WO2023165573A1 - 用于激活整体抗肿瘤免疫系统的培养基配方物及制备激动剂激活的整体免疫效应细胞的方法 - Google Patents

用于激活整体抗肿瘤免疫系统的培养基配方物及制备激动剂激活的整体免疫效应细胞的方法 Download PDF

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WO2023165573A1
WO2023165573A1 PCT/CN2023/079384 CN2023079384W WO2023165573A1 WO 2023165573 A1 WO2023165573 A1 WO 2023165573A1 CN 2023079384 W CN2023079384 W CN 2023079384W WO 2023165573 A1 WO2023165573 A1 WO 2023165573A1
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cells
agonist
activated
immune effector
wast
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French (fr)
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苏盛
黄劲松
于和鸣
宋亚丽
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北京市希波生物医学技术有限责任公司
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    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • 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
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes

Definitions

  • the invention relates to tumor immunotherapy and a method for regulating body immunity. Specifically, the present invention relates to an immune cell preparation that can be used to treat tumors or regulate body immunity.
  • adoptive immune cell therapy includes chimeric antigen receptor T cell (CAR-T) therapy and neoantigen tumor Infiltrating lymphocyte (TIL) and cytotoxic T lymphocyte (CTL) therapy; immune checkpoint inhibitor therapy includes PD1/PDL1 inhibitor therapy, CTLA-4 inhibitor therapy, etc.
  • CAR-T chimeric antigen receptor T cell
  • TIL tumor Infiltrating lymphocyte
  • CTL cytotoxic T lymphocyte
  • immune checkpoint inhibitor therapy includes PD1/PDL1 inhibitor therapy, CTLA-4 inhibitor therapy, etc.
  • CRS cytokine release syndrome
  • Neurotoxicity can cause clinical symptoms such as confusion, aphasia, and cerebral edema.
  • Off-target effects can accidentally injure normal tissue cells expressing the same target antigen, which in turn can cause normal tissue damage or immunodeficiency diseases, and can even cause death in severe cases.
  • Another shortcoming of CAR-T therapy is that the therapeutic effect needs to be improved.
  • the third disadvantage is that CAR-T therapy has poor or no therapeutic effect on solid tumors, because it is difficult for CAR-T to enter solid tumor lesions and infiltrate inside the tumor; even if it can infiltrate into solid tumors, it will Faced with the suppression of the tumor microenvironment, T cells cannot exert anti-tumor effects.
  • the fourth shortcoming is that the currently approved CAR-T products all use their own T cells as the starting material, which is difficult to industrialize, and the product price is as high as hundreds of thousands of dollars, which is not conducive to market promotion.
  • Neoantigens are polypeptides obtained through transcription, translation and processing of non-synonymous mutations in tumor cells. Since tumor antigens are not expressed in normal cells, neoantigen-specific immune responses do not undergo central and peripheral tolerance mechanisms, so theoretically, neoantigens have application value as therapeutic targets for immunotherapy. With the development of high-throughput gene sequencing technology, whole genome and exome sequencing technology can help researchers obtain mutation information on the genome (including point mutations, insertion mutations, etc.). How to quickly and accurately identify candidate neoantigens from these data and screen out highly immunogenic neoantigens is an urgent problem in the field of tumor immunotherapy.
  • TMB tumor gene burden
  • the present invention proposes that based on the pattern recognition receptor (PRR) theory targeting innate immunity, through the joint application of multiple new agonists and immunostimulators, innate immunity is activated, and then adaptive immunity is activated, resulting in the overall immune system cell activation, so as to achieve the purpose of killing and eliminating tumors.
  • PRR pattern recognition receptor
  • the present invention provides an agonist-activated global immune effector cell.
  • the present invention provides a culture medium formulation for preparing agonist-activated overall immune effector cells in the first aspect of the present invention.
  • the present invention provides a method for preparing the agonist-activated overall immune effector cells of the first aspect of the present invention.
  • the present invention provides an agonist-activated overall immune effector cell preparation.
  • the present invention provides a method for treating tumors, comprising administering the agonist-activated overall immune effector cell preparation of the fourth aspect of the present invention to a subject in need of treatment.
  • the present invention provides a method for regulating the body's immunity, comprising administering the agonist-activated overall immune effector cell preparation of the fourth aspect of the present invention to a subject in need.
  • WAST used in this article is the English abbreviation of "Whole Agonist Stimulation”, which means the whole immune response activated by agonist.
  • WAST cells or “WAST effector cells”, it is meant the overall immune effector cells activated by the agonists of the present invention; when referring to “WAST precursor cells”, it is meant to refer to the agonistic Agent-activated overall immune precursor cells.
  • the term “the proportion of cells” refers to the proportion of the type of cells in the total lymphocyte population.
  • the term “the ratio of V ⁇ 9 ⁇ 2 cells” refers to the ratio of V ⁇ 9 ⁇ 2 cells to the CD4-CD8Low- cell population in the total lymphocytes.
  • the above-mentioned ratios refer to the ratios detected by those skilled in the art using conventional methods for detecting peripheral blood lymphocyte subpopulations, for example, referring to detection by conventional flow cytometry methods for detecting peripheral blood lymphocyte subpopulations The ratio obtained.
  • the proportion of total immune effector cells activated by the agonist is greater than 70% total T lymphocytes, 45%-80% killer T cells, and 7.5%-25% NKT cells , the ratio of V ⁇ 9 ⁇ 2 cells is 3%-50%.
  • the proportion of total T lymphocytes is greater than 90%, the proportion of killer T cells is 45%-70%, and the proportion of V ⁇ 9 ⁇ 2 cells is 5%-50% .
  • the proportion of agonist-activated overall immune effector cells secreting granzyme B is 15%-55%
  • the proportion of perforin-secreting cells is 15%-50%
  • secreting granzyme The ratio of B and perforin cells is 10%-45%.
  • the overall immune effector cells activated by the agonist are obtained by applying the agonist to the peripheral blood of the donor, wherein the agonist comprises a TLR3 agonist, a TLR9 agonist and a stimulator of ⁇ T cells .
  • the TLR3 agonist is PolyI:C.
  • the TLR9 agonist is CpG ODN.
  • the stimulator of ⁇ T cells is a phosphine antigen.
  • the TLR3 agonist is PolyI:C
  • the TLR9 agonist is CpG ODN
  • the stimulator of ⁇ T cells is a phosphine antigen.
  • a culture medium formulation for preparing agonist-activated global immune effector cells comprises a TLR3 agonist, a TLR9 agonist, and a stimulator of ⁇ T cells.
  • the TLR3 agonist is PolyI:C.
  • the TLR9 agonist is CpG ODN.
  • the stimulator of ⁇ T cells is a phosphine antigen.
  • a medium formulation for preparing agonist-activated whole body immune effector cells comprises PolyI:C, CpG ODN, and a phosphine antigen.
  • the medium formulation further comprises IL-2, IL-15, anti-CD3 antibody, anti-CD28 antibody.
  • the culture medium formulation for preparing agonist-activated global immune effector cells comprises IL-2 at a concentration of 100-500 IU/mL, IL-15 at 5-50 ng/mL, IL-15 at a concentration of 0.5-5 ⁇ g/mL Anti-CD3 antibody, 0.5-5 ⁇ g/mL anti-CD28 antibody, 2-20 ⁇ g/mL PolyI:C, 2-20 ⁇ g/mL CpG ODN, 10-100 ⁇ mol/L zoledronic acid.
  • the culture medium formulation for preparing agonist-activated global immune effector cells comprises IL-2 at a concentration of 300-500 IU/mL, IL-15 at 10-50 ng/mL, IL-15 at 1-3 ⁇ g/mL Anti-CD3 antibody, 1-3 ⁇ g/mL anti-CD28 antibody, 5-15 ⁇ g/mL PolyI:C, 5-15 ⁇ g/mL CpG ODN, 10-75 ⁇ mol/L zoledronic acid.
  • the culture medium formulation for preparing agonist-activated global immune effector cells comprises IL-2 at a concentration of 300 IU/mL, IL-15 at 15 ng/mL, anti-CD3 antibody at 1 ⁇ g/mL, 1 ⁇ g The anti-CD28 antibody of /mL, the PolyI:C of 10 ⁇ g/mL, the CpG ODN of 10 ⁇ g/mL, the zoledronic acid of 50 ⁇ mol/L.
  • the method of making an agonist-activated global immune effector cell comprises:
  • step (3) continue to induce the immature dendritic cells obtained in step (2) to obtain mature dendritic cells;
  • step (1) (4) culturing the non-adherent suspension cells obtained in step (1) in a medium formulation to obtain agonist-activated whole immune precursor cells;
  • the cytokines in step (2) are granulocyte-macrophage colony-stimulating factor and interleukin-4.
  • interleukin-2, interleukin-33 and tumor necrosis factor- ⁇ are used for induction culture in step (3).
  • the donor is a human.
  • an agonist-activated global immune effector cell preparation comprises an agonist-activated global immune effector cell and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is selected from physiological saline, cell cryopreservation fluid or cell cryoprotectant.
  • the agonist-activated global immune effector cell preparation is an injection.
  • methods of treating tumors comprise administering an agonist-activated preparation of global immune effector cells to a subject in need thereof.
  • the subject in need of treatment is a human.
  • the tumor is a solid tumor.
  • the solid tumor is selected from lung cancer, liver cancer, gastric cancer, breast cancer, or colon cancer.
  • the cell preparation is administered to a subject in need of treatment by intravenous injection, intratumoral injection, and/or peritumoral injection.
  • the cell preparation is administered in combination with other therapeutic agents or treatment means selected from one of chemotherapy, targeted therapy, cytokines, surgery, radiotherapy, immunotherapy, and traditional Chinese medicine treatment. one or more species.
  • the method of modulating immunity in a body comprises administering to a subject in need thereof an agonist-activated preparation of global immune effector cells.
  • the subject in need thereof is selected from an immunocompromised or immunologically imbalanced population.
  • the WAST cell preparation is composed of immune cells with a variety of different killing mechanisms, which can be used alone or in combination with existing tumor treatment methods and drugs in clinical practice. Combined application will produce higher effectiveness;
  • the various agonists and immunostimulants used have good safety and effectiveness, the main starting material used is the patient's own PBMC or cryopreserved PBMC, the cell culture and expansion reagents used are in compliance with GMP regulations, so WAST cell preparations have good safety;
  • WAST cells are activated immune cells, which can avoid the defects of using various T cells in the prior art and adverse reactions;
  • WAST cells are broad-spectrum tumor-killing preparations, which can be widely used in the treatment of various solid tumors;
  • WAST cell preparations do not involve complex preparation processes such as genetic modification and gene recombination, and the preparation cycle is short.
  • WAST cell preparations can be used in combination with chemotherapy, targeted therapy, cytokines, surgery, radiotherapy, immunotherapy and traditional Chinese medicine to increase its curative effect through synergistic effects; (7) TNM stage I or II cases treated with WAST cell preparations after early surgery can reduce the recurrence rate.
  • the invention also has the beneficial effect of improving the body's overall immune response (innate immunity+adaptive immunity).
  • the application of the WAST cells of the present invention can regulate the immunity of the body, especially for the population with existing immune imbalance in the body, it can regulate the immune imbalance and realize the immune balance.
  • Figure 1 outlines the preparation method of the WAST cell preparation of the present invention and its anti-tumor mechanism.
  • Figure 2 shows the proportion of cells secreting granzyme B and perforin before and after induction, where l represents cells secreting granzyme B, m represents cells secreting perforin, n represents cells secreting granzyme B and perforin at the same time; error bars Refers to the 95% confidence interval (CI) of the measured values of various cells.
  • Figure 3 shows the measured values of the percentages of various types of cells before and after induction with agonists and immunostimulants.
  • a percentage of total T lymphocytes
  • b percentage of helper T cells
  • c percentage of killer T cells
  • e percentage of total B lymphocytes
  • f percentage of NK cells
  • g percentage of NKT cells
  • h percentage of CD25+ cells
  • i percentage of monocytes
  • j percentage of HLA-DR+ cells
  • k percentage of V ⁇ 9 ⁇ 2 (CD4-CD8low-); error bars refer to the 95% confidence interval (CI) of the measured values of various cells.
  • CI 95% confidence interval
  • Agonists and immune induction reagents used in the examples of the present invention (1) AIM-V cell culture medium, purchased from ThermoFisher Company; (2) Human lymphocyte separation tube (ready-to-use), purchased from Dakowi Company; (3) IL-4, GM-CSF, TNF- ⁇ , IL-2, IL-15, IL-33, anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody were all purchased from Peprotech Company; (4) PolyI:C, CpG ODN (5'-tcgtcgttttcgcg-3') was purchased from InvivoGen; (5) Zoledronic acid was purchased from Sinopharm.
  • Example 1 The PBMCs obtained in Example 1 were resuspended in AIM-V medium, added to a 75 cm 2 cell culture flask, and cultured at 37° C., 5% CO 2 .
  • AIM-V medium containing 500 IU/ml IL-4 and 500 IU/ml GM-CSF was added to the culture flask.
  • AIM-V cell culture medium was added with a final concentration of 10 ng/ml TNF- ⁇ , 300 IU/ml IL-2, and 10 ng/ml IL-33.
  • Example 2 (1) Obtain suspension cells through step (2) of Example 2, resuspend them in AIM-V medium, adjust the cell concentration to 1 ⁇ 10 6 cells/ml, and this suspension is rich in T lymphocytes.
  • this suspension add the IL-2 of final concentration 300IU/ml, the IL-15 of 15ng/ml, the anti-CD3 monoclonal antibody of 1 ⁇ g/ml, the anti-CD28 monoclonal antibody of 1 ⁇ g/ml, the PolyI:C of 10 ⁇ g/ml, 10 ⁇ g/ml of CpG ODN, 50 ⁇ mol/L of zoledronic acid, cultured at 37°C, 5% CO 2 for 8 days, half of the medium was changed every 3 days to obtain WAST precursor cells;
  • WAST effector cells According to the preparation method of WAST effector cells described in Examples 1-3, fresh WAST effector cells from 6 healthy people were obtained. Carry out cell phenotype analysis with flow cytometer, measure before and after induction using the culture medium formulation of the present invention (before induction: PBMC, represented by 1 to 6 respectively; After induction: WAST effector cells, represented by 1' to 6' respectively ) changes in the percentages of various types of cells, the results are shown in the table below.
  • Fig. 3 the percentages of cells related to tumor killing among the WAST effector cells obtained after induction were all significantly increased.
  • the percentage of total T lymphocytes it was 57.83% before induction and 98.78% after induction; for the percentage of killer T cells, it was 13.97% before induction and 45.35% after induction; for the percentage of NKT cells, after induction It was 2.54% before induction and 24.63% after induction; for the percentage of V ⁇ 9 ⁇ 2 (CD4-CD8Low-) cells, it was 3.42% before induction and 12.18% after induction.
  • These cells are the main killer cells among WAST effector cells, and their ratios are significantly increased after induction.
  • the ability of the fresh WAST effector cells prepared in Example 4 to secrete granzyme B and perforin was detected by flow cytometry.
  • the table below shows the changes in the secretory ability of cells before and after induction (before induction: PBMC, respectively indicated by 1 to 6; after induction: WAST effector cells, respectively indicated by 1' to 6').
  • Example 6 Determination of in vitro direct killing ability of WAST effector cells on 5 different tumor cell lines
  • WAST effector cells were obtained according to the preparation methods in Examples 2-3, which were respectively named WAST -121061203C, WAST-121030702C, WAST-121050801C.
  • WAST -121061203C human non-small cell lung cancer
  • MCF-7 human breast cancer cells
  • HepG2 human liver cancer cells
  • HGC-27 human gastric cancer cells
  • HCT116 human colon cancer cells
  • CCK8 Cell Counting Kit-8
  • control groups respectively: blank control group, only add 200 ⁇ l AIM-V medium to each well; target cell control group, without adding effector cells, add 100 ⁇ l AIM-V medium to each well, and then add 100 ⁇ l target cells ; effector cell control group, no target cell For cells, add 100 ⁇ l AIM-V medium to each well, and then add 100 ⁇ l effector cells; set up three replicate wells for each group, and culture them at 37°C, 5% CO2 for 24 hours;
  • target cells ratio of immediate effect target
  • Killing rate (%) [1-(A experimental group-A effector cell control group)/(A target cell control group-A blank control group)] ⁇ 100%.
  • A549 human non-small cell lung cancer
  • MCF-7 human breast cancer cells
  • HepG2 human liver cancer cells
  • HGC-27 human gastric cancer cells
  • HCT116 human colon cancer cells
  • the killing rates of WAST-121061203C were 41.94% and 53.18% when the effect-to-target ratio was 20:1 and 30:1, respectively;
  • the killing rate of WAST-121030702C was 36.37% and 47.36% when the effect-target ratio was 20:1 and 30:1; the killing rate of WAST-121050801C was 80.37% and 96.33% when the effect-target ratio was 20:1 and 30:1.
  • the killing rates of WAST-121061203C were 9.17% and 17.20% when the effect-target ratio was 20:1 and 30:1; the killing rates of WAST-121030702C were 17.83% and 32.67% when the effect-target ratio was 20:1 and 30:1;
  • the effective target ratios of WAST-121050801C were 11.94% and 20.82% for 20:1 and 30:1.
  • the killing rates of WAST-121061203C were 17.81% and 28.90% when the effect-to-target ratio was 20:1 and 30:1, respectively;
  • the killing rate of WAST-121030702C was 19.33% and 31.98% when the effect-to-target ratio was 20:1 and 30:1; the killing rate of WAST-121050801C was 25.51% and 44.24% when the effect-target ratio was 20:1 and 30:1, respectively.
  • the killing rates of WAST-121061203C were 81.45% and 92.63% when the effect-to-target ratio was 20:1 and 30:1, respectively;
  • the killing rate of WAST-121030702C was 40.39% and 47.34% at the target ratio of 20:1 and 30:1 respectively; the killing rate of WAST-121050801C was 76.19% and 87.62% at the target ratio of 20:1 and 30:1 respectively.
  • the killing rates of WAST-121061203C were 54.49% and 47.41% when the effect-to-target ratio was 20:1 and 30:1, respectively;
  • the killing rates of WAST-121030702C were 69.83% and 53.31% when the effect-to-target ratio was 20:1 and 30:1, respectively.
  • Example 7 Determination of in vitro indirect killing ability of WAST effector cells on lung cancer A549 cell line
  • the WAST effector cells WAST-121061203C, WAST-121030702C and WAST-121050801C prepared in Example 6 were used as effector cells, and the A549 (human non-small cell lung cancer) cell line was used as the target cell to carry out cell double chamber Culture killing assay.
  • Blank group medium only (blank control group)
  • the lower chamber A549, the upper chamber contains WAST-121061203C 300000 pieces/well
  • the lower chamber is A549, and the upper chamber contains simple medium (target cell control group)
  • Killing rate (%) [1-(A experimental group-A blank control group)/(A target cell control group-A blank control group)] ⁇ 100%.
  • WAST effector cells and A549 cells can inhibit the proliferation of A549 cells after double-chamber culture, and the inhibition rate will increase with the increase of the number of WAST effector cells. This proves that WAST effector cells have an indirect killing effect on tumor cells.
  • Example 8 Drug efficacy experiment of WAST cells killing solid tumors in mice (taking non-small cell lung cancer tumor-bearing model as an example)
  • the CDX lung cancer A549 tumor-bearing mouse model—NPI immunodeficiency mice (this experiment was entrusted to Beijing Admore Biotechnology Co., Ltd.) was used, with an age of 6-8 weeks and a body weight of 20-30 g.
  • the human WAST cells were injected into the tail vein and intratumoral peritumor in groups according to the time points, and the tumor growth was observed and the tumor size was measured.
  • the injection frequency of WAST effector cells was once a week for 3 weeks.
  • the first injection was regarded as day 0, and WAST was performed on day 0 (D0), day 7 (D7), and day 14 (D14).
  • the effector cell injection was carried out 3 times in total; the experiment was divided into 3 groups, namely: normal saline group, intratumoral peritumoral injection group (I.T.) (this group injected WAST-121030702C cells), intravenous injection group (I.V.) (this group Inject WAST-121030702C cells), 3 CDX tumor-bearing mice in each group;
  • the tumor-bearing volume of the mice was measured on the 25th day.
  • the results of tumor volume measurement in the three groups of mice are as follows:
  • the tumor-bearing volume of the mice was measured on the 25th day, and the average tumor volume of the mice in the intratumoral peritumoral injection group and the intravenous injection group was smaller than that in the normal saline group.
  • the immune cell phenotype flow cytometry was performed on the peripheral blood of the mice by flow cytometry, and the results of the CD8+CD69+ flow cytometry detection on the peripheral blood of the mice were as follows:
  • WAST effector cells were activated in both the intravenous injection group and the intratumoral peritumoral injection group. After three injections of WAST effector cells, it can be concluded from the analysis of the above results that no matter intratumoral peritumoral injection or intravenous injection of WAST Effector cells can play a role in tumor suppression.
  • Example 9 Drug efficacy experiment of WAST cells killing solid tumors in humanized mice (taking non-small cell lung cancer tumor-bearing model as an example)
  • the CDX lung cancer A549 tumor-bearing mouse model Human-NPI immunodeficient mice after administration of gemcitabine (this experiment was entrusted to Beijing Admo Biotechnology Co., Ltd. to implement), the age of the week was 37 weeks, and the body weight was 19 -24g.
  • the tumors in the mice grew to 70-120mm3
  • the groups were injected with human WAST cells through the intratumoral peritumor once, and the tumor growth was observed and the tumor size was measured.
  • WAST effector cells were obtained according to the preparation method of Example 2-3, named WAST-22010517H. Before injection, the cryopreserved WAST effector cells WAST-22010517H were recovered;
  • mice tumor infiltrating lymphocytes TIL
  • the proportion of CD8+ cells to all CD3+ cells was detected by flow cytometry, and the results are as follows:
  • the results of flow cytometric detection of tumor-infiltrating lymphocytes and immune cells in mice showed that the tumor TIL infiltration (total CD45+) in the peritumoral injection group was significantly higher than that in the saline group, and among the infiltrating CD3+ T cells, CD8+ was positive The ratio was significantly higher than that of the first group, indicating that WAST cells proliferated and persisted significantly in vivo, and at the same time, they were mainly killer cells in tumor infiltration.
  • WAST effector cells are activated in the intratumoral peritumoral injection group of mice. After a single injection of WAST effector cells, WAST effector cells can continue to proliferate in vivo after intratumoral peritumoral injection, and can inhibit tumors.
  • WAST cell preparations are unmodified immune cells, clinically used to treat solid tumors, and the route of administration is intravenous infusion.
  • WAST cell preparations were given intravenously to B-NDG mice and observed for 14 days to observe the nature, degree and reversibility of the possible toxic reactions caused by WAST cell preparations, and speculate on the target organs and tissues of toxicity, which is clinically safe. Provide reference information.
  • the undertaking unit of this experiment is: Guoke Saifu Hebei Pharmaceutical Co., Ltd. (GLP).
  • mice purchased from Biocytogen Jiangsu Gene Biotechnology Co., Ltd.
  • the weight of female mice is 18.84g-22.01g
  • the weight of male mice is 26.14g-28.89g
  • the weight of individual mice of the same sex is within the range of average body weight ⁇ 20%.
  • the mice were about 7 weeks old when the groups were administered.
  • the WAST effector cells were WAST-121030702C mentioned above.
  • the volume is 20ml/kg body weight, and the dosage is 2 ⁇ 108 cells/kg body weight and 8.5 ⁇ 108 cells/kg body weight respectively, which is about 2.4-10 times of the clinical human dose.
  • the solvent control group was given the same dose of cell cryopreservation premix (purchased from Nanjing Sunshine Biotechnology Co., Ltd., main components: dextran, glucose, process water).
  • mice 90 B-DNG mice were randomly divided into 3 groups, 30 in each group, half male and half male: vehicle control group, WAST effector cell low dose group (2 ⁇ 108 cells/kg), WAST effector cell high dose group (8.5 ⁇ 108 cells/kg).
  • the administration volume of each group was 20ml/kg.
  • For the low-dose WAST effector cell group 1 ⁇ 107 cells/ml was injected through the tail vein; for the high-dose WAST effector cell group, 4.25 ⁇ 107 cells/ml was injected through the tail vein; for the solvent control group, the same volume was injected through the tail vein cell protection solution.
  • the day of administration was the first day of the experiment (ie D1).
  • mice in each group After administration, the general state of the mice in each group was observed every day, and the body weight and food intake were measured respectively; at the end of the observation period on D15, the mice in each group were weighed and euthanized, and hematology, blood coagulation, blood biochemical tests, Gross anatomical observation and weighing of major organs, and histopathological examination of abnormal organs found in gross anatomy.
  • mice were given a single intravenous injection of WAST effector cells, and recovered after drug withdrawal for 14 days.
  • the general state, body weight and food intake of the mice in each group had no obvious abnormal changes related to the injection of WAST effector cells. No animal death or severe toxic reaction was observed.
  • the maximum tolerated dose (MTD) of mice to WAST effector cells was 8.5 ⁇ 108 cells/kg.
  • the individual WAST effector cells were given to each patient through intravenous infusion, and the dosage was 5 ⁇ 108-5 ⁇ 109 cells, and the WAST effector cells were given continuously for 3-6 times. No adverse reactions were observed during the treatment. All cases were observed for 6-12 months and showed stable disease, tumor shrinkage, cancer cell reduction or metastatic cancer number reduction, and quality of life was improved. Clinical research is still in progress.
  • WAST cell preparation intervention was carried out under the following conditions: 1. Animal experiments confirmed safety; 2. Subjects voluntarily requested to do WAST cell preparation intervention; 3. No treatment fee will be charged.
  • WAST cell preparation The following 1 subject was intervened with WAST cell preparation: the subject received immune function flow cytometry cell phenotype detection on day 0. On the 39th, 43rd, 57th, 326th, 340th, and 362th days, WAST cell preparations were injected intravenously once, a total of 6 times, each dose ⁇ 5 ⁇ 108 cells.
  • the preparation method of the WAST cell preparation is as follows: extract the peripheral blood mononuclear cells of the subject, obtain the WAST effector cells according to the method of Examples 1-3, and the carrier is physiological saline. On the 406th day, the immune function flow cytometry cell phenotype was tested again.
  • test results of the subject's immune function flow cytometry on day 0 and day 406 are as follows:
  • the subject was diagnosed with right transdermal seborrheic keratinization on day 0, which was characterized by the discovery of irregular painless and non-itching vegetation; between the 242nd day and the 272nd day of treatment, the subject The patient reported that the vegetation disappeared.
  • the subject On the 8th day of treatment, the subject underwent cervical vascular color Doppler ultrasound, and plaques in the enlarged bilateral carotid arteries were found; on the 426th day, the subject underwent neck vascular color Doppler ultrasound again, and it was found that the original carotid artery plaque disappeared.

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Abstract

一种用于激活整体抗肿瘤免疫系统的培养基配方物，其包含PolyI:C、CpG ODN 和膦抗原。还提供了一种制备 WAST效应细胞的方法，包括分离供者自体 PBMC 并使用PolyI:C、CpG ODN和膦抗原对其进行活化诱导，从而获得激活机体整体抗肿瘤免疫系统的WAST效应细胞。所述WAST效应细胞具有较强的IFN-Y、颗粒酶B和穿孔素分泌能力，并且在体外和体内均表现出肿瘤细胞杀伤活性。

Description

用于激活整体抗肿瘤免疫系统的培养基配方物及制备激动剂激活的整体免疫效应细胞的方法 技术领域
本发明涉及肿瘤的免疫疗法和调节机体免疫力的方法。具体而言，本发明涉及一种可用于治疗肿瘤或调节机体免疫力的免疫细胞制剂。
背景技术
十多年来，肿瘤免疫疗法已取得迅猛的发展，研究人员研发出了一些创新性的免疫治疗方法，其中过继免疫细胞疗法包括嵌合抗原受体T细胞(CAR-T)疗法以及新生抗原肿瘤浸润淋巴细胞(TIL)与细胞毒性T淋巴细胞(CTL)疗法；免疫检查点抑制剂疗法包括PD1/PDL1抑制剂疗法、CTLA-4抑制剂疗法等。这些治疗方法已在临床应用，解决了肿瘤治疗中的不少难题，但也显现了一些局限性。
CAR-T疗法最大的不足之处是在治疗过程中会出现严重的毒副作用。目前已知的严重毒副作用是细胞因子释放综合征(CRS)，也称为细胞因子风暴，严重者会导致患者死亡。其次为神经毒性与脱靶效应。神经毒性会引起意识混乱、失语、脑水肿等临床症状，脱靶效应会误伤表达同样靶点抗原的正常组织细胞，进而引起正常组织损伤或免疫缺陷病，严重时也可引起死亡。CAR-T疗法的另一个不足之处是治疗效果尚待提高，它对淋巴细胞性白血病与淋巴瘤的短期疗效十分显著，但治疗后几个月或更长时间会出现复发。第三个不足之处是CAR-T疗法对实体瘤疗效不佳或没有治疗效果，原因是CAR-T难于进入实体瘤病灶内，浸润于肿瘤内部；即使其能浸润到实体瘤内部，也会面临肿瘤微环境的抑制，使T细胞不能发挥抗肿瘤作用。第四个不足是目前已获批的CAR-T产品均以自身T细胞为起始材料，难以产业化，并且产品价格高达几十万美元，不利于市场推广。
新生抗原是肿瘤细胞中的非同义突变经过转录翻译及加工得到的多肽。由于正常细胞中不会表达肿瘤抗原，新抗原特异性免疫反应不会经历中枢耐受机制和外周耐受机制，所以从理论上来说，新生抗原具有作为免疫疗法治疗靶点的应用价值。随着高通量基因测序技术的发展，全基因组与外显子测序技术可以帮助研究者获取基因组上的突变信息(包括点突变、插入性突变等)。如何从这些数据中快速准确的鉴定候选新抗原，并筛选出高免疫原性的新抗原是肿瘤免疫治疗领域一个亟待解决的问题。目前已有的工具方法如pVAC-seq、MuPeXI、neopepsee等在一定程度上解决了新抗原的鉴定问题，但对候选抗原的有效筛选和排序仍有不足。因此，目前在新抗原TIL或CTL制备的核心技术中，尚存在许多技术瓶颈，造成了靶抗原准确率低，导致新生抗原TIL及CTL疗法的临床疗效不令人满意，加上其制备周期长，成本高，绝大多数患者无法接受，因而这种抗肿瘤免疫细胞疗法未能在临床上广泛使用。
近年来，愈来愈多的临床医生应用免疫检查点抑制剂治疗各种肿瘤。就目前临床应用最广泛的免疫检查点来说，也还存在总体应答率低，以及治疗过程中出现不良反应与耐药等问题。例如PD-1抑制剂在未经选择实体瘤患者中，有效率只有10-30％，但如果以肿瘤基因负荷(TMB)作为免疫检查点抑制剂疗效相关的标志物选择治疗对象，则发现TMB高的有效率为62％，然而免疫检查点抑制剂疗效相关的标志物目前还在研究当中，其具体机制与预测的准确性有待大规模的前瞻性临床研究的验证。此外，免疫检查点抑制剂带来的免疫治疗相关不良反应可累及全身各个脏器，引起多个脏器损伤，而且长期使用免疫检查点抑制剂会产生获得性耐药。
2019年10月，Oliver Demaria等人在《自然(Nature)》上发表了一篇题目为“Harnessing innate immunity in cancer therapy”(利用固有免疫治疗肿瘤)的综述。在这个综述中，提出了目前肿瘤的免疫治疗已发生了重大的发展，但现有的治疗方法(如上文所述各种免疫疗法)均聚焦于MHC限制性特异性T细胞。虽然这些抗肿瘤疗法取得了相当大的成功，但只有部分病人对治疗有反应，总体治疗响应率较低。近年来，肿瘤免疫治疗的关注点已逐渐向固有免疫方向转移。
发明内容
本发明提出了以靶向固有免疫的模式识别受体(PRR)理论为基础，通过新的多种激动剂及免疫刺激剂联合应用，激活固有免疫，进而活化适应性免疫，导致整体免疫系统细胞的活化，从而达到杀伤与消除肿瘤的目的。
第一方面，本发明提供了一种激动剂激活的整体免疫效应细胞。
第二方面，本发明提供了一种用于制备本发明第一方面的激动剂激活的整体免疫效应细胞的培养基配方物。
第三方面，本发明提供了一种制备本发明第一方面的激动剂激活的整体免疫效应细胞的方法。
第四方面，本发明提供了一种激动剂激活的整体免疫效应细胞制剂。
第五方面，本发明提供了一种肿瘤的治疗方法，包括给需要治疗的对象施用本发明第四方面的激动剂激活的整体免疫效应细胞制剂。
第六方面，本发明提供了一种调节机体免疫力的方法，包括给需要的对象施用本发明第四方面的激动剂激活的整体免疫效应细胞制剂。
在本文中使用的术语“WAST”是英文“Whole Agonist Stimulation”的英文缩写，意指由激动剂激活的整体免疫反应。在本文中，当提及“WAST细胞”或“WAST效应细胞”时，意指本发明的激动剂激活的整体免疫效应细胞；当提及“WAST前体细胞”时，意指本发明的激动剂激活的整体免疫前体细胞。
在本文中，除非特别注明，术语“细胞的比例”是指该种类细胞占总淋巴细胞群的比例。特别地，除非特别注明，术语“Vγ9δ2细胞的比例”是指Vγ9δ2细胞占总淋巴细胞中CD4-CD8Low-细胞群的比例。除非特别注明，上述比例是指根据本领域技术人员检测外周血淋巴细胞亚群的常规方法检测得到的比例，例如，是指根据常规的流式细胞术检测外周血淋巴细胞亚群的方法检测得到的比例。
在某些实施方案中，激动剂激活的整体免疫效应细胞中，总T淋巴细胞的比例大于70％，杀伤性T细胞的比例为45％-80％，NKT细胞的比例为7.5％-25％，Vγ9δ2细胞的比例为3％-50％。
在某些实施方案中，激动剂激活的整体免疫效应细胞中，总T淋巴细胞的比例大于90％，杀伤性T细胞的比例为45％-70％，Vγ9δ2细胞的比例为5％-50％。
在某些实施方案中，激动剂激活的整体免疫效应细胞中，分泌颗粒酶B的细胞的比例为15％-55％，分泌穿孔素的细胞的比例为15％-50％，同时分泌颗粒酶B和穿孔素的细胞的比例为10％-45％。
在某些实施方案中，通过将激动剂作用于供者的外周血得到所述激动剂激活的整体免疫效应细胞，其中，所述激动剂包含TLR3激动剂、TLR9激动剂和γδT细胞的刺激剂。
在某些实施方案中，TLR3激动剂是PolyI:C。
在某些实施方案中，TLR9激动剂是CpG ODN。
在某些实施方案中，γδT细胞的刺激剂是膦抗原。
在某些实施方案中，TLR3激动剂是PolyI:C，TLR9激动剂是CpG ODN且γδT细胞的刺激剂是膦抗原。
在某些实施方案中，制备激动剂激活的整体免疫效应细胞的培养基配方物包含TLR3激动剂、TLR9激动剂和γδT细胞的刺激剂。
在某些实施方案中，TLR3激动剂是PolyI:C。
在某些实施方案中，TLR9激动剂是CpG ODN。
在某些实施方案中，γδT细胞的刺激剂是膦抗原。
在某些实施方案中，制备激动剂激活的整体免疫效应细胞的培养基配方物包含PolyI:C、CpG ODN和膦抗原。
在某些实施方案中，培养基配方物还包含IL-2、IL-15、抗CD3抗体、抗CD28抗体。
在某些实施方案中，制备激动剂激活的整体免疫效应细胞的培养基配方物包含浓度为100-500IU/mL的IL-2，5-50ng/mL的IL-15，0.5-5μg/mL的抗CD3抗体，0.5-5μg/mL的抗CD28抗体，2-20μg/mL的PolyI:C，2-20μg/mL的CpG ODN，10-100μmol/L的唑来膦酸。
在某些实施方案中，制备激动剂激活的整体免疫效应细胞的培养基配方物包含浓度为300-500IU/mL的IL-2，10-50ng/mL的IL-15，1-3μg/mL的抗CD3抗体，1-3μg/mL的抗CD28抗体，5-15μg/mL的PolyI:C，5-15μg/mL的CpG ODN，10-75μmol/L的唑来膦酸。
在某些实施方案中，制备激动剂激活的整体免疫效应细胞的培养基配方物包含浓度为300IU/mL的IL-2，15ng/mL的IL-15，1μg/mL的抗-CD3抗体，1μg/mL的抗-CD28抗体，10μg/mL的PolyI:C，10μg/mL的CpG ODN，50μmol/L的唑来膦酸。
在某些实施方案中，制备激动剂激活的整体免疫效应细胞的方法包括：
(1)分离供者的外周抗凝血，并从中分离外周血单个核细胞；培养外周血单个核细胞，得到贴壁细胞和非贴壁的悬浮细胞；
(2)收集贴壁细胞，经细胞因子诱导培养，获得非成熟树突状细胞；
(3)继续诱导培养步骤(2)中获得的非成熟树突状细胞，以获得成熟树突状细胞；
(4)将步骤(1)中得到的非贴壁的悬浮细胞在培养基配方物中培养，得到激动剂激活的整体免疫前体细胞；
(5)将步骤(3)制得的成熟树突状细胞与步骤(4)得到的激动剂激活的整体免疫前体细胞混合并培养，以得到激动剂激活的整体免疫效应细胞。
在某些实施方案中，步骤(2)中的细胞因子为粒细胞-巨噬细胞集落刺激因子与白细胞介素-4。
在某些实施方案中，步骤(3)中使用白细胞介素-2、白细胞介素-33和肿瘤坏死因子α进行诱导培养。
在某些实施方案中，供者是人。
在某些实施方案中，激动剂激活的整体免疫效应细胞制剂包含激动剂激活的整体免疫效应细胞和药学上可接受的载体。
在某些实施方案中，药学上可接受的载体选自生理盐水、细胞冻存液或细胞冷冻保护剂。
在某些实施方案中，激动剂激活的整体免疫效应细胞制剂是注射剂。
在某些实施方案中，肿瘤的治疗方法包括给需要治疗的对象施用激动剂激活的整体免疫效应细胞制剂。
在某些实施方案中，需要治疗的对象是人。
在某些实施方案中，肿瘤为实体瘤。
在某些实施方案中，实体瘤选自肺癌、肝癌、胃癌、乳腺癌或结肠癌。
在某些实施方案中，细胞制剂通过静脉注射、瘤内注射和/或瘤周注射给予需要治疗的对象。
在某些实施方案中，细胞制剂与其他治疗剂或治疗手段组合施用，其他治疗剂或治疗手段选自化疗、靶向疗法、细胞因子、外科手术、放射治疗、免疫疗法和中医治疗中的一种或多种。
在某些实施方案中，调节机体免疫力的方法包括给需要的对象施用激动剂激活的整体免疫效应细胞制剂。
在某些实施方案中，需要的对象选自免疫力低下或免疫力失衡人群。
相比于现有的抗肿瘤免疫疗法，本发明具有以下优势：(1)WAST细胞制剂由具有多种不同杀伤机制的免疫细胞构成，其在临床上单独使用或与现有肿瘤治疗方法及药物联合应用，都会产生较高的有效性；(2)由于所用的多种激动剂与免疫刺激剂具有良好的安全性与有效性，所用的主要起始材料是患者自体PBMC或符合规定的冻存PBMC，所用的细胞培养及扩增试剂都符合GMP规定，因此WAST细胞制剂具有很好的安全性；(3)WAST细胞属于活化的免疫细胞，可以避免现有技术中使用各种T细胞的缺陷与不良反应；(4)WAST细胞是广谱肿瘤杀伤制剂，可广泛的用于多种实体肿瘤的治疗；(5)WAST细胞制剂不涉及基因改造、基因重组等复杂制备过程，制备周期短，安全性高，易于临床推广应用；(6)WAST细胞制剂可以与化疗、靶向疗法、细胞因子、外科手术、放射治疗、免疫疗法和中医治疗联合应用，通过协同作用增加其疗效；(7)TNM分期为I期或II期病例早期手术后加用WAST细胞制剂治疗，可减少复发率。
本发明还具有提高机体整体免疫应答(固有免疫+适应性免疫)的有益效果。此外，通过应用本发明的WAST细胞能够起到调节机体免疫的作用，特别对于机体已经存在免疫失衡的人群，有调节免疫失衡，实现免疫平衡作用。
附图说明
图1概述了本发明的WAST细胞制剂的制备方法及其抗肿瘤机制。
图2表示诱导前后分泌颗粒酶B及穿孔素的细胞的比例，其中l表示分泌颗粒酶B的细胞，m表示分泌穿孔素的细胞，n表示同时分泌颗粒酶B和穿孔素的细胞；误差线是指各类细胞测量值的95％置信区间(CI)。
图3表示经激动剂和免疫刺激剂诱导前后各类细胞百分比的测量值。a：总T淋巴细胞百分比；b：辅助性T细胞百分比；c：杀伤性T细胞百分比；e：总B淋巴细胞百分比；f：NK细胞百分比；g：NKT细胞百分比；h：CD25+细胞百分比；i：单核细胞百分比；j：HLA-DR+细胞百分比；k：Vγ9δ2(CD4-CD8low-)百分比；误差线是指各类细胞测量值的95％置信区间(CI)。
具体实施方式
以下结合实施例对本发明进行详细描述。下述实施例中的实验方法，未注明具体技术或条件者，则按照常规实验条件如《ATCC细胞培养手册》中所述条件，而在实施例中明确提及有试剂公司说明书时，则参考说明书所建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买获得常规产品。
本发明实施例中所用的激动剂及免疫诱导试剂：(1)AIM-V细胞培养基，购自ThermoFisher公司；(2)人淋巴细胞分离管(即用型)，购自达科为公司；(3)IL-4、GM-CSF、TNF-α、IL-2、IL-15、IL-33、抗CD3单抗、抗CD28单抗，均购自Peprotech公司；(4)PolyI:C、CpG ODN(5’-tcgtcgttttcgcg-3’)，均购自InvivoGen公司；(5)唑来膦酸，购自国药集团。
实施例1提取和培养外周血单个核细胞(PBMC)
(1)分离捐赠的来自健康人的外周抗凝血50ml。在两支50ml分离管中各加入25ml外周血。
(2)离心管配平，以水平离心机1700rpm，慢升慢降速，室温离心20min。
(3)吸出最上层部分多余的血浆。用2ml无菌吸管轻轻吸出淋巴细胞层，移入新的50ml离心管中，将所有管中的淋巴细胞层吸入同一个50ml离心管中。加入0.9％无菌生理盐水至50ml，充分混匀。
(4)离心管配平，以水平离心机1700rpm，快升快降速，室温离心10min；取出试管，完全弃去上清液。
(5)从37℃培养箱中取出预热的AIM-V培养基，从中取5ml将细胞沉淀充分混匀，注意轻柔操作，避免产生气泡，随后加入AIM-V培养基至1×106细胞/ml细胞浓度，混匀细胞。
(6)将混匀的细胞悬液移入75cm2细胞培养瓶，轻轻晃动混匀，放置于37℃培养箱中培养。
实施例2树突状细胞(DC)的制备
(1)将实施例1中得到的PBMC以AIM-V培养基重悬，加入75cm2细胞培养瓶中，37℃，5％CO2培养。
(2)3小时后将培养瓶取出，轻轻晃动，吸出悬浮细胞。
(3)向培养瓶中加入含500IU/ml的IL-4、500IU/ml的GM-CSF的AIM-V培养基。
(4)分别在培养后第3天、第5天半量换液，加入新鲜含500IU/ml的IL-4、500IU/ml的GM-CSF的AIM-V培养基。
(5)培养第7天加入终浓度为10ng/ml的TNF-α、300IU/ml的IL-2、10ng/ml的IL-33的AIM-V细胞培养基。
(6)第8天收获成熟树突状细胞。
实施例3 WAST效应细胞的制备
(1)通过实施例2步骤(2)得到悬浮细胞，将其重悬于AIM-V培养基中，调节细胞浓度至1×106个/ml，此悬液中富含T淋巴细胞。向该悬液中加入终浓度300IU/ml的IL-2，15ng/ml的IL-15，1μg/ml的抗CD3单抗，1μg/ml的抗CD28单抗，10μg/ml的PolyI:C，10μg/ml的CpG ODN，50μmol/L的唑来膦酸，37℃，5％CO2培养8天，每3天半量换液，以获得WAST前体细胞；
(2)第8天，将实施例2制备的成熟树突状细胞与WAST前体细胞按照成熟树突状细胞:WAST前体细胞＝1:10的比例混合后，继续培养2天；
(3)第10天收集细胞，即得WAST效应细胞。
实施例4 WAST效应细胞表型分析
按照实施例1-3中所述的WAST效应细胞的制备方法，获得6例健康人新鲜的WAST效应细胞。用流式细胞仪进行细胞表型分析，测定使用本发明的培养基配方物诱导前后(诱导前：PBMC，分别用1至6表示；诱导后：WAST效应细胞，分别用1’至6’表示)各类细胞百分比的变化，结果如下表所示。

使用数据分析软件SPSS19.0对数据进行配对T检验统计学数据分析，所有检验均为双侧检验，检验水准α＝0.05。分析结果见图3。
从图3可见，诱导后获得的WAST效应细胞中与肿瘤杀伤相关的细胞的百分比均显著升高。以样品3为例：对于总T淋巴细胞百分比，诱导前为57.83％，诱导后为98.78％；对于杀伤性T细胞百分比，诱导前为13.97％，诱导后为45.35％；对于NKT细胞百分比，诱导前为2.54％，诱导后为24.63％；对于Vγ9δ2(CD4-CD8Low-)细胞百分比，诱导前为3.42％，诱导后为12.18％。这些细胞均为WAST效应细胞中的主要杀伤细胞，其在诱导后比例都有显著升高。
实施例5 WAST效应细胞的颗粒酶B(Granzyme B)和穿孔素(Perforin)分泌能力检测
通过流式细胞术检测实施例4制备的新鲜的WAST效应细胞分泌颗粒酶B、穿孔素的能力。下表说明诱导前后的细胞(诱导前：PBMC，分别用1至6表示；诱导后：WAST效应细胞，分别用1’至6’表示)分泌能力的变化。
使用数据分析软件SPSS19.0对数据进行配对T检验统计学数据分析，所有检验均为双侧检验，检验水准α＝0.05。分析结果见图2。
如图2所示，诱导后分泌颗粒酶B或穿孔素或同时分泌颗粒酶B和穿孔素的细胞数量显著增多，表明WAST效应细胞颗粒酶B、穿孔素分泌能力较PBMC更强。
实施例6 WAST效应细胞对5种不同肿瘤细胞系的体外直接杀伤能力测定
在本实施例中，分别以3例外购PBMC(均购自上海妙顺生物技术公司)为起始材料，按照实施例2-3中的制备方法获得WAST效应细胞，将其分别为命名为WAST-121061203C、WAST-121030702C、WAST-121050801C。将这三种细胞作为效应细胞，以A549(人非小细胞肺癌)、MCF-7(人乳腺癌细胞)、HepG2(人肝癌细胞)、HGC-27(人胃癌细胞)和HCT116(人结肠癌细胞)为靶细胞，效靶比分别为20:1和30:1，进行体外直接杀伤试验。
用流式细胞仪进行细胞表型分析，测定三种新鲜的WAST效应细胞中各类细胞的比例如下表所示：
通过流式细胞术检测三种WAST效应细胞分泌颗粒酶B、穿孔素的能力。
使用CCK8(细胞计数试剂盒(Cell Counting Kit)-8)检测释放法，检测WAST细胞对A549、MCF-7、HepG2、HGC-27和HCT116细胞的体外杀伤能力，该方法的具体操作方法如下：
(1)调整靶细胞浓度至5×104个/ml；
(2)分别设置以下对照组：空白对照组，每孔只加200μl AIM-V培养基；靶细胞对照组，不加效应细胞，向每孔加100μl AIM-V培养基，再加入100μl靶细胞；效应细胞对照组，不加靶细 胞，向每孔加100μl AIM-V培养基，再加入100μl效应细胞；每组设定三个复孔，并且均在37℃，5％CO2培养24小时；
(3)对于实验组，将靶细胞按照100μl/孔转移至96孔板中，每组设定三个复孔，37℃，5％CO2培养24小时(此时靶细胞数量增加到10000个/孔)；然后向每个实验孔中加入效应细胞100μl，效应细胞:靶细胞(即效靶比)分别为20:1和30:1；
(4)将上述对照组和实验组分别于37℃，5％CO2培养4-6小时；
(5)每孔加入10μl CCK8试剂，37℃培养2小时；
(6)酶标仪测定各孔吸光值OD450，取复孔平均值A计算杀伤活性，其计算公式为：
杀伤率(％)＝[1-(A实验组-A效应细胞对照组)/(A靶细胞对照组-A空白对照组)]×100％。
以下各表列出了分别以A549(人非小细胞肺癌)、MCF-7(人乳腺癌细胞)、HepG2(人肝癌细胞)、HGC-27(人胃癌细胞)和HCT116(人结肠癌细胞)作为靶细胞的体外杀伤试验中各对照组和实验组测定的吸光值OD450及计算的杀伤率。
WAST效应细胞对肺癌细胞系A549体外杀伤结果
WAST效应细胞对乳腺癌细胞系MCF-7体外杀伤结果
WAST效应细胞对肝癌细胞系HepG2体外杀伤结果

WAST效应细胞对胃癌细胞系HGC-27体外杀伤结果

WAST效应细胞对结肠癌细胞系HCT116体外杀伤结果
(1)WAST效应细胞对A549(人非小细胞肺癌)直接杀伤结果
WAST-121061203C在效靶比20:1、30:1时杀伤率分别为41.94％和53.18％；
WAST-121030702C在效靶比20:1、30:1时杀伤率分别为36.37％和47.36％；WAST-121050801C在效靶比20:1、30:1为80.37％和96.33％。
(2)WAST效应细胞对MCF-7(人乳腺癌细胞)直接杀伤结果
WAST-121061203C在效靶比20:1、30:1时杀伤率分别为9.17％和17.20％；WAST-121030702C在效靶比20:1、30:1时杀伤率分别为17.83％和32.67％；WAST-121050801C在效靶比20:1、30:1为11.94％和20.82％。
(3)WAST效应细胞对HepG2(人肝癌细胞)直接杀伤结果
WAST-121061203C在效靶比20:1、30:1时杀伤率分别为17.81％和28.90％；
WAST-121030702C在效靶比20:1、30:1时杀伤率分别为19.33％和31.98％；WAST-121050801C在效靶比20:1、30:1分别为25.51％和44.24％。
(4)WAST效应细胞对HGC-27(人胃癌细胞)直接杀伤结果
WAST-121061203C在效靶比20:1、30:1时杀伤率分别为81.45％和92.63％；
WAST-121030702C在效靶比20:1、30:1时杀伤率分别为40.39％和47.34％；WAST-121050801C在效靶比20:1、30:1分别为76.19％和87.62％。
(5)WAST效应细胞对HCT116(人结肠癌细胞)直接杀伤结果
WAST-121061203C在效靶比20:1、30:1时杀伤率分别为54.49％和47.41％；
WAST-121030702C在效靶比20:1、30:1时杀伤率分别为69.83％和53.31％。
上述三种WAST效应细胞对5种不同肿瘤细胞系的体外直接杀伤结果均表现出针对肿瘤细胞有体外杀伤效果，证明了WAST效应细胞的抗肿瘤活性。
实施例7WAST效应细胞对肺癌A549细胞系的体外间接杀伤能力测定
在本实施例中，以实施例6中制得的WAST效应细胞WAST-121061203C、WAST-121030702C和WAST-121050801C作为效应细胞，以A549(人非小细胞肺癌)细胞系作为靶细胞进行细胞双室培养杀伤试验。
具体方法如下：
(1)将A549和效应细胞从液氮中取出，复苏细胞；对A549细胞进行传代培养。
(2)取对数生长期、生长状态良好的A549细胞，5x103个/孔接入24孔板，同时设空白组。37℃培养过夜待用。
(3)取复苏18-24h后的A549细胞上清液，用含IL-2的RPMI 1640完全培养基于37℃、5％CO2饱和湿度条件培养24h，离心并收集上清。用无IL-2的RPMI 1640完全培养基重悬,计数待用。
(4)用洁净锻子将细胞共培小室置入含有A549的24孔板中，然后按照如下分组将效应T细胞稀释到合适浓度并加入细胞共培小室中，并将上室和下室每孔培养基体积补充至500μL：
1)空白组：仅培养基(空白对照组)
2)下室A549，无上室空白孔
3)下室A549，上室含WAST-121061203C 200000个/孔
4)下室A549，上室含WAST-121061203C 300000个/孔
5)下室A549，上室含WAST-121030702C 200000个/孔
6)下室A549，上室含WAST-121030702C 300000个/孔
7)下室A549，上室含WAST-121050801C 200000个/孔
8)下室A549，上室含WAST-121050801C 300000个/孔
9)下室A549，上室含单纯培养基(靶细胞对照组)
(5)作用24h后，移除上室，将下室培养基更换为300μL含有10％MTT的完全培养基，37℃培养4h。吸出培养基，加入400μL DMSO震荡10min。用酶标仪测定各孔吸光值OD568。 取复孔平均值A计算杀伤活性，其计算公式为：
杀伤率(％)＝[1-(A实验组-A空白对照组)/(A靶细胞对照组-A空白对照组)]×100％。
下表列出了以A549作为靶细胞的体外双室培养杀伤实验中，各实验组计算得到的杀伤率：
上述三种WAST效应细胞与A549细胞进行双室培养后，均能抑制A549细胞的增殖，且随着WAST效应细胞数量增加，抑制率会增加。这证明了WAST效应细胞具有对肿瘤细胞的间接杀伤作用。
实施例8 WAST细胞在小鼠体内杀伤实体肿瘤的药效实验(以非小细胞肺癌荷瘤模型为例)
本实施例中使用CDX肺癌A549荷瘤小鼠模型——NPI免疫缺陷小鼠(本试验委托北京艾德摩生物技术有限公司实施)，周龄为6-8周，体重为20-30g。待小鼠体内肿瘤长至80-150mm3时，按时间点分组分次经尾静脉及瘤内瘤周注射人WAST细胞，观察肿瘤生长情况并测量肿瘤大小。
具体操作方法如下：
(1)将实施例6中的WAST效应细胞WAST-121030702C，悬浮于生理盐水中备用；
(2)WAST效应细胞注射频率为每周1次，持续3周，以首次注射为第0天，分别在第0天(D0)、第7天(D7)、第14天(D14)进行WAST效应细胞注射，共进行3次；实验分为3组，分别为：生理盐水组，瘤内瘤周注射组(I.T.)(该组注射WAST-121030702C细胞)、静脉注射组(I.V.)(该组注射WAST-121030702C细胞)，每组3只CDX荷瘤小鼠；
(3)确定WAST细胞的注射剂量：以60kg的成人为基准，WAST细胞在临床预期使用剂量为5×108-5×109个细胞(即0.8×107-0.8×108个细胞/kg)。以CDX小鼠体重30g为基准，以其通常最大耐受剂量1×107个细胞(该剂量是目前细胞产品的小鼠动物试验中使用的最高剂量)为高剂量组；
(4)注射剂量：生理盐水组，每只小鼠注射生理盐水200μl；瘤内瘤周注射组，每只小鼠每次注射1×107个细胞；静脉注射组，每只小鼠每次注射1×107个细胞；
完成3次注射后，第25天对小鼠荷瘤体积进行测量。使用卡尺测量肿瘤的长径及短径，肿瘤的体积(TV)计算公式为：TV＝0.5a×b2(a＝长径长度，b＝短径长度)。3组小鼠肿瘤体积测量结果如下：
由上表可知，连续注射WAST效应细胞后，第25天对小鼠荷瘤体积进行测量，瘤内瘤周注射组及静脉注射组小鼠肿瘤平均体积均小于生理盐水组。
另外，第17天经流式细胞仪对小鼠外周血进行免疫细胞表型流式检测，其中对小鼠外周血CD8+CD69+流式细胞检测结果如下：
第17天小鼠外周血免疫细胞表型流式检测结果显示，静脉注射组CD8+CD69+百分比为74.11％，而生理盐水组CD8+CD69+百分比为0％，瘤内瘤周注射组CD8+CD69+百分比为11.92％。因给药途径不同，静脉注射组与瘤内瘤周注射组免疫细胞被激活的状态存在差异。
结论：WAST效应细胞在静脉注射组及瘤内瘤周注射组小鼠体内均被激活，3次WAST效应细胞注射后，从上述结果分析可得出，不管是瘤内瘤周注射还是静脉注射WAST效应细胞均能起到抑制肿瘤的作用。
实施例9 WAST细胞在人源化小鼠体内杀伤实体肿瘤的药效实验(以非小细胞肺癌荷瘤模型为例)
本实施例中使用CDX肺癌A549荷瘤小鼠模型——吉西他滨用药后的Hu-NPI免疫缺陷小鼠(本试验委托北京艾德摩生物技术有限公司实施)，周龄为37周，体重为19-24g。待小鼠体内肿瘤长至70-120mm3时，分组单次经瘤内瘤周注射人WAST细胞，观察肿瘤生长情况并测量肿瘤大小。
具体操作方法如下：
(1)以Hu-NPI小鼠的脾细胞为起始原料，按照实施例2-3的制备方法获得WAST效应细胞，命名为WAST-22010517H。注射前，将冻存的WAST效应细胞WAST-22010517H复苏；
用流式细胞仪进行细胞表型分析，测定WAST效应细胞中各类细胞的比例如下表所示：
(2)单次注射WAST效应细胞，以首次注射为第0天；实验分为2组，分别为：生理盐水组、瘤内瘤周注射组(I.T.)(该组注射WAST-22010517H细胞)，每组3只CDX荷瘤小鼠；
(3)WAST细胞的注射剂量：每只小鼠注射1.33×107个细胞作为单次给药高剂量组；
(4)注射剂量：生理盐水组，每只小鼠注射生理盐水80μl；瘤内瘤周注射组，每只小鼠注射1.33×107个细胞；完成注射后，第58天对小鼠荷瘤体积进行测量。使用卡尺测量肿瘤的长径及短径，肿瘤的体积(TV)计算公式为：TV＝0.5a×b2(a＝长径长度，b＝短径长度)。2组小鼠肿瘤体积测量结果如下：
由上表可知，单次高剂量注射WAST效应细胞后，第58天对小鼠荷瘤体积进行测量，瘤内瘤周注射组小鼠肿瘤平均体积、中位体积小于生理盐水组。
第59天时采集小鼠肿瘤，经流式细胞仪对小鼠肿瘤浸润淋巴细胞(TIL)进行免疫细胞表型流式检测，其中对小鼠肿瘤浸润淋巴细胞中CD45+细胞占全部活细胞比例及CD4-CD8+细胞占全部CD3+细胞比例进行流式细胞检测，结果如下：
第59天小鼠肿瘤浸润淋巴细胞免疫细胞表型流式检测结果显示，瘤内瘤周注射组肿瘤TIL浸润(总CD45+)显著比生理盐水组高，同时浸润的CD3+T细胞中，CD8+阳性比例显著高于第一组，说明WAST细胞在体内有显著增殖和存续，同时在肿瘤浸润中主要为杀伤性细胞。
结论：WAST效应细胞在瘤内瘤周注射组小鼠体内被激活。单次WAST效应细胞注射后，瘤内瘤周注射后WAST效应细胞能够持续在体内增殖，且能起到抑制肿瘤的作用。
实施例10 B-NDG小鼠静脉注射WAST效应细胞制剂单次给药毒性动物试验
试验目的
WAST细胞制剂是未经修饰的免疫细胞，临床用于治疗实体肿瘤，给药途径为静脉输注。本实验通过WAST细胞制剂经静脉单次给予B-NDG小鼠后观察14天，观察WAST细胞制剂可能引起的毒性反应的性质、程度和可逆性，推测毒性靶器官靶组织，为临床的安全性提供参考信息。本实验的承接单位为：国科赛赋河北医药有限公司(GLP)。
本实验使用SPF级B-DNG小鼠(购自百奥赛图江苏基因生物技术有限责任公司)。雌鼠体重为18.84g-22.01g，雄鼠体重为26.14g-28.89g，同性别小鼠个体体重在平均体重±20％范围内。分组给药时小鼠约7周龄。
剂量设计
本次试验采用最大给药量法，WAST效应细胞为上述WAST-121030702C，WAST效应细胞的低、高剂量组给药浓度分别设计为1×107细胞/ml、4.25×107细胞/ml，给药体积为20ml/kg体重，给药剂量分别为2×108细胞/kg体重、8.5×108细胞/kg体重，约为临床人用剂量的2.4-10倍。溶剂对照组给予等剂量的细胞冷藏保护预混液(购自南京三生生物技术股份有限公司，主要成分：右旋糖酐、葡萄糖、工艺用水)。
实验方法
将90只B-DNG小鼠随机分为3组，每组30只，雌雄各半：溶剂对照组、WAST效应细胞低剂量组(2×108细胞/kg)、WAST效应细胞高剂量组(8.5×108细胞/kg)。各组给药体积为均20ml/kg。对于WAST效应细胞低剂量组，经尾静脉注射给予1×107细胞/ml；对于WAST效应细胞高剂量组，经尾静脉注射给予4.25×107细胞/ml；溶剂对照组经尾静脉注射给予等体积的细胞保护液。给药当天为实验的第1天(即D1)。
给药后，每天观察各组小鼠的一般状态，分别测定体重、摄食量；D15时观察期结束，对各组小鼠称重后实施安乐死，并进行血液学、血凝、血液生化检测、大体解剖观察及主要脏器称重，对大体解剖发现的异常脏器进行组织病理学检查。
结果
在本试验条件下，B-DNG小鼠单次静脉注射WAST效应细胞，停药恢复14天。试验期间，各组小鼠一般状态、体重、摄食量均未见与注射WAST效应细胞相关的明显异常改变。未见动物死亡和严重毒性反应。小鼠对WAST效应细胞的最大耐受剂量(MTD)为8.5×108细胞/kg。
实施例11 WAST细胞制剂对肿瘤患者的临床初步预试
为了观察WAST细胞制剂在治疗肿瘤中的安全性及其可能的有效性，在下述条件下：1、动物试验证实安全；2、患者志愿请求做WAST细胞制剂治疗；3、不收取治疗费。
对下列5例肿瘤患者进行1-2个疗程WAST效应细胞治疗。5例患者的临床病理诊断及TNM分期如下：
具体治疗过程如下：
(1)分离上述5例患者的外周抗凝血，并根据实施例1-3所述，制备各患者的WAST效应细胞，保存备用。
(2)向各患者给予制备得到的各自的WAST效应细胞。
在治疗过程中通过静脉回输给予各患者各自的WAST效应细胞，给药剂量为5×108-5×109个细胞，连续给予3-6次WAST效应细胞。在治疗过程中没有观察到任何不良反应。所有病例观察6-12个月均显示病情稳定，并且肿瘤缩小、癌细胞减少或转移癌数量减少，生活质量上调。目前临床研究仍在进行中。
实施例12 WAST细胞制剂对免疫低下人群的临床初步预试
为了观察WAST细胞制剂调节机体免疫力的安全性及其可能的有效性，在下述条件下进行了WAST细胞制剂干预：1.动物试验证实安全；2.受试者志愿请求做WAST细胞制剂干预；3.不收取治疗费。
对以下1例受试者进行了WAST细胞制剂干预：受试者在第0天接受了免疫功能流式细胞仪细胞表型检测。在第39、43、57、326、340、362天，分别进行了一次WAST细胞制剂静脉注射，共6次，每次剂量≥5×108个细胞。WAST细胞制剂的制备方法是：提取受试者的外周血单个核细胞，按照实施例1-3的方法获得WAST效应细胞，载体为生理盐水。在第406天再次接受免疫功能流式细胞仪细胞表型检测。
受试者第0天和第406天免疫功能流式细胞仪细胞表型检测结果如下：
此外，受试者在第0天确诊有右侧透皮脂溢性角质化，表现为发现有不规则的无痛、无痒的赘生物；在治疗第242天-第272天之间，受试者报告该赘生物消失。治疗第8天受试者进行了颈部血管彩超，发现双侧颈动脉膨大处斑块；第426天再次进行了颈部血管彩超，发现原有颈动脉斑块消失。受试者报告，每次WAST细胞制剂干预后无任何不适，并感受到体力、精力改善。
目前临床研究仍在进行中。

Claims (31)

  1. 一种激动剂激活的整体免疫效应细胞，其特征在于，所述激动剂激活的整体免疫效应细胞中，总T淋巴细胞的比例大于70％，杀伤性T细胞的比例为45％-80％，NKT细胞的比例为7.5％-25％，Vγ9δ2细胞的比例为3％-50％。
  2. 根据权利要求1所述的激动剂激活的整体免疫效应细胞，其特征在于，所述总T淋巴细胞的比例大于90％，所述杀伤性T细胞的比例为45％-70％，Vγ9δ2细胞的比例为5％-50％。
  3. 一种激动剂激活的整体免疫效应细胞，其特征在于，所述激动剂激活的整体免疫效应细胞中，分泌颗粒酶B的细胞的比例为15％-55％，分泌穿孔素的细胞的比例为15％-50％，同时分泌颗粒酶B和穿孔素的细胞的比例为10％-45％。
  4. 一种激动剂激活的整体免疫效应细胞，其特征在于，通过将激动剂作用于供者的外周血得到所述激动剂激活的整体免疫效应细胞，其中，所述激动剂包含TLR3激动剂、TLR9激动剂和γδT细胞的刺激剂。
  5. 根据权利要求4所述的激动剂激活的整体免疫效应细胞，其特征在于，所述TLR3激动剂是PolyI：C。
  6. 根据权利要求4所述的激动剂激活的整体免疫效应细胞，其特征在于，所述TLR9激动剂是CpG ODN。
  7. 根据权利要求4所述的激动剂激活的整体免疫效应细胞，其特征在于，所述γδT细胞的刺激剂是膦抗原。
  8. 一种用于制备激动剂激活的整体免疫效应细胞的培养基配方物，包含TLR3激动剂、TLR9激动剂和γδT细胞的刺激剂。
  9. 根据权利要求8所述的培养基配方物，其特征在于，所述TLR3激动剂是PolyI：C。
  10. 根据权利要求8所述的培养基配方物，其特征在于，所述TLR9激动剂是CpG ODN。
  11. 根据权利要求8所述的培养基配方物，其特征在于，所述γδT细胞的刺激剂是膦抗原。
  12. 根据权利要求8所述的培养基配方物，其特征在于，所述培养基配方物包含PolyI：C、CpG ODN和膦抗原唑来膦酸。
  13. 根据权利要求12所述的培养基配方物，其特征在于，所述培养基配方物还包含IL-2、IL-15、抗CD3抗体、抗CD28抗体。
  14. 根据权利要求13所述的培养基配方物，其特征在于，所述培养基配方物包含浓度为100-500IU/mL的IL-2，5-50ng/mL的IL-15，0.5-5μg/mL的抗CD3抗体，0.5-5μg/mL的抗CD28抗体，2-20μg/mL的PolyI:C，2-20μg/mL的CpG ODN，10-100μmol/L的唑来膦酸。
  15. 根据权利要求14所述的培养基配方物，其特征在于，所述培养基配方物包含浓度为300-500IU/mL的IL-2，10-50ng/mL的IL-15，1-3μg/mL的抗CD3抗体，1-3μg/mL的抗CD28抗体，5-15μg/mL的PolyI:C，5-15μg/mL的CpG ODN，10-75μmol/L的唑来膦酸。
  16. 根据权利要求15所述的培养基配方物，其特征在于，所述培养基配方物包含浓度为300IU/mL的IL-2，15ng/mL的IL-15，1μg/mL的抗-CD3抗体，1μg/mL的抗-CD28抗体，10μg/mL的PolyI:C，10μg/mL的CpG ODN，50μmol/L的唑来膦酸。
  17. 一种制备如权利要求1-7中任一项所述的激动剂激活的整体免疫效应细胞的方法，包括：
    (1)分离供者的外周抗凝血，并从中分离外周血单个核细胞；培养外周血单个核细胞，得到贴壁细胞和非贴壁的悬浮细胞；
    (2)收集贴壁细胞，经细胞因子诱导培养，获得非成熟树突状细胞；
    (3)继续诱导培养步骤(2)中获得的非成熟树突状细胞，以获得成熟树突状细胞；
    (4)将步骤(1)中得到的非贴壁的悬浮细胞在权利要求8-16中任一项的培养基配方物中培养，得到激动剂激活的整体免疫前体细胞；
    (5)将步骤(3)制得的成熟树突状细胞与步骤(4)得到的激动剂激活的整体免疫前体细胞混合并培养，以得到激动剂激活的整体免疫效应细胞。
  18. 根据权利要求17所述的方法，其特征在于，步骤(2)中的细胞因子为粒细胞-巨噬细胞集落刺激因子与白细胞介素-4。
  19. 根据权利要求17所述的方法，其特征在于，步骤(3)中使用白细胞介素-2、白细胞介素-33和肿瘤坏死因子α进行诱导培养。
  20. 根据权利要求17-19中任一项所述的方法，其特征在于，所述供者是人。
  21. 一种激动剂激活的整体免疫效应细胞制剂，包含如权利要求1-7中任一项所述的激动剂激活的整体免疫效应细胞和药学上可接受的载体。
  22. 根据权利要求21所述的激动剂激活的整体免疫效应细胞制剂，其特征在于，所述药学上可接受的载体选自生理盐水、细胞冻存液或细胞冷冻保护剂。
  23. 根据权利要求21或22所述的激动剂激活的整体免疫效应细胞制剂，其特征在于，所述制剂是注射剂。
  24. 一种肿瘤的治疗方法，包括给需要治疗的对象施用权利要求21-23中任一项所述的激动剂激活的整体免疫效应细胞制剂。
  25. 根据权利要求24所述的治疗方法，其特征在于，所述需要治疗的对象是人。
  26. 根据权利要求25所述的治疗方法，其特征在于，所述肿瘤为实体瘤。
  27. 根据权利要求26所述的治疗方法，其特征在于，所述实体瘤选自肺癌、肝癌、胃癌、乳腺癌或结肠癌。
  28. 根据权利要求24-27中任一项所述的治疗方法，其特征在于，所述细胞制剂通过静脉注射、瘤内注射和/或瘤周注射给予需要治疗的对象。
  29. 根据权利要求24-28中任一项所述的治疗方法，其特征在于，所述细胞制剂与其他治疗剂或治疗手段组合施用，所述其他治疗剂或治疗手段选自化疗、靶向疗法、细胞因子、外科手术、放射治疗、免疫疗法和中医治疗中的一种或多种。
  30. 一种调节机体免疫力的方法，包括给需要的对象施用权利要求21-23中任一项所述的激动剂激活的整体免疫效应细胞制剂。
  31. 根据权利要求30所述的方法，其特征在于，所述需要的对象选自免疫力低下或免疫力失衡人群。
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