生长、延长生存期，具有显著的抗肿瘤作用。一个病毒，同时整合多种独特的抗肿瘤机制于一身，具有预料不到的抗肿瘤效果。可以用来制备抗肿瘤药物。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，本发明要求保护范围由所附的权利要求书、说明书及其等效物界定。

Claims (14)

  1. 一种兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白，其特征在于：所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白的两端分别为结合PD-L1的PD1和结合CD137的CD137L，PD1和CD137L之间通过linker序列连接。
  2. 如权利要求1所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白，其特征在于：所述的可溶性融合蛋白为sPD1CD137L，sPD1CD137L的蛋白序列和氨基酸序列分别如序列表SEQ ID NO:1和SEQ ID NO:6所示。
  3. 权利要求1或2所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备活化抗肿瘤免疫药物中的应用。
  4. 权利要求1或2所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备刺激IFN-γ表达药物中的应用。
  5. 权利要求1或2所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备抗肿瘤药物中的应用。
  6. 如权利要求5所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的可溶性融合蛋白在制备抗肿瘤药物中的应用，其特征在于：所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
  7. 一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒，其特征在于：所述的复制型溶瘤腺病毒在肿瘤细胞内复制，并且表达和分泌可溶性融合蛋白，所述的可溶性融合蛋白的两端分别为结合PD-L1的PD1和结合CD137的CD137L，PD1和CD137L之间通过linker序列连接。
  8. 如权利要求7所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒，其特征在于：所述的可溶性融合蛋白为sPD1CD137L，sPD1CD137L的蛋白序列的氨基酸序列如序列表SEQ ID NO:1所示。
  9. 权利要求7或8所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒，其特征在于：所述的复制型溶瘤腺病毒能够溶瘤。
  10. 权利要求7或8所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备活化抗肿瘤免疫药物中的应用。
  11. 权利要求7或8所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备刺激IFN-γ表达药物中的应用。
  12. 权利要求7或8所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用。
  13. 如权利要求12所述的兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒在制备抗肿瘤药物中的应用，其特征在于：所述的肿瘤为肝癌、腹水癌、黑色素瘤或乳腺癌。
  14. 一种兼具激活免疫共刺激信号通路和阻断免疫检查点的复制型溶瘤腺病毒AD5 sPD1CD137L的构建方法，其特征在于
    包括如下步骤：(1)AD5 sPD1CD137L全长质粒构建：将构建好的穿梭载体AD5-pShuttle-sPD1-CD137L用PmeI线性化后转入感受态pAdEasy-BJ5183中，使用含50ug/ml卡那霉素LB平板的进行筛选，挑取阳性克隆培养鉴定，鉴定正确的克隆质粒重新转化DH5a感受态进行二次筛选鉴定，鉴定正确后进行质粒大提获得AD5-sPD1-CD137L全长质粒；
    (2)AD5 sPD1CD137L病毒拯救：AD5 sPD1CD137L全长质粒使用PacI线性化，纯化后6孔板中1ug/well转染293T细胞，5％CO2、37℃培养，2天后将细胞消化后转入10cm平皿，2-3天换液，至80％细胞出现病变，使用10ml培养基将细胞吹下收集至15ml离心管，反复冻融2次，3000rpm/min离心15min，收集病毒上清-80℃保存做为毒种；
    (3)病毒扩增：取病毒种液50ul加入60％293T细胞10cm平皿中，5％CO 237℃培养，细胞密度至90％以上，按照1传3比例传代，直至80％细胞出现病变，大约有10个平皿细胞，按上述方法收病毒，使用氯化铯密度梯度离心纯化 病毒；使用TCID50方法进行滴度测定。
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CN113832111A (zh) * 2020-06-23 2021-12-24 南京大学 一种类外泌体技术用于制备新型溶瘤病毒的方法
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CN112941039A (zh) * 2021-02-01 2021-06-11 南京大学 一种新型类囊泡溶瘤病毒及其在制备抗肿瘤药物上的应用
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017118866A1 (en) * 2016-01-08 2017-07-13 Replimune Limited Engineered virus
CN108165536A (zh) * 2017-12-11 2018-06-15 浙江大学 一种重组溶瘤痘苗病毒及其制备方法与应用
CN108728488A (zh) * 2017-04-19 2018-11-02 生命序有限公司 溶瘤病毒构建体、溶瘤病毒及其应用
CN109069561A (zh) * 2016-01-11 2018-12-21 图恩斯通有限合伙公司 溶瘤病毒和检查点抑制剂组合疗法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102174522B (zh) * 2011-02-24 2013-11-06 杭州师范大学 一种4-1bbl蛋白的制备方法
EP2964241A1 (en) * 2013-03-05 2016-01-13 Baylor College Of Medicine Oncolytic virus
FI3909972T3 (fi) * 2015-06-19 2024-05-03 Sebastian Kobold Pd1-cd28-fuusioproteiineja ja niiden käyttö lääkkeessä
SG10201913576RA (en) * 2015-10-01 2020-02-27 Heat Biologics Inc Compositions and methods for adjoining type i and type ii extracellular domains as heterologous chimeric proteins
DK3565579T3 (da) * 2017-01-05 2023-09-04 Kahr Medical Ltd Pd1-41bbl-fusionsprotein og fremgangsmåder til anvendelse deraf

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017118866A1 (en) * 2016-01-08 2017-07-13 Replimune Limited Engineered virus
CN109312309A (zh) * 2016-01-08 2019-02-05 雷普利穆内有限公司 工程化的病毒
CN109069561A (zh) * 2016-01-11 2018-12-21 图恩斯通有限合伙公司 溶瘤病毒和检查点抑制剂组合疗法
CN108728488A (zh) * 2017-04-19 2018-11-02 生命序有限公司 溶瘤病毒构建体、溶瘤病毒及其应用
CN108165536A (zh) * 2017-12-11 2018-06-15 浙江大学 一种重组溶瘤痘苗病毒及其制备方法与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, YONGHUI ET AL.: "Recombinant Adenovirus Expressing a Soluble Fusion Protein PD-1/ CD 137L Subverts the Suppression of CD 8+ T Cells in HCC", MOLECULAR THERAPY, vol. 27, no. 11, 5 August 2019 (2019-08-05) *

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