WO2022222845A1 - 混合的固有淋巴细胞、制备方法及其应用 - Google Patents

混合的固有淋巴细胞、制备方法及其应用 Download PDF

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WO2022222845A1
WO2022222845A1 PCT/CN2022/086903 CN2022086903W WO2022222845A1 WO 2022222845 A1 WO2022222845 A1 WO 2022222845A1 CN 2022086903 W CN2022086903 W CN 2022086903W WO 2022222845 A1 WO2022222845 A1 WO 2022222845A1
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cells
mixed
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李建强
刘莹
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河北森朗生物科技有限公司
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Definitions

  • the invention belongs to the field of immunotherapy, and particularly relates to mixed innate lymphocytes, a preparation method and applications thereof.
  • Innate lymphocytes include natural killer (NK) cells, ⁇ T cells, cytokine-induced killer (CIK) cells, etc., and have innate immune defense functions.
  • ⁇ T cells are T cells that perform innate immune functions, and their TCR consists of ⁇ and ⁇ chains. Such T cells are mainly distributed in mucosa and subcutaneous tissues such as intestinal respiratory tract and urogenital tract, and only account for 0.5%-1% of CD3+ T cells in peripheral blood. Its TCR lacks diversity and can directly recognize some complete polypeptide antigens.
  • the types of antigens recognized by ⁇ T cells are limited: 1 HSP; 2 lipid antigens extracted from CD1 molecules on the surface of infected cells; 3 certain viral proteins or viral proteins expressed on the surface of infected cells; 4 phosphorylated antigens in bacterial lysates.
  • ⁇ T cells are immune cells that can not only kill cancer cells, tumor stem cells, but also recognize cancer antigens. Therefore, it is mainly used to kill cancer cells and assist DC cells to recognize and discover cancer cell antigens, and then kill or transmit these antigens to other cells. At the same time, ⁇ T cells are mainly distributed in the skin and mucosal tissues, so they have outstanding therapeutic effects on mucosal cancers, such as those in the digestive tract, respiratory tract, and reproductive system.
  • ⁇ T cells mature mainly in the thymus and generate the ⁇ T cell receptor (TCR) through V(D)J gene recombination. Differentiation from a common lymphoid precursor (CLP) to T cell lines expressing ⁇ receptors and ⁇ receptors through specific gene rearrangements. ⁇ T cells are not easily affected by the loss of antigen processing and presentation, so ⁇ T cells have high potential application value in clinical tumor immunotherapy. ⁇ T cells play an important role in tumor immune surveillance and antitumor immune responses.
  • CLP common lymphoid precursor
  • ⁇ T cells are amplified from peripheral blood mononuclear cells, but the amplification fold is low, and the cell purity and cell quantity are not high. It is difficult for the expanded ⁇ T cells to meet the clinical needs. Even if the single ⁇ T cells expanded by optimizing various induction conditions and expansion methods have been applied in corresponding immune diseases and tumor diseases, they have not reached the desired level after application. people's desired effect.
  • NK cell immunotherapy is one of the most promising tumor treatment methods at present. It can be amplified or transformed in vitro and then infused into the patient to kill tumor cells. other illnesses.
  • NK cell immunotherapy has received more and more attention.
  • NK cells account for 5-15% of human peripheral blood lymphocytes, and their phenotype is generally defined as CD3-CD56+.
  • NK cells can be further subdivided into two main subgroups: CD56highCD16- cells with immunomodulatory functions and cells with Toxic activity of CD56dimCD16+ cells.
  • NK cells play an important immune surveillance function in the early immune response against viral infection and anti-tumor. NK cells can directly and rapidly exert cytotoxic activity without recognizing tumor-specific antigens. It is particularly important that NK cells can effectively eliminate tumor stem cell-like cells in the body and inhibit tumor growth and metastasis.
  • NK cells NK cells, ⁇ T cells, CIK cells, etc.
  • CIK cells CIK cells
  • PBMCs from umbilical cord blood or peripheral blood are sorted by CD4 and CD8 to obtain CD4-CD8- cells, and cultured to prepare mixed innate lymphocytes (Mixed Innate lymphocytes, MILS) containing NK cells and ⁇ T cells.
  • MILS Mixed innate lymphocytes
  • the purpose of the present invention is to provide a method that is practical, efficient, low-cost, simple in technology, and can massively expand NK cells and ⁇ T cells at the same time.
  • the present invention provides a cytokine combination comprising OK432, IFN- ⁇ , IL-2, IL-15, and IL-21.
  • the present invention provides an activation medium comprising the following combination of cytokines: OK432, IFN- ⁇ , IL-2, IL-15, IL-21.
  • the final concentration of OK432 is 100ng/ml
  • the final concentration of IFN- ⁇ is 1000IU/ml
  • the final concentration of IL-2 is 1000IU/ml
  • the final concentration of IL-15 is 100ng/ml
  • the final concentration of IL-21 is 100ng/ml .
  • the activation medium also includes: KBM581, autologous plasma or human AB serum.
  • the proportion of autologous plasma or human AB serum is 10%.
  • the present invention provides a method for preparing mixed innate lymphocytes (MILS), the preparation method comprising the steps of:
  • step 2) MILS cell expansion: The cells obtained in step 2) are cultured using expansion medium.
  • the expansion medium includes KBM581, IL-2, autologous plasma or human AB serum.
  • the final concentration of IL-2 is 1000 IU/ml, and the proportion of autologous plasma or human AB serum is 5%.
  • step 1) comprises the following steps:
  • the buffy coat cells are isolated from blood; more preferably, the blood includes umbilical cord blood or peripheral blood.
  • the step of separating buffy coat cells includes: centrifuging the blood to obtain autologous plasma; resuspending the centrifuged blood cell pellet with physiological saline to obtain diluted blood, adding a mononuclear cell separation solution, and the volume ratio of the diluted blood to the mononuclear cell separation solution.
  • the buffy coat cells were collected by centrifugation at a ratio of 2:1 and washed twice with normal saline.
  • the step of isolating buffy coat cells comprises:
  • the step of lysing red blood cells includes: adding red blood cell lysing solution to lyse the buffy coat cells obtained in step a), discarding the supernatant, adding normal saline to resuspend and wash, centrifuging and discarding the supernatant to obtain mononuclear cells, and resuspending the cells with normal saline.
  • the step of lysing red blood cells includes: adding 1 ⁇ red blood cell lysing solution for lysing for 10 min, centrifuging at 2000 rpm for 5 min, discarding the supernatant, adding normal saline for resuspending and washing, centrifuging at 2000 rpm for 5 min, and discarding the supernatant. That is, mononuclear cells, resuspend the cells with 5ml of normal saline, count, and take samples to detect the ratio of CD3, CD4, and CD8.
  • CD4-/CD8-T cell sorting include:
  • iiiiiii) Prepare the LD magnetic separation column, rinse the column with buffer, add the cell suspension obtained in iiii) to sort the cells, and then add the buffer to wash the column, the sorted negative cells are CD4-/CD8- cells .
  • the sorting buffer is phosphate buffered saline containing 0.5% HAS.
  • step ii) The specific scheme of step ii) is as follows: centrifuge the mononuclear cell suspension at 2000 rpm/min for 5 min, discard the supernatant, and add 80 ⁇ l sorting buffer, 20 ⁇ l CD4 MicroBeads human and 20 ⁇ l CD8 MicroBeads human per 1 ⁇ 10 7 cells to mix. Incubate at 4°C for 15 min, shaking every 5 min.
  • step of MILS cell induction in step 2) also includes antibody coating, and the antibody coating includes:
  • the primary antibody is anti-human TCR ⁇ / ⁇ .
  • the secondary antibody is Goat Anti-mouse IgG.
  • the secondary antibody is coated; AffiniPure F(ab ⁇ )2 Fragment Goat Anti-mouse IgG, Fcy Fragment Specific working concentration is 10 ⁇ g/ml, each bottle of T25 cell culture flask is 3ml of antibody dilution, incubated at 37°C for 1h, discarded Antibody, add DPBS to wash the T25 cell culture flask once;
  • the primary antibody is coated: LEAF TM Purified anti-human TCR ⁇ / ⁇ working concentration is 100ng/ml, 3ml antibody dilution solution per flask of T25 cell culture flask, incubated at room temperature for 1h, discard the primary antibody, add DPBS to soak the T25 cell culture Spare bottle.
  • MILS cell induction CD4-/CD8-T cells were cultured for 6-8 days using the previously described activation medium.
  • the MILS cell expansion steps are as follows: from the 7th to 9th day of culture, the activation medium is gradually replaced with the expansion medium by means of half-change medium.
  • the present invention provides that MILS obtained by the aforementioned preparation method are mainly ⁇ T-NK mixed lymphocytes.
  • the subgroups of ⁇ T cells are mainly composed of three subgroups, v ⁇ 1, v ⁇ 2 and v ⁇ 1 - v ⁇ 2-, of which v ⁇ 1 has the highest proportion in umbilical cord blood, followed by v ⁇ 1 - v ⁇ 2- , and v ⁇ 2 has the lowest proportion, with the proportions of v ⁇ 1 and v ⁇ 1- respectively .
  • the proportions of v ⁇ 2 - and v ⁇ 2 were 60 ⁇ 10%, 35 ⁇ 10% and 3 ⁇ 2%, respectively.
  • the proportion of v ⁇ 2 in peripheral blood ⁇ T cells was the highest, followed by v ⁇ 1 - v ⁇ 2 - , and the proportion of v ⁇ 1 was the lowest, the proportions of v ⁇ 2, v ⁇ 1 - v ⁇ 2 - and v ⁇ 1 were 75 ⁇ 10%, 15 ⁇ 5%, 10 ⁇ 5% .
  • the present invention provides a kit or a medicament comprising the aforementioned MILS.
  • the present invention provides a method for preventing and/or treating a tumor or autoimmune disease in a subject, the method comprising administering to a subject in need thereof an effective amount of the foregoing The MILS described, or the drug described earlier.
  • the route of administration of the aforementioned MILS, or the aforementioned pharmaceutical composition of the present invention may be parenterally, intramuscularly, subcutaneously, intradermally, intraperitoneally, intranasally, intravenously, via afferent lymphatics, or by treating Other ways of treating the tumor and the condition of the patient are appropriate.
  • MILS hereinbefore described, or the pharmaceutical compositions hereinbefore described, are administered in an amount that is "effective" to result in the desired therapeutic effect.
  • effective doses generally lie in the range of 105 to 1011 cells, including allogeneic lymphocytes and (if present) from the patient to be treated Tumor cells and other cells.
  • about 106 to 1010 cells are used; more preferably, about 1 ⁇ 107 to 2 ⁇ 109 cells are used; more preferably, about 5 ⁇ 107 to 2 x 109 cells; more preferably, about 1 x 108 to 1 x 109 cells are used.
  • Each of the multiple doses also falls within the defined range of an effective amount when used in admixture to achieve the desired effect Inside.
  • the MILS described above, or the pharmaceutical composition described above, of the present invention may be administered after, before, or concurrently with other treatments in the patient involved.
  • the patient may be previously or concurrently treated with chemotherapy, radiotherapy or other forms of immunotherapy and adoptive metastases.
  • the timing of administration of the aforementioned MILS, or the aforementioned pharmaceutical compositions of the present invention is at the discretion of the clinician and depends on the clinical condition of the patient, the purpose of the treatment, and other concurrently administered treatments.
  • the present invention provides an application of the aforementioned MILS, the application comprising any one of the following:
  • the tumors of the present invention include non-solid tumors and solid tumors. Treatable tumors include tumors that are not vascularized or substantially not vascularized, as well as tumors that are vascularized. Tumors may include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may include solid tumors. Tumor types treated with the MILS of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemic or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and pediatric tumors/cancers.
  • Hematological cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastoid, promyelocytic, myelomonocytic type) , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (myeloid) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such
  • Solid tumors are abnormal masses of tissue that typically do not contain cysts or areas of fluid. Different types of solid tumors are named after the cell type that forms them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer, and any other cancer now known or later discovered (see, eg, Rosenberg (1996) " Annals of Medicine 47:481-491, the entire contents of which are incorporated herein by reference).
  • Autoimmune diseases of the present invention include, but are not limited to, acquired immunodeficiency syndrome (AIDS), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, Autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac disease sprue) - dermatitis herpetiformis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), scarring pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's Enn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, idiopathic mixed cryoglobulinemia, fibromyalgia-
  • the targeting molecules of the chimeric antigen receptors of the present invention include tumor surface antigens and tumor-associated antigens.
  • targeting molecules for chimeric antigen receptors of the present invention include, but are not limited to, CD5, CD19, CD123, CD22; CD30, CD171, CS1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variants III.
  • Ganglioside G2 Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), TNF receptor family member B cell maturation antigen (BCMA), Tn antigen ((Tn Ag) or (GalNAc ⁇ -Ser/Thr)), prostate specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine Kinase 3 (FLT3), tumor-associated glycoprotein 72 (TAG72), CD38, CD44v6, glycosylated CD43 epitope expressed on acute leukemia or lymphoma but not on hematopoietic progenitors, expressed on non-hematopoietic cancers Glycosylated CD43 epitope, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM), B7H3 (CD276), KIT (
  • the medicament of the present invention also includes a pharmaceutically acceptable carrier and/or excipient.
  • the medicine also includes modified mixed innate lymphocytes obtained after the MILS prepared in the present invention is modified.
  • Modified MILS comprise insertions/deletions of one or more of the following genes: targeting patterns, receptors, signaling molecules, transcription factors, drug target candidates, immune response modulation and modulation, or inhibition of the MILS or derivatives thereof Proteins for cell transplantation, transport, homing, viability, self-renewal, persistence and/or survival.
  • modified MILS achieves functional improvements in:
  • the medicament of the present invention also includes other active pharmaceutical agents, and other active pharmaceutical agents include other immune cells, other anti-tumor drugs, and other drugs for treating autoimmune diseases.
  • the present invention provides a method for preparing mixed innate lymphocytes comprising ⁇ T cells and NK cells, the preparation method is simple to operate, and a large number of expanded ⁇ T cells and NK cells can be obtained after induction and expansion.
  • the preparation method of the present invention adopts the same induction and culture conditions, and the ⁇ T cells and NK cells can stimulate each other to stimulate growth and expansion. Compared with the preparation of ⁇ T cells alone, the ⁇ T cells in the mixed innate lymphocytes obtained by the preparation method of the present invention are in ⁇ T cells. Cell expansion efficiency is greatly increased.
  • Figure 1 shows a graph of cell expansion curves
  • Figure 2 shows a graph of the ratio change of NK and ⁇ T cells
  • FIG. 3 shows the results of the effect of antibody secondary stimulation on cell expansion efficiency
  • Figure 4 shows a graph of expansion curves of MILS cells
  • Figure 5 shows the change in the proportion of each cell in MILS during the culture process
  • Figure 6 shows a statistical graph of the proportion of ⁇ T cell subsets
  • Figure 7 shows the results of the killing effect of MILS on AML primary cells
  • Figure 8 shows the results of the killing effect of MILS on K562 cells.
  • the centrifuged blood cell pellet Resuspend the centrifuged blood cell pellet with 2-3 times the volume (the volume of blood) of normal saline, and slowly add the diluted blood to the human mononuclear cell separation solution (purchased from Dongfang Huahui, product number: 25810). In the upper layer, the volume ratio of diluted blood and human mononuclear cell isolation solution is 2:1. Then increase 7 to 4, 2000 rpm, centrifuge for 20 min, collect buffy coat cells, wash twice with normal saline.
  • LEAF TM Purified anti-human TCR ⁇ / ⁇ (concentration 1mg/ml) working concentration is 100ng/ml, 3ml antibody dilution solution per bottle of T25 cell culture flask, incubate at room temperature for 1h, discard the primary antibody, add DPBS to soak T25 cell culture flask for use.
  • the sorted CD4-CD8- cells were seeded into antibody-coated T25 flasks and cultured in a 37°C, 5% CO 2 incubator.
  • the cell culture medium used in the first 6-8 days of cell culture is activation medium (SLAM): KBM581+OK432(100ng/ml)+IFN- ⁇ (1000IU/ml)+IL-2(1000IU/ml)+ IL-15 (100ng/ml) + IL-21 (100ng/ml) + 10% autologous plasma (inactivated); from the 7th to 9th day of culture, the medium was gradually changed to expansion by half-change medium Medium: KBM581+IL-2 (1000IU/ml)+5% autologous plasma (inactivated).
  • CD4-CD8- cells Different numbers of CD4-CD8- cells (1.5 ⁇ 10 6 , 3 ⁇ 10 6 , 6 ⁇ 10 6 , 9 ⁇ 10 6 ) were seeded into T25 flasks coated with antibodies, and cells cultured at different cell plating densities Amplification curves are shown in Figure 1. Cells in 6 ⁇ 10 6 /T25 flasks grew the fastest.
  • the sorted CD4-CD8- cells (6 ⁇ 10 6 ) were inoculated into T25 flasks coated with antibodies, the medium used was activation medium (SLAM), and cultured at 37°C, 5% CO 2 Cultivated in the box.
  • the cells cultured on days 6 to 8 were seeded into culture flasks re-coated with anti-human TCR ⁇ / ⁇ antibodies, and the cell expansion curve was shown in Figure 3.
  • the results in Figure 3 show that secondary stimulation with antibodies can significantly improve the expansion efficiency and growth rate of cells, and increase the number of cells.
  • the sorted CD4-CD8- cells (6 ⁇ 10 6 ) were inoculated into T25 flasks coated with antibodies, the medium used was activation medium (SLAM), and cultured at 37°C, 5% CO 2 Cultivated in the box.
  • the cells were seeded into culture flasks re-coated with anti-human TCR ⁇ / ⁇ antibodies on days 6 to 8 for secondary stimulation, and the cells were harvested on days 25-30, and the harvested cells were subjected to in vitro functional assays.
  • the expansion curve of MILS cells is shown in Figure 4. MILS cells were expanded from 6E6 to 1.84E10 within 30 days of culture, and the expansion was more than 3000 times. The changes in the proportion of cells in MILS during the culture process are shown in Figure 5.
  • the MILS cells were initially mainly composed of NK cells, CD3-CD56- cells and a small amount of ⁇ T cells. Cells and NK cells continue to expand.
  • the proportion of ⁇ T cell subsets is shown in Figure 6.
  • the ⁇ T of umbilical cord blood-derived MILS (CB-MILS) is mainly v ⁇ 1-type ⁇ T and some v ⁇ 1 - v ⁇ 2 - type ⁇ T, and the proportions of v ⁇ 1, v ⁇ 1 - v ⁇ 2- , and v ⁇ 2 are respectively 63.12 ⁇ 8.5%, 34.44 ⁇ 7.53%, 2.44 ⁇ 1.63%.
  • the ⁇ T of peripheral blood-derived MILS was mainly v ⁇ 2 type ⁇ T, and the proportions of v ⁇ 2, v ⁇ 1 - v ⁇ 2 - and v ⁇ 1 were 75.87 ⁇ 4.37%, 14.43 ⁇ 2.14% and 9.59 ⁇ 5.56%, respectively.
  • PBMCs peripheral blood of acute myeloid leukemia (AML), and PBMCs were stained with CFSE as target cells.
  • MILS cells were added to the target cells according to different effect-to-target ratios (2:1, 10:1, 50:1) and mixed. After 4 hours of incubation, cell killing was detected by flow cytometry. MILS had a killing effect on AML primary cells (Fig. 7), the killing rate was 8.2% with an effector-target ratio of 2:1, 28.13% with 10:1, and 65.36% with 50:1.
  • K562 cells stained with CFSE were used as target cells (stained at a ratio of 2 ⁇ 10 6 cells to 100 ⁇ l of 2 ⁇ M CFSE working solution). 1 ⁇ 10 5 cells/100 ⁇ l K562 cells per well were added to a 96-well plate. Then, an appropriate amount of MILS effector cells was added to the target cells according to the effector-target ratio of 1:1, mixed and incubated for 4 hours, and the cell killing ratio was detected by flow cytometry. MILS had a killing effect on K562 cells (Fig. 8), and the killing rate was 80.0%.

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Abstract

本发明公开了混合的淋巴细胞、制备方法及其应用。本发明利用同一培养体系同时诱导扩增γδT细胞和NK细胞。

Description

混合的固有淋巴细胞、制备方法及其应用 技术领域
本发明属于免疫治疗领域，具体涉及混合的固有淋巴细胞、制备方法及其应用。
背景技术
固有淋巴细胞包括自然杀伤(NK)细胞、γδT细胞、细胞因子诱导的杀伤(CIK)细胞等，具有天然免疫防御功能。
γδT细胞是执行固有免疫功能的T细胞，其TCR由γ和δ链组成。此类T细胞主要分布于肠道呼吸道以及泌尿生殖道等黏膜和皮下组织，在外周血中只占CD3+T细胞的0.5％-1％。其TCR缺乏多样性，可直接识别某些完整的多肽抗原。γδT细胞所识别的抗原种类有限：①HSP；②感染细胞表面CD1分子提成的脂类抗原；③某些病毒蛋白或表达于感染细胞表面的病毒蛋白；④细菌裂解产物中的磷酸化抗原。
γδT细胞是一种既能杀伤癌细胞，肿瘤干细胞，又能识别癌抗原的免疫细胞，它的杀伤性较强，但肿瘤干细胞杀伤不如NK细胞。因此，它主要用于杀伤癌细胞与协助DC细胞识别发现癌细胞抗原，然后将这些抗原进行杀伤或是传递给其他细胞。同时，γδT细胞主要分布于皮肤和黏膜组织上，因此对于黏膜方面的癌症治疗效果突出，比如消化道、呼吸道、生殖系统方面的癌症效果显著。
γδT细胞主要在胸腺中发育成熟，通过V(D)J基因重组产生γδT细胞受体(TCR)。通过特异性的基因重排，从一个共同淋巴细胞前体(common lymphoidprecursor,CLP)分化为表达αβ受体和γδ受体的T细胞系。γδT细胞不易受抗原加工和呈递缺失的影响，因此γδT细胞具有很高的临床肿瘤免疫治疗潜在应用价值。γδT细胞在肿瘤免疫监视和抗肿瘤免疫反应中发挥重要作用。
由于γδT细胞在外周血中的含量极低，大大限制了γδT细胞作为过继免疫细胞在临床上的应用。目前从外周血单个核细胞扩增γδT细胞，扩增倍数低，细胞纯度及细胞量不高。扩增出的γδT细胞很难满足临床需求，即使通过优化各种诱导条件及扩增方法扩增出的单一γδT细胞在相应的免疫性疾病和肿瘤疾病上有所应用，但是应用后并未达到人们理想中的效果。
细胞免疫治疗是目前最具有前景的肿瘤治疗方式之一，通过体外扩增或改造后回输到病人体内达到杀伤肿瘤细胞的目的或者通过活化机体免疫系统，增强肿瘤患者自身免疫功能从而抵抗肿瘤或其他疾病。目前，NK细胞免疫治疗受到越来越多的重视。NK细胞占人外周血淋巴细胞的5-15％，一般定义其表型为CD3-CD56+，NK细胞又可以进一步细分为两个主要的亚群：具有免疫调节功能的CD56highCD16-细胞和具有细胞毒活性的CD56dimCD16+细胞。NK细胞在抗病毒感染和抗肿瘤的早期免疫反应中发挥着重要的免疫监视功能，NK细胞不需要识别肿瘤特异性抗原，便可直接、快速的发挥细胞毒活性。特别重要的是NK细胞能够有效地清除机体内的肿瘤干细胞样细胞，抑制肿瘤的生长和转移。
目前大部分固有免疫应答细胞治疗产品多为单一种类细胞制剂(NK细胞，γδT细胞，CIK细胞等)对肿瘤的治疗较为局限。或者是将单一种类细胞培养后混合一起的多细胞免疫制剂，操作复杂。
本申请将来源于脐带血或者外周血中的PBMC通过CD4和CD8分选获得CD4-CD8-的细胞，通过培养制备成含有NK细胞和γδT细胞的混合的固有淋巴细胞(Mixed Innate lymphocytes，MILS)，通过将NK细胞和γδT细胞同时诱导培养使得γδT细胞扩增效率大大提高。
发明内容
本发明的目的在于提供一种实用、高效、成本低、技术简单、可同时大量扩增NK细胞和γδT细胞的方法。
为了实现上述目的，本发明采用了如下技术方案：
根据本发明的一个方面，本发明提供了一种细胞因子组合，所述细胞因子组合包括OK432、IFN-γ、IL-2、IL-15、IL-21。
根据本发明的另一个方面，本发明提供了一种活化培养基，所述活化培养基包括以下细胞因子组合：OK432、IFN-γ、IL-2、IL-15、IL-21。
优选地，OK432终浓度为100ng/ml、IFN-γ终浓度为1000IU/ml、IL-2终浓度为1000IU/ml、IL-15终浓度为100ng/ml、IL-21终浓度为100ng/ml。
进一步，所述活化培养基还包括：KBM581、自体血浆或人AB血清。
优选地，自体血浆或人AB血清比例为10％。
根据本发明的又一个方面，本发明提供了一种混合的固有淋巴细胞(MILS)的制备方法，所述制备方法包括如下步骤：
1)CD4-/CD8-T细胞分选；
2)MILS细胞诱导：使用前面所述的活化培养基培养CD4-/CD8-T细胞；
3)MILS细胞扩增：使用扩增培养基培养经步骤2)获得的细胞。
进一步，所述扩增培养基包括KBM581、IL-2、自体血浆或人AB血清。
优选地，所述IL-2终浓度为1000IU/ml、自体血浆或人AB血清比例为5％。
进一步，所述步骤1)包括以下步骤：
a)分离白膜层细胞；
b)红细胞裂解；
c)CD4-/CD8-T细胞分选。
进一步，从血液中分离白膜层细胞；更优选地，所述血液包括脐带血或外周血。
进一步，分离白膜层细胞的步骤包括：将血液离心获得自体血浆；将离心后的血细胞沉淀用生理盐水重悬获得稀释血，加入单个核细胞分离液，稀释血和单个核细胞分离液体积比为2:1，然后离心收集白膜层细胞，生理盐水清洗两次。
在本发明的具体实施方案中，分离白膜层细胞的步骤包括：
将血液到入无菌50ml离心管，升9降7，750g离心15min。收集上层血浆于50ml离心管中，56℃孵育30min，4℃放置30min，1000g离心10min，收集上清4℃保存备用。
将离心后的血细胞沉淀用2-3倍体积(血液的体积)的生理盐水重悬混匀，将稀释血缓缓加入到人单个核细胞分离液的上层，稀释血和人单个核细胞分离液体积比为2:1。然后升7降4，2000rpm，离心20min，收集白膜层细胞，生理盐水清洗两次。
进一步，红细胞裂解的步骤包括：加入红细胞裂解液裂解步骤a)获得的白膜层细胞，弃上清，加入生理盐水重悬清洗，离心弃上清获得单个核细胞，用生理盐水重悬细胞。
在本发明的具体实施方案中，红细胞裂解的步骤包括：加入1x红细胞裂解液裂解10min，2000rpm，5min离心，弃去上清，加入生理盐水重悬清洗，2000rpm，5min离心，弃去上清。即为单个核细胞，用5ml生理盐水重悬细胞，计数，并取样检测CD3、CD4、CD8的比例。
进一步，CD4-/CD8-T细胞分选的步骤包括：
i)配置分选缓冲液；
ii)将单个核细胞悬液离心，弃上清，加入分选缓冲液、CD4 MicroBeads human和CD8 MicroBeads human混匀，低温孵育；
iii)加入分选缓冲液清洗细胞，离心弃上清；
iiii)加入分选缓冲液重悬细胞；
iiiii)准备LD磁力分选柱，用缓冲液润洗柱子，加入经iiii)获得的细胞悬液分选细胞，再加入缓冲液清洗柱子，分选出的阴性细胞即为CD4-/CD8-细胞。
进一步，所述分选缓冲液是含有0.5％HAS的磷酸盐缓冲液。
步骤ii)的具体方案如下：将单个核细胞悬液2000rpm/min，离心5min，弃上清，按每1×10 7个细胞加入80μl分选缓冲液、20μl CD4 MicroBeads human和20μl CD8 MicroBeads human混匀，4℃孵育15min，每5min摇匀一次。
进一步，步骤2)中MILS细胞诱导的步骤还包括抗体包被，抗体包被包括：
二抗包被和一抗包被；
优选地，一抗是anti-human TCRγ/δ。
优选地，二抗是Goat Anti-mouse lgG。
优选地，二抗包被；AffiniPure F(ab`)2Fragment Goat Anti-mouse lgG，Fcy Fragment Specific工作浓度为10μg/ml，T25细胞培养瓶每瓶3ml抗体稀释液，37℃孵育1h，弃掉二抗，加入DPBS清洗T25细胞培养瓶一次；
优选地，一抗包被：LEAF TM Purified anti-human TCRγ/δ工作浓度为100ng/ml，T25细胞培养瓶每瓶3ml抗体稀释液，室温孵育1h，弃掉一抗，加入DPBS浸泡T25细胞培养瓶备用。
进一步，MILS细胞诱导：使用前面所述的活化培养基培养CD4-/CD8-T细胞6-8天。
进一步，MILS细胞扩增步骤如下：从培养的第7-9天开始通过半换液的方式将活化培养基逐步换为扩增培养基。
作为扩增的一种方式：从培养的第7-9天开始，将培养6-8天的细胞接种到不包被anti-human TCRγ/δ抗体的培养瓶中，通过半换液的方式将活化培养基逐步换为扩增培养基。
作为扩增的另一种更优选的方式：从培养的第7-9天开始，将培养6-8天的细胞接种到重新包被anti-human TCRγ/δ抗体的培养瓶中，通过半换液的方式将活化培养基逐步换为扩增培养基。
根据本发明的又一个方面，本发明提供了利用前面所述的制备方法获得的MILS主要为γδT-NK混合淋巴细胞。其中γδT细胞的亚群主要由vδ1，vδ2和vδ1 -vδ2 -三个亚群组成，其中脐带血中vδ1占比最高，其次是vδ1 -vδ2 -，vδ2占比最低，比例分别为vδ1、vδ1 -vδ2 -、vδ2比例分别为60±10％、35±10％、3±2％。外周血γδT细胞中vδ2占比最高，其次是vδ1 -vδ2 -，vδ1占比最低，比例分别为vδ2、vδ1 -vδ2 -、vδ1比例分别为75±10％、15±5％、10±5％。
根据本发明的又一个方面，本发明提供了一种试剂盒或药物，所述试剂盒或药物包括前面所述的MILS。
根据本发明的又一个方面，本发明提供了用于在受试者中预防和/或治疗肿瘤或自身免疫性疾病的方法，所述方法包括向有此需要的受试者施用有效量的前面所述的MILS，或前面所述的药物。
本发明前面所述的MILS，或前面所述的药物组合物的施用途径可以是胃肠外、肌肉内、皮下、皮内、腹膜内、鼻内、静脉内、经过传入淋巴管或通过对待治疗肿瘤和患者的病情适当的其它途径。
本发明前面所述的MILS，或前面所述的药物组合物施用的剂量为导致所需治疗效应的“有效的”量。对于本发明前面所述的MILS，或前面所述的药物组合物，有效的剂量一般位于10 5到10 11个细胞的范围，包括同种异体淋巴细胞和(如果存在的话〕来自待治疗患者的肿瘤细胞和其它细胞。优选地，使用大约10 6到10 10个细胞；更为优选地，使用大约1×10 7到2×10 9个细胞；更为优选地，使用大约5×10 7到2×10 9个细胞；更为优选地，使用大约1×10 8到1×10 9个细胞。当混合使用以达到所需效应时，多重剂量的每一种剂量也位于有效量的定义范围内。
本发明前面所述的MILS，或前面所述的药物组合物可以在涉及患者中其它治疗之后、之前或同时施用。例如，该患者可以在之前或同时通过化疗、放疗或其它形式的免疫治疗和继承性转移加以治疗。
本发明前面所述的MILS，或前面所述的药物组合物施用时间的选择依据临床医生的判断，并且依赖于病人的临床病情、治疗的目的和同时施用的其它治疗。
根据本发明的又一个方面，本发明提供了前面所述的MILS的应用，所述应用包括以下任一项：
1)在制备治疗肿瘤或自身免疫性疾病的药物中的应用；
2)在制备表达嵌合抗原受体的免疫细胞中的应用；
3)在制备前面所述的试剂盒中的应用。
本发明的肿瘤包括非实体瘤、实体瘤。可治疗的肿瘤包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。肿瘤可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的MILS治疗的肿瘤类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌、卵巢癌以及任何其他现在已知或以后发现的癌症(参见，例如Rosenberg(1996)《医学年鉴》47：481-491，其全部内容通过引用并入本文)。
本发明的自身免疫性疾病包括但不限于，获得性免疫缺陷综合征(AIDS)、斑秃、强直性脊柱炎、抗磷脂综合征、自身免疫性阿狄森氏病、自身免疫性溶血性贫血、自身免疫性肝炎、自身免疫性内耳疾病(AIED)、自身免疫性淋巴细胞增生综合征(ALPS)、自身免疫性血小板减少性紫癜(ATP)、贝切特氏病、心肌病、乳糜泻(celiac sprue)-疱疹样皮炎、慢性疲劳免疫功能障碍综合征(CFIDS)、慢性炎性脱髓鞘性多发性神经病(CIPD)、疤痕性类天疱疮、冷凝集素疾病、CREST综合征、克罗恩病、德戈斯氏病、青少年型皮肌炎(dermatomyositis-juvenile)、盘状狼疮、特发性混合型冷沉淀球蛋白血症、纤维肌痛-纤维肌炎、格雷夫斯氏病、格-巴二氏综合征、桥本氏甲状腺炎、特发性肺纤维变性、特发性血小板减少性紫癜(ITP)、IgA肾病、胰岛素依赖型糖尿病、青少年慢性关节炎(斯提耳氏病)、青少年型类风湿性关节炎、美尼尔氏病、混合结缔组织病、多发性硬化、重症肌无力、恶性贫血(pernacious anemia)、结节性多动炎症、多软骨炎、多腺体综合征、风湿性多肌痛、多发性肌炎和皮肌炎、原发性无丙种球蛋白血症、原发性胆汁性肝硬变、牛皮癣、牛皮癣性关节炎、雷诺氏现象、莱特尔综合征、风湿热、类风湿性关节炎、结节病、硬皮病(进行性全身性硬化症(PSS)，也称为全身性硬化症(SS))、斯耶格伦氏综合征、僵体综合征、系统性红斑狼疮、高安氏动脉炎、 颞动脉炎/巨细胞动脉炎、溃疡性结肠炎、眼葡萄膜炎、白斑病、韦格纳氏肉芽肿病和其任意组合。
本发明的嵌合抗原受体的靶向分子包括肿瘤表面抗原、肿瘤相关抗原。
本发明的嵌合抗原受体的靶向分子的实例包括但不限于CD5、CD19、CD123、CD22；CD30、CD171、CS1、C型凝集素样分子-1、CD33、表皮生长因子受体变体III、神经节苷脂G2、神经节苷脂GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer)、TNF受体家族成员B细胞成熟抗原(BCMA)、Tn抗原((Tn Ag)或(GalNAcα-Ser/Thr))、前列腺特异性膜抗原(PSMA)、受体酪氨酸激酶样孤儿受体1(ROR1)、Fms样酪氨酸激酶3(FLT3)、肿瘤相关糖蛋白72(TAG72)、CD38、CD44v6、在急性白血病或淋巴瘤上表达但在造血祖细胞上不表达的糖基化CD43表位、在非造血系统癌症上表达的糖基化CD43表位、癌胚抗原(CEA)；上皮细胞粘附分子(EPCAM)、B7H3(CD276)、KIT(CD117)、白介素13受体亚基α-2(IL-13Ra2或CD213A2)、间皮素、白介素11受体α(IL-11Ra)、前列腺干细胞抗原(PSCA)、蛋白酶丝氨酸21(睾蛋白或PRSS21)、血管内皮生长因子受体2(VEGFR2)、路易斯(Y)抗原、CD24；血小板衍生的生长因子受体β(PDGFR-β)、阶段特异性胚胎抗原4(SSEA-4)、CD20、叶酸受体α(FRa或FR1)、叶酸受体β(FRb)、受体酪氨酸蛋白激酶ERBB2(Her2/neu)、细胞表面相关粘蛋白1(MUC1)、表皮生长因子受体(EGFR)、神经细胞粘附分子(NCAM)、前列腺酶、前列腺酸性磷酸酶(PAP)、突变的延伸因子2(ELF2M)、肝配蛋白B2、成纤维细胞激活蛋白α(FAP)、胰岛素样生长因子1受体(IGF-I受体)、碳酸酐酶IX(CAIX)、糖蛋白100(gp100)、酪氨酸酶、肝配蛋白A型受体2(EphA2)、唾液酸化的路易斯粘附分子(sLe)、转谷氨酰胺酶5(TGS5)、高分子量黑色素瘤相关抗原(HMWMAA)、O-乙酰基GD2神经节苷脂(OAcGD2)、肿瘤内皮标志物1(TEM1/CD248)、肿瘤内皮标志物7相关的(TEM7R)、紧密连接蛋白6(CLDN6)、甲状腺刺激激素受体(TSHR)、G蛋白偶联受体C类5组，成员D(GPRC5D)、X染色体开放阅读框61(CXORF61)、CD97、CD179a、间变性淋巴瘤激酶(ALK)、聚唾液酸、胎盘特异性1(PLAC1)、globoH糖神经酰胺的六糖部分(GloboH)、乳腺分化抗原(NY-BR-1)、尿溶蛋白2(UPK2)、甲型肝炎病毒细胞受体1(HAVCR1)、肾上腺素受体β3(ADRB3)、泛连接蛋白3(PANX3)、G蛋白偶联受体20(GPR20)、 淋巴细胞抗原6复合物，基因座K 9(LY6K)、嗅觉受体51E2(OR51E2)、TCRγ交替阅读框蛋白(TARP)、肾母细胞瘤蛋白(Wilms tumor protein，WT1)、癌症/睾丸抗原1(NY-ESO-1)、癌症/睾丸抗原2(LAGE-1a)、黑色素瘤相关抗原1(MAGE-A1)、位于12p染色体上的ETS易位变异基因6(ETV6-AML)、精子蛋白17(SPA17)、X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)、黑色素瘤睾丸抗原-1(MAD-CT-1)、黑色素瘤睾丸抗原2(MAD-CT-2)、Fos相关抗原1、肿瘤蛋白p53(p53)、p53突变体、前列腺特异素(prostein)、生存素、端粒酶、前列腺癌肿瘤抗原-1(PCT A-1或半乳凝素8)、T细胞识别的黑色素瘤抗原1(MelanA或MARTI)、大鼠肉瘤(Ras)突变体、人端粒酶逆转录酶(hTERT)、肉瘤易位断点、黑色素瘤细胞凋亡抑制剂(ML-IAP)、ERG(跨膜蛋白酶，丝氨酸2(TMPRSS2)ETS融合基因)、N-乙酰氨基葡萄糖转移酶V(NA17)、配对盒蛋白Pax-3(PAX3)、雄激素受体、细胞周期蛋白B1、v-myc禽骨髓细胞瘤病毒癌基因神经母细胞瘤衍生同源物(MYCN)、Ras同源家族成员C(RhoC)、酪氨酸酶相关蛋白2(TRP-2)、细胞色素P450 1B 1(CYP1B 1)、CCCTC结合因子(锌指蛋白)样(BORIS或印记位点调节因子样蛋白)、T细胞识别的鳞状细胞癌抗原3(SART3)、配对盒蛋白Pax-5(PAX5)、前顶体素结合蛋白sp32(OY-TES1)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)、激酶锚蛋白4(AKAP-4)、滑膜肉瘤X断点2(SSX2)、晚期糖基化终产物受体(RAGE-1)、肾泛素1(RU1)、肾泛素2(RU2)、豆荚蛋白、人乳头瘤病毒E6(HPV E6)、人乳头瘤病毒E7(HPV E7)、肠羧基酯酶、突变的热激蛋白70-2(mut hsp70-2)、CD79a、CD79b、CD72、白细胞相关的免疫球蛋白样受体1(LAIR1)、IgA受体的Fc片段(FCAR或CD89)、白细胞免疫球蛋白样受体亚家族A成员2(LILRA2)、CD300分子样家族成员f(CD300LF)、C型凝集素结构域家族12成员A(CLEC12A)、骨髓基质细胞抗原2(BST2)、含有EGF样模块的粘蛋白样激素受体样2(EMR2)、淋巴细胞抗原75(LY75)、磷脂酰肌醇蛋白聚糖-3(GPC3)、Fc受体样5(FCRL5)、和免疫球蛋白λ样多肽1(IGLL1)、MPL、生物素、c-MYC表位标签、CD34、LAMP1TROP2、GFRα4、CDH17、CDH6、NYBR1、CDH19、CD200R、Slea(CA19.9；唾液酸化的路易斯抗原)；岩藻糖基GM1、PTK7、gpNMB、CDH1-CD324、DLL3、CD276/B7H3、IL11Ra、IL13Ra2、CD179b-IGLl1、TCRγ-δ、NKG2D、CD32(FCGR2A)、Tn ag、Tim1-/HVCR1、CSF2RA(GM-CSFR-α)、TGFβR2、LewsAg、TCR-β1链、TCR-β2链、TCR-γ链、TCR-δ链、FITC、促 黄体生成激素受体(LHR)、促卵泡素受体(FSHR)、促性腺激素受体(CGHR或GR)、CCR4、GD3、SLAMF6、SLAMF4、HIV1包膜糖蛋白、HTLV1-Tax、CMVpp65、EBV-EBNA3c、KSHV K8.1、KSHV-gH、甲型流感血凝素(HA)、GAD、PDL1、胍基环化酶C(GCC)、抗桥粒芯糖蛋白3(Dsg3)的自身抗体、抗桥粒芯糖蛋白1(Dsg1)的自身抗体、HLA、HLA-A、HLA-A2、HLA-B、HLA-C、HLA-DP、HLA-DM、HLA-DOA、HLA-DOB、HLA-DQ、HLA-DR、HLA-G、IgE、CD99、Ras G12V、组织因子1(TF1)、AFP、GPRC5D、紧密连接蛋白18.2(CLD18A2或CLDN18A.2)、P-糖蛋白、STEAP1、Liv1、粘连蛋白-4、Cripto、gpA33、BST1/CD157、低电导氯离子通道、TNT抗体识别的抗原。
本发明的药物除了包括本发明制备的MILS外，还包括药学上可接受的载体和/或赋形剂。
进一步，所述药物还包括本发明制备的MILS经改造后获得的修饰型的混合的固有淋巴细胞。
修饰型MILS包含以下一种或多种基因的插入/缺失：靶向模式、受体、信号传导分子、转录因子、药物标靶候选物、免疫反应调控和调节，或抑制所述MILS或其衍生细胞移植、运输、归巢、活力、自我更新、存留和/或存活的蛋白质。
与从外周血、脐带血或任何其它供体组织中获得的其天然对应细胞相比，修饰型MILS在以下方面获得了功能性改进：
1)存留和/或存活改善；
2)对天然免疫细胞的抗性增加；
3)细胞毒性增加；
4)肿瘤渗透改善；
5)ADCC增强或获得；
6)其他免疫细胞迁移到肿瘤部位和/或激活或募集到肿瘤部位的能力增强；
7)使降低肿瘤免疫抑制的能力增强；
8)挽救肿瘤抗原逃逸的能力提高；和
9)自杀减少。
进一步，本发明的所述药物还包括其他药物活性剂，其他药物活性剂包括其他免疫细胞、其他抗肿瘤药物、其他治疗自身免疫性疾病的药物。
本发明的优点和有益效果：
(1)本发明提供了一种制备包含γδT细胞和NK细胞的混合的固有淋巴细胞的方法，制备方法操作简单，诱导扩增后可获得大量扩增的γδT细胞和NK细胞。
(2)相较于独立培养两种免疫细胞而言大幅度降低培养成本，也降低了培养技术难度，简化了在临床中治疗过程，也降低了治疗成本。
(3)本发明的制备方法采用相同的诱导培养条件，γδT细胞和NK细胞可相互促进刺激生长和扩增，相比单独制备γδT细胞，本发明的制备方法获得的混合的固有淋巴细胞中γδT细胞扩增效率大大增加。
(4)本发明共培养出的MILS毒性/活性强于使用单一的免疫细胞。
附图说明
图1显示细胞扩增曲线图；
图2显示NK和γδT细胞的比例变化图；
图3显示抗体二次刺激对细胞扩增效率的影响结果图；
图4显示MILS细胞的扩增曲线图；
图5显示培养过程中MILS中各细胞占比变化图；
图6显示γδT细胞亚群的占比统计图；
图7显示MILS对AML原代细胞的杀伤作用结果图；
图8显示MILS对K562细胞的杀伤作用结果图。
具体实施方式
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得可实施。
实施例 MILS制备
1、CD4-/CD8-的获得
(1)获得自体血浆并灭活
将血液到入无菌50ml离心管，升9降7，750g离心15min。收集上层血浆于50ml离心管中，56℃孵育30min，4℃放置30min，1000g离心10min，收集上清4℃保存备用。
(2)分离白膜层细胞
将离心后的血细胞沉淀用2-3倍体积(血液的体积)的生理盐水重悬混匀，将稀释血缓缓加入到人单个核细胞分离液(购自东方华辉，货号：25810)的上层，稀释血和人单个核细胞分离液体积比为2:1。然后升7降4，2000rpm，离心20min，收集白膜层细胞，生理盐水清洗两次。
(3)红细胞裂解
加入1x红细胞裂解液(购自BD公司，货号：555899)裂解10min，2000rpm，5min离心，弃去上清，加入生理盐水重悬清洗，2000rpm，5min离心，弃去上清。即为单个核细胞，用5ml生理盐水重悬细胞，计数，并取样检测CD3、CD4、CD8的比例。
(4)CD4-/CD8-细胞分选
a.配置0.5％HAS分选缓冲液，配方为1.25ml HSA+48.75ml DPBS；
b.将单个核细胞悬液2000rpm/min，离心5min，弃上清，按每1×10 7个细胞加入80μl分选缓冲液、20μl CD4 MicroBeads human和20μl CD8 MicroBeads human混匀，4℃孵育15min，每5min摇匀一次；
c.每1×10 7个细胞加入1～2ml分选缓冲液清洗细胞，2000rpm/min，离心5min，弃上清；
d.细胞数量低于1×10 8个加入500μl分选缓冲液，数量高于1×10 8个，按比例增加分选缓冲液，重悬细胞；
e.准备LD磁力分选柱，用2ml缓冲液润洗柱子1次，加入500μl细胞悬液分选细胞，再加入1ml缓冲液清洗柱子2次，分选出的阴性细胞即为CD4-/CD8-细胞。
2、抗体包被
二抗包被；AffiniPure F(ab`)2Fragment Goat Anti-mouse lgG，Fcy Fragment Specific(浓度1.3μg/μl)工作浓度为10μg/ml，T25细胞培养瓶每瓶3ml抗体稀释液，37℃孵育1h，弃掉二抗，加入DPBS清洗T25细胞培养瓶一次。
一抗包被：LEAF TM Purified anti-human TCRγ/δ(浓度1mg/ml)工作浓度为100ng/ml，T25细胞培养瓶每瓶3ml抗体稀释液，室温孵育1h，弃掉一抗，加入DPBS浸泡T25细胞培养瓶备用。
3、细胞培养
将分选得到的CD4-CD8-的细胞接种到已包被抗体的T25瓶中，放入37℃，5％CO 2培养箱中进行培养。细胞培养过程中前6-8天所使用的细胞培养基为活化培养基(SLAM)：KBM581+OK432(100ng/ml)+IFN-γ(1000IU/ml)+IL-2(1000IU/ml)+IL-15(100ng/ml)+IL-21(100ng/ml)+10％自体血浆(灭活)；从培养的第7-9天开始通过半换液的方式将培养基逐步换为扩增培养基：KBM581+IL-2(1000IU/ml)+5％自体血浆(灭活)。
4、结果
1)铺瓶的细胞密度影响细胞后期生长
将不同数量的CD4-CD8-的细胞(1.5×10 6、3×10 6、6×10 6、9×10 6)接种到已包被抗体的T25瓶中，不同细胞铺瓶密度培养的细胞扩增曲线图见图1所示。6×10 6/T25瓶的细胞生长最快。
2)铺瓶的细胞密度影响NK和γδT细胞的比例变化
将不同数量的CD4-CD8-的细胞(1.5×10 6、3×10 6、6×10 6、9×10 6)接种到已包被抗体的T25瓶中，不同细胞铺瓶密度培养的细胞数中NK和γδT细胞的 比例变化见图2所示，随每T25瓶中细胞数目的增加，γδT细胞的比例越高。
3)anti-human TCRγ/δ二次刺激对细胞生长的影响
将分选得到的CD4-CD8-的细胞(6×10 6)接种到已包被抗体的T25瓶中，使用的培养基是活化培养基(SLAM)，放入37℃，5％CO 2培养箱中进行培养。将培养到第6到8天的细胞接种到重新包被anti-human TCRγ/δ抗体的培养瓶中，细胞的扩增曲线见图3。图3结果显示利用抗体二次刺激可以显著提高细胞的扩增效率和生长速度，提高细胞数量。
4)细胞的生长、表型的变化
将分选得到的CD4-CD8-的细胞(6×10 6)接种到已包被抗体的T25瓶中，使用的培养基是活化培养基(SLAM)，放入37℃，5％CO 2培养箱中进行培养。将培养到第6到8天细胞接种到重新包被anti-human TCRγ/δ抗体的培养瓶中进行二次刺激，细胞培养至25-30天进行收获，并对收获的细胞进行体外功能检测。MILS细胞的扩增曲线见图4，MILS细胞在培养30天内由6E6个扩增到1.84E10个，扩增3000多倍。培养过程中MILS中各细胞占比变化见图5，MILS细胞培养初始主要由NK细胞，CD3-CD56-细胞及少量的γδT细胞组成，随着培养CD3-CD56-的细胞逐渐降低直至消失，γδT细胞和NK细胞不断扩增。γδT细胞亚群的占比见图6，脐带血来源的MILS(CB-MILS)的γδT主要是vδ1型的γδT和部分vδ1 -vδ2 -型的γδT，vδ1、vδ1 -vδ2 -、vδ2比例分别为63.12±8.5％、34.44±7.53％、2.44±1.63％。外周血来源的MILS(PB-MILS)的γδT主要是vδ2型的γδT，vδ2、vδ1 -vδ2 -、vδ1比例分别为75.87±4.37％、14.43±2.14％、9.59±5.56％。
5)对AML原代肿瘤细胞具有杀伤作用
取急性髓细胞白血病(AML)的外周血分离出PBMC，用CFSE染色PBMC作为靶细胞，根据不同效靶比(2:1，10：1，50：1)向靶细胞中添加MILS细胞，混合后孵育4小时，流式细胞术检测细胞杀伤。MILS对AML原代细胞具有杀伤作用(图7)，效靶比为2：1的杀伤率为8.2％，10：1的杀伤率为28.13％，50：1的杀伤率为65.36％。
6)对肿瘤细胞系的杀伤作用
用CFSE染色的K562细胞作为靶细胞(按照2×10 6个细胞添加100μl的2μM CFSE工作液的比例进行染色)。按每孔1×10 5个/100μl K562细胞加入96孔 板中。然后根据效靶比1:1向靶细胞中添加适量的MILS效应细胞，混合后孵育4小时，流式细胞术检测细胞杀伤比例。MILS对K562细胞具有杀伤作用(图8)，杀伤率为80.0％。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (11)

  1. 一种活化培养基，其特征在于，所述活化培养基包括以下细胞因子组合：OK432、IFN-γ、IL-2、IL-15、IL-21；优选地，OK432终浓度为100ng/ml、IFN-γ终浓度为1000IU/ml、IL-2终浓度为1000IU/ml、IL-15终浓度为100ng/ml、IL-21终浓度为100ng/ml；
    优选地，所述活化培养基还包括：KMB581、自体血浆或人AB血清；优选地，自体血浆或人AB血清比例为10％。
  2. 一种混合的固有淋巴细胞的制备方法，其特征在于，所述制备方法包括如下步骤：
    1)CD4-/CD8-T细胞分选；
    2)混合的固有淋巴细胞诱导：使用权利要求1的活化培养基培养CD4-/CD8-T细胞；
    3)混合的固有淋巴细胞扩增：使用扩增培养基培养经步骤2)获得的细胞；
    优选地，所述扩增培养基包括KMB581、IL-2、自体血浆或人AB血清；优选地，所述IL-2终浓度为1000IU/ml、自体血浆或人AB血清比例为5％。
  3. 根据权利要求2所述的制备方法，其特征在于，所述步骤1)包括以下步骤：
    a)分离白膜层细胞；
    b)红细胞裂解；
    c)CD4-/CD8-T细胞分选；
    优选地，从血液中分离白膜层细胞；更优选地，所述血液包括脐带血或外周血；
    优选地，分离白膜层细胞的步骤包括：将血液离心获得自体血浆；将离心后的血细胞沉淀用生理盐水重悬获得稀释血，加入单个核细胞分离液，稀释血和单个核细胞分离液体积比为2:1，然后离心收集白膜层细胞，生理盐水清洗两次；
    优选地，红细胞裂解的步骤包括：加入红细胞裂解液裂解步骤a)获得的白膜层细胞，弃上清，加入生理盐水重悬清洗，离心弃上清获得单个核细胞，用生理盐水重悬细胞；
    优选地，CD4-/CD8-T细胞分选的步骤包括：
    i)配置分选缓冲液；
    ii)将单个核细胞悬液离心，弃上清，加入分选缓冲液、CD4 MicroBeads human和CD8 MicroBeads human混匀，低温孵育；
    iii)加入分选缓冲液清洗细胞，离心弃上清；
    iiii)加入分选缓冲液重悬细胞；
    iiiii)准备LD磁力分选柱，用缓冲液润洗柱子，加入经iiii)获得的细胞悬液分选细胞，再加入缓冲液清洗柱子，分选出的阴性细胞即为CD4-/CD8-细胞。
  4. 根据权利要求2所述的制备方法，其特征在于，步骤2)中混合的固有淋巴细胞诱导的步骤还包括抗体包被，抗体包被包括：二抗包被和一抗包被；
    优选地，一抗包被使用的是anti-human TCRγ/δ包被；
    优选地，二抗包被使用的是Goat Anti-mouse lgG包被；
    优选地，二抗包被；AffiniPure F(ab`)2 Fragment Goat Anti-mouse lgG，Fcy Fragment Specific工作浓度为10μg/ml，T25细胞培养瓶每瓶3ml抗体稀释液，37℃孵育1h，弃掉二抗，加入DPBS清洗T25细胞培养瓶一次；
    优选地，一抗包被：LEAF TMPurified anti-human TCRγ/δ工作浓度为100ng/ml，T25细胞培养瓶每瓶3ml抗体稀释液，室温孵育1h，弃掉一抗，加入DPBS浸泡T25细胞培养瓶备用。
  5. 根据权利要求2所述的制备方法，其特征在于，混合的固有淋巴细胞诱导：使用权利要求1的活化培养基培养CD4-/CD8-T细胞6-8天。
  6. 根据权利要求2所述的制备方法，其特征在于，混合的固有淋巴细胞扩增步骤如下：从培养的第7-9天开始通过半换液的方式将活化培养基逐步换为扩增培养基；
    优选地，从培养的第7-9天开始，将培养6-8天的细胞接种到不包被或包被anti-human TCRγ/δ抗体的培养瓶中，通过半换液的方式将活化培养基逐步换为扩增培养基。
  7. 根据权利要求1-6中任一项所述的制备方法获得的混合的固有淋巴细胞；
    优选地，所述混合的固有淋巴细胞中主要为γδT细胞和NK细胞组成的混合淋巴细胞，其中γδT细胞的亚群主要由vδ1，vδ2和vδ1 -vδ2 -三个亚群组成；
    更优选地，以脐带血为来源获得的混合的固有淋巴细胞中vδ1占比最高，其次是vδ1 -vδ2 -，vδ2占比最低；优选地，vδ1、vδ1 -vδ2 -、vδ2比例分别为60±10％、35±10％、3±2％；
    更优选地，以外周血为来源获得的混合的固有淋巴细胞中vδ2占比最高，其次是vδ1 -vδ2 -，vδ1占比最低；优选地，vδ2、vδ1 -vδ2 -、vδ1比例分别为75±10％、15±5％、10±5％。
  8. 一种试剂盒或药物，其特征在于，所述试剂盒或药物包括权利要求7所述的混合的固有淋巴细胞。
  9. 一种用于在受试者中预防和/或治疗肿瘤或自身免疫性疾病的方法，其特征在于，所述方法包括向有此需要的受试者施用有效量的权利要求7所述的混合的固有淋巴细胞、权利要求8所述的药物。
  10. 根据权利要求9所述的方法，其特征在于，所述肿瘤包括非实体瘤、实体瘤。
  11. 权利要求7所述的混合的固有淋巴细胞的应用，其特征在于，所述应用包括以下任一项：
    1)在制备预防和/或治疗肿瘤或自身免疫性疾病的药物中的应用；
    2)在制备表达嵌合抗原受体的免疫细胞中的应用；
    3)在制备权利要求8所述的试剂盒中的应用。
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