CN113088490A - Mixed innate lymphocytes, methods of preparation and uses thereof - Google Patents

Mixed innate lymphocytes, methods of preparation and uses thereof Download PDF

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CN113088490A
CN113088490A CN202110438381.1A CN202110438381A CN113088490A CN 113088490 A CN113088490 A CN 113088490A CN 202110438381 A CN202110438381 A CN 202110438381A CN 113088490 A CN113088490 A CN 113088490A
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李建强
刘莹
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Hebei Senlang Biotechnology Co ltd
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Hebei Senlang Biotechnology Co ltd
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Priority to PCT/CN2022/086903 priority patent/WO2022222845A1/en
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Abstract

The invention discloses a mixed lymphocyte, a preparation method and application thereof. The invention utilizes the same culture system to simultaneously induce and amplify the gamma delta T cells and the NK cells, the gamma delta T cells and the NK cells can mutually promote the stimulated growth and the amplification, and compared with the independent preparation of the gamma delta T cells, the amplification efficiency of the gamma delta T cells in the mixed lymphocytes obtained by the preparation method is greatly increased. The preparation method of the invention greatly reduces the culture cost, simplifies the treatment process in clinic, reduces the treatment cost and is suitable for large-scale popularization.

Description

Mixed innate lymphocytes, methods of preparation and uses thereof
Technical Field
The invention belongs to the field of immunotherapy, and particularly relates to mixed intrinsic lymphocytes, a preparation method and application thereof.
Background
The innate lymphocytes include Natural Killer (NK) cells, γ δ T cells, cytokine-induced killer (CIK) cells, and the like, and have a natural immune defense function.
γ δ T cells are T cells that perform innate immune functions, the TCR of which consists of γ and δ chains. The T cells are mainly distributed in mucous membranes and subcutaneous tissues such as intestinal tracts, urogenital tracts and the like, and only account for 0.5-1% of CD3+ T cells in peripheral blood. The TCR lacks diversity and can directly recognize certain intact polypeptide antigens. γ δ T cells recognize a limited number of antigens: HSP; ② lipid antigen extracted from CD1 molecule on the surface of infected cell; ③ certain viral proteins or viral proteins expressed on the surface of infected cells; and fourthly, phosphorylation antigen in the bacterial lysate.
The gamma delta T cell is an immune cell which can kill cancer cells and tumor stem cells and can recognize cancer antigens, and the killing property of the gamma delta T cell is stronger, but the killing property of the tumor stem cells is not better than that of NK cells. Therefore, it is mainly used for killing cancer cells and assisting DC cells to recognize and find cancer cell antigens, and then killing or transferring the antigens to other cells. Meanwhile, the gamma delta T cells are mainly distributed on the skin and mucosal tissues, so that the treatment effect on cancers on the aspects of the mucosa, such as cancers on the aspects of digestive tract, respiratory tract and reproductive system, is remarkable.
γ δ T cells develop and mature primarily in the thymus, producing γ δ T Cell Receptors (TCRs) through v (d) J gene recombination. Through specific gene rearrangement, a Common Lymphodepursor (CLP) is differentiated into a T cell line expressing an α β receptor and a γ δ receptor. The gamma delta T cells are not easily affected by antigen processing and presentation deficiency, so the gamma delta T cells have high potential application value in clinical tumor immunotherapy. γ δ T cells play an important role in tumor immune surveillance and anti-tumor immune responses.
Because the content of the gamma delta T cells in peripheral blood is extremely low, the clinical application of the gamma delta T cells as adoptive immune cells is greatly limited. At present, gamma delta T cells are amplified from peripheral blood mononuclear cells, the amplification times are low, and the cell purity and the cell quantity are not high. The amplified gamma delta T cells hardly meet clinical requirements, and even if the single gamma delta T cells amplified by optimizing various induction conditions and amplification methods are applied to corresponding immune diseases and tumor diseases, the ideal effect of people cannot be achieved after the application.
Cellular immunotherapy is one of the most promising tumor therapy methods at present, and achieves the purpose of killing tumor cells by in vitro amplification or transformation and then infusing the tumor cells back to a patient body, or enhances the autoimmune function of the tumor patient by activating the immune system of the body so as to resist tumors or other diseases. At present, NK cell immunotherapy is receiving more and more attention. NK cells account for 5-15% of human peripheral blood lymphocytes, and their phenotype is generally defined as CD3-CD56+, which can be further subdivided into two major subgroups: CD56highCD 16-cells with immunoregulatory function and CD56dimCD16+ cells with cytotoxic activity. NK cells play an important immune monitoring function in early immune response of resisting virus infection and tumors, and can directly and quickly play cytotoxic activity without identifying tumor specific antigens. Particularly important is that NK cells can effectively eliminate tumor stem cell-like cells in organisms and inhibit the growth and metastasis of tumors.
At present, most of inherent immune response cell therapy products are single cell preparations (NK cells, gamma delta T cells, CIK cells and the like) which are limited to the treatment of tumors. Or a multi-cell immune preparation in which single-type cells are cultured and then mixed, is complicated in operation.
The application obtains cells of CD4-CD 8-from PBMC derived from umbilical cord blood or peripheral blood by sorting the cells through CD4 and CD8, prepares Mixed Intrinsic Lymphocytes (MILS) containing NK cells and gamma delta T cells through culture, and greatly improves the expansion efficiency of the gamma delta T cells by simultaneously inducing the culture of the NK cells and the gamma delta T cells.
Disclosure of Invention
The purpose of the present invention is to provide a practical, efficient, low-cost, technically simple method capable of simultaneously amplifying a large number of NK cells and γ δ T cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
according to one aspect of the invention, the invention provides a cytokine combination comprising OK432, IFN-gamma, IL-2, IL-15, IL-21.
According to another aspect of the invention, the invention provides an activation medium comprising the following cytokine combinations: OK432, IFN-gamma, IL-2, IL-15, IL-21.
Preferably, OK432 final concentration is 100ng/ml, IFN-gamma final concentration is 1000IU/ml, IL-2 final concentration is 1000IU/ml, IL-15 final concentration is 100ng/ml, IL-21 final concentration is 100 ng/ml.
Further, the activation medium further comprises: KBM581, autologous plasma or human AB serum.
Preferably, the ratio of autologous plasma or human AB serum is 10%.
According to yet another aspect of the present invention, there is provided a method for preparing Mixed Innate Lymphocytes (MILS), the method comprising the steps of:
1) CD4-/CD8-T cell sorting;
2) MILS cell induction: culturing CD4-/CD8-T cells using the activation medium described previously;
3) MILS cell expansion: culturing the cells obtained by step 2) using an amplification medium.
Further, the amplification medium includes KBM581, IL-2, autologous plasma, or human AB serum.
Preferably, the final IL-2 concentration is 1000IU/ml, and the ratio of autologous plasma or human AB serum is 5%.
Further, the step 1) comprises the following steps:
a) separating the white membrane layer cells;
b) red blood cell lysis;
c) CD4-/CD8-T cell sorting.
Further, separating the leucocyte layer cells from the blood; more preferably, the blood comprises umbilical cord blood or peripheral blood.
Further, the step of isolating the buffy coat cells comprises: centrifuging the blood to obtain autologous plasma; and (3) resuspending the centrifuged blood cell sediment with physiological saline to obtain diluted blood, adding the mononuclear cell separation solution, wherein the volume ratio of the diluted blood to the mononuclear cell separation solution is 2:1, centrifuging to collect leucocyte layer cells, and washing twice with the physiological saline.
In a specific embodiment of the present invention, the step of isolating the buffy coat cells comprises:
the blood was put into a sterile 50ml centrifuge tube, lifted 9 and lowered 7, and centrifuged at 750g for 15 min. Collecting supernatant plasma in 50ml centrifuge tube, incubating at 56 deg.C for 30min, standing at 4 deg.C for 30min, centrifuging at 1000g for 10min, collecting supernatant, and storing at 4 deg.C for use.
Resuspending and mixing the centrifuged blood cell sediment with 2-3 times of normal saline, slowly adding the diluted blood into the upper layer of the human mononuclear cell separating medium, wherein the volume ratio of the diluted blood to the human mononuclear cell separating medium is 2: 1. Then, the speed is increased and decreased by 4 degrees, 2000rpm is carried out, centrifugation is carried out for 20min, leucocyte layer cells are collected, and the leucocyte layer cells are washed twice by normal saline.
Further, the step of lysing the red blood cells comprises: adding a red blood cell lysate to lyse the white membrane layer cells obtained in the step a), discarding the supernatant, adding physiological saline to carry out heavy suspension cleaning, centrifuging and discarding the supernatant to obtain mononuclear cells, and carrying out heavy suspension on the cells by using the physiological saline.
In a particular embodiment of the invention, the step of lysing the red blood cells comprises: adding 1 Xerythrocyte lysate for lysis for 10min, centrifuging at 2000rpm for 5min, discarding the supernatant, adding physiological saline for resuspension and cleaning, centrifuging at 2000rpm for 5min, and discarding the supernatant. The mononuclear cells are obtained, the cells are resuspended by using 5ml of physiological saline, counted, sampled and tested for the proportion of CD3, CD4 and CD 8.
Further, the step of sorting CD4-/CD8-T cells comprises:
i) preparing a sorting buffer solution;
ii) centrifuging the single nuclear cell suspension, removing the supernatant, adding a sorting buffer solution, CD4 MicroBeads human and CD8 MicroBeads human, uniformly mixing, and incubating at low temperature;
iii) adding a sorting buffer solution to wash the cells, centrifuging and removing a supernatant;
iiii) resuspending the cells by adding sorting buffer;
iiii) preparing LD magnetic sorting column, rinsing the column with buffer solution, adding the cell suspension obtained by iiii) to sort cells, and then adding buffer solution to clean the column, wherein the negative cell sorted out is CD4-/CD 8-cell.
Further, the sorting buffer was phosphate buffer containing 0.5% HAS.
The specific scheme of step ii) is as follows: centrifuging the mononuclear cell suspension at 2000rpm/min for 5min, discarding the supernatant, and adding the supernatant at 1 × 107The cells were mixed with 80. mu.l of sorting buffer, 20. mu.l of CD4 MicroBeads human and 20. mu.l of CD8 MicroBeads human, incubated at 4 ℃ for 15min and shaken every 5 min.
Further, the step of inducing the MILS cells in step 2) further comprises antibody coating, the antibody coating comprising:
secondary antibody coating and primary antibody coating;
preferably, the primary antibody is anti-human TCR γ/δ.
Preferably, the secondary antibody is Goat Anti-mouse lgG.
Preferably, a secondary antibody coating; affinipure F (ab') 2Fragment Goat Anti-mouse lgG, the working concentration of Fcy Fragment specificity is 10 mug/ml, each bottle of T25 cell culture bottle is 3ml of antibody diluent, the incubation is carried out for 1h at 37 ℃, secondary antibody is discarded, and DPBS is added to wash the T25 cell culture bottle for one time;
preferably, the primary antibody coating: LEAFTMThe working concentration of the Purified anti-human TCR gamma/delta is 100ng/ml, 3ml of antibody diluent is added into each bottle of T25 cell culture bottles, the mixture is incubated for 1h at room temperature, primary antibody is removed, and DPBS is added to soak the T25 cell culture bottles for later use.
Further, MILS cells induced: CD4-/CD8-T cells were cultured for 6-8 days using the previously described activation medium.
Further, the MILS cell expansion step was as follows: the activation medium was gradually changed to the amplification medium by half-liquid change from day 7 to day 9 of the culture.
As a means of amplification: starting from 7-9 days of culture, cells cultured for 6-8 days are inoculated into culture flasks which are not coated with anti-human TCR gamma/delta antibody, and the activation medium is gradually changed into the amplification medium by half-liquid change.
As another more preferred mode of amplification: starting from 7-9 days of culture, cells cultured for 6-8 days are inoculated into culture bottles re-coated with anti-human TCR gamma/delta antibody, and the activation medium is gradually changed into the amplification medium by half liquid change.
According to still another aspect of the present invention, the present invention provides that the MILS obtained by the aforementioned preparation method is mainly gamma delta T-NK mixed lymphocytes. Wherein the sub-population of γ δ T cells consists mainly of v δ 1, v δ 2 and v δ 1-vδ2-Three subgroups consisted of cord blood with the highest ratio of v δ 1 followed by v δ 1-vδ2-The ratio of v delta 2 is the lowest, and the ratios are v delta 1 and v delta 1 respectively-vδ2-And v delta 2 ratios are 60 + -10%, 35 + -10% and 3+ -2%, respectively. The highest ratio of v δ 2 among gamma δ T cells in peripheral blood, followed by v δ 1-vδ2-The ratio of v delta 1 is the lowest, and the ratios are v delta 2 and v delta 1 respectively-vδ2-And v.delta.1 ratios of 75 + -10%, 15 + -5% and 10 + -5%, respectively.
According to a further aspect of the invention there is provided a kit or medicament comprising the MILS as hereinbefore described.
According to a further aspect of the invention, there is provided a method for preventing and/or treating a tumor or an autoimmune disease in a subject, the method comprising administering to a subject in need thereof an effective amount of an MILS as described above, or a medicament as described above.
According to a further aspect of the invention, there is provided a use of the MILS as described above, the use comprising any one of:
1) the application in preparing the medicine for treating tumor or autoimmune disease;
2) the application of the chimeric antigen receptor in preparing immune cells expressing the chimeric antigen receptor;
3) use in the preparation of a kit as hereinbefore described.
The tumor of the present invention includes non-solid tumor and solid tumor. Treatable tumors include tumors that are not vascularized or have not substantially been vascularized, as well as vascularized tumors. The tumor may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. Tumor types treated with the MILS of the present invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.
Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.
A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid regions. Different types of solid tumors are named for the cell types that form 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 now known or later discovered cancer (see, e.g., Rosenberg (1996) Ann. Med. 47: 481-491, the entire contents of which are incorporated herein by reference).
Autoimmune diseases of the 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 (celiac sprue) -dermatitis herpetiformis, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos's disease, juvenile dermatomyositis (dermatatis-juvenile), discoid lupus, idiopathic mixed cryoprecipitated globulinemia, fibromyalgia-fibromyositis, Graves ' disease, guillain-barre syndrome, hashimoto's thyroiditis, idiopathic pulmonary fibrosis, Idiopathic Thrombocytopenic Purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (stipuls disease), juvenile rheumatoid arthritis, meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia (pernicious anemia), nodular hyperactive inflammation, polychondritis, polyglandular syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, raynaud's phenomenon, reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as Systemic Sclerosis (SS)), sjogren's syndrome, stiff body syndrome, systemic lupus erythematosus, takayasu's arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligo, Wegener's granulomatosis, and any combination thereof.
The targeting molecule of the chimeric antigen receptor comprises a tumor surface antigen and a tumor-associated antigen.
Examples of targeting molecules for chimeric antigen receptors of the invention include, but are not limited to, CD5, CD19, CD123, CD 22; CD30, CD171, CS1, C-type lectin-like molecule-1, CD33, epidermal growth factor receptor variant 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) or (GalNAc α -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 cells, glycosylated CD43 expressed on nonhematopoietic cancer, embryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM), B7H3(CD276), KIT (CD117), interleukin 13 receptor subunit alpha-2 (IL-13Ra2 or CD213a2), mesothelin, interleukin 11 receptor alpha (IL-11Ra), Prostate Stem Cell Antigen (PSCA), protease serine 21 (testosterone protein or PRSS21), vascular endothelial growth factor receptor 2(VEGFR2), lewis (Y) antigen, CD 24; platelet-derived growth factor receptor beta (PDGFR-beta), stage-specific embryonic antigen 4(SSEA-4), CD20, folate receptor alpha (FRa or FR1), folate receptor beta (FRb), receptor tyrosine protein kinase ERBB2(Her2/neu), cell surface-associated mucin 1(MUC1), Epidermal Growth Factor Receptor (EGFR), neuronal adhesion molecule (NCAM), prostatase, Prostatic Acid Phosphatase (PAP), mutated elongation factor 2(ELF2M), ephrin B2, fibroblast activation protein alpha (FAP), insulin-like growth factor 1 receptor (IGF-I receptor), Carbonic Anhydrase IX (CAIX), glycoprotein 100(gp100), tyrosinase, ephrin A type A receptor 2(EphA2), sialylated Lewis adhesion molecule (sLe), transglutaminase 5(TGS5), High Molecular Weight Melanoma Associated Antigen (HMWMAA), O-acetyl GD2 ganglioside (OAcGD2), tumor endothelial marker 1(TEM1/CD248), tumor endothelial marker 7 related (TEM7R), claudin 6(CLDN6), Thyroid Stimulating Hormone Receptor (TSHR), group 5G protein-coupled receptors, member D (GPRC5D), X chromosome open reading frame 61(CXORF61), CD97, CD179a, Anaplastic Lymphoma Kinase (ALK), polysialic acid, placenta-specific 1(PLAC1), the hexasaccharide portion of globoH glycoceramide (GloboH), mammary gland differentiation antigen (NY-BR-1), urolytic protein 2(UPK2), hepatitis A virus cell receptor 1(HAVCR1), adrenoreceptor beta 3(ADRB3), pannexin 3(PANX3), G protein receptor 20(GPR20), lymphocyte antigen 6, gene locus 638 (TCR 636K 2), olfactory receptor complex 6351 (OR 51), and alternate reading frame 51E 51 protein, Wilms tumor protein (WT 1), cancer/testis antigen 1(NY-ESO-1), cancer/testis antigen 2(LAGE-1A), melanoma-associated antigen 1(MAGE-a1), ETS translocation variant gene 6(ETV6-AML) located on chromosome 12p, sperm protein 17(SPA17), X antigen family member 1A (XAGE 1); angiogenin binds to cell surface receptor 2(Tie2), melanoma testis antigen-1 (MAD-CT-1), melanoma testis antigen 2(MAD-CT-2), Fos-associated antigen 1, tumor protein p53(p53), p53 mutant, prostacyclin (prostein), survivin, telomerase, prostate cancer tumor antigen-1 (PCT A-1 or galectin 8), T cell recognized melanoma antigen 1(MelanA or MARTI), rat sarcoma (Ras) mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoint, melanoma apoptosis inhibitor (ML-IAP), ERG (transmembrane protease, serine 2(TMPRSS2) ETS fusion gene), N-acetylglucosamine transferase V (NA17), paired box protein Pax-3(PAX3), androgen receptor, gene, protein, and so-2, Cyclin B1, v-myc avian myelocytoma oncogene neuroblastoma derived homolog (MYCN), Ras cognate family member C (RhoC), tyrosinase related protein 2(TRP-2), cytochrome P4501B (CYP1B 1), CCTC binding factor (zinc finger protein) -like (BORIS or imprinted site regulatory factor-like protein), T cell recognized squamous cell carcinoma antigen 3(SART3), paired box protein Pax-5(PAX5), pre-apical voxel binding protein sp32(OY-TES1), lymphocyte specific protein tyrosine kinase (LCK), kinase ankyrin 4(AKAP-4), synovial sarcoma X breakpoint 2(SSX2), late glycosylation product receptor (RAGE-1), renal ubiquitin 1(RU1), renal cell 2(RU2), legumain, human papilloma virus E6(HPV E6), human papilloma virus E7(HPV E685 2), Intestinal carboxylesterase, mutant heat shock protein 70-2(mut hsp70-2), CD79a, CD79b, CD72, leukocyte-associated immunoglobulin-like receptor 1(LAIR1), Fc fragment of IgA receptor (FCAR or CD89), leukocyte immunoglobulin-like receptor subfamily A member 2(LILRA2), CD300 molecular-like family member f (CD300LF), C-type lectin domain family 12 member A (CLEC12A), bone marrow stromal cell antigen 2(BST2), mucin-like hormone receptor-like 2 containing EGF-like modules (EMR2), lymphocyte antigen 75(LY75), glypican-3 (GPC3), fc receptor-like 5(FCRL5), and immunoglobulin lambda-like polypeptide 1(IGLL1), MPL, biotin, c-MYC epitope tag, CD34, LAMP1 TROP2, GFR α 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA 19.9; sialylated Lewis antigen); fucosyl GM1, PTK7, gpNMB, CDH1-CD324, DLL3, CD276/B7H3, IL11Ra, IL13Ra2, CD179B-IGLl1, TCR γ - δ, NKG2D, CD32(FCGR2A), Tn ag, Tim1-/HVCR1, CSF 21 (GM-CSFR- α), TGF β R1, LewsAg, TCR- β 1 chain, TCR- β 2 chain, TCR- γ chain, TCR- δ chain, FITC, Luteinizing Hormone Receptor (LHR), follitropin receptor (FSHR), gonadotropin receptor (CGHR or GR), SL3672, GD 1, CCR AMF 1, HIV1 envelope glycoprotein, HTLV1-Tax, Vpv 36p 72, EBV-363, EBV-1, HA 3-gHV 72, influenza HA-gHA (GAsDNA) receptor), anti-IgG 1, anti-HLA-IgG 1, anti-TNF-IgG 1, anti-IgG (IgG) antibody (KSHV-IgG) IgG 1), IgG 3, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, tissue factor 1(TF1), AFP, GPRC5D, claudin 18.2(CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, Liv1, fibronectin-4, Cripto, gpA33, BST1/CD157, low conductivity chloride channel, TNT antibody-recognized antigen.
The medicament of the invention comprises pharmaceutically acceptable carriers and/or excipients besides the MILS prepared by the invention.
Further, the medicament also comprises modified mixed intrinsic lymphocytes obtained by modifying MILS prepared by the invention.
Modified MILS comprise insertions/deletions of one or more of the following genes: targeting modalities, receptors, signaling molecules, transcription factors, drug target candidates, immune response modulation and modulation, or proteins that inhibit the transplantation, trafficking, homing, viability, self-renewal, persistence and/or survival of the MILS or derived cells thereof.
The modified MILS achieved functional improvements compared to its natural counterpart cells obtained from peripheral blood, umbilical cord blood or any other donor tissue in the following respects:
1) improved survival and/or survival;
2) increased resistance to innate immune cells;
3) increased cytotoxicity;
4) improved tumor penetration;
5) ADCC enhancement or acquisition;
6) enhanced ability of other immune cells to migrate to and/or activate or recruit to the tumor site;
7) enhancing the ability to reduce tumor immunosuppression;
8) the ability to rescue escape of tumor antigens is improved; and
9) suicide is reduced.
Furthermore, the medicine also comprises other medicinal active agents, and the other medicinal active agents comprise other immune cells, other anti-tumor medicines and other medicines for treating autoimmune diseases.
The invention has the advantages and beneficial effects that:
(1) the invention provides a method for preparing mixed inherent lymphocytes containing gamma delta T cells and NK cells, which is simple to operate and can obtain a large amount of amplified gamma delta T cells and NK cells after induced amplification.
(2) Compared with the independent culture of two types of immune cells, the method greatly reduces the culture cost, also reduces the difficulty of the culture technology, simplifies the treatment process in clinic and also reduces the treatment cost.
(3) The preparation method adopts the same induction culture conditions, the gamma delta T cells and the NK cells can mutually promote the stimulated growth and the amplification, and compared with the single preparation of the gamma delta T cells, the amplification efficiency of the gamma delta T cells in the mixed inherent lymphocytes obtained by the preparation method is greatly increased.
(4) The toxicity/activity of the co-cultured MILS of the present invention is stronger than that of using a single immune cell.
Drawings
FIG. 1 shows a graph of cell expansion;
FIG. 2 shows a graph of the proportional change of NK and γ δ T cells;
FIG. 3 is a graph showing the effect of secondary stimulation with antibodies on cell expansion efficiency;
FIG. 4 shows a graph of the expansion of MILS cells;
FIG. 5 shows a graph showing the change in the ratio of each cell in MILS during the culture;
figure 6 shows a histogram of the proportion of γ δ T cell subpopulations;
FIG. 7 shows the results of the killing effect of MILS on AML primary cells;
FIG. 8 shows the results of the killing effect of MILS on K562 cells.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiments.
Examples MILS preparation
1. Acquisition of CD4-/CD8-
(1) Obtaining autologous plasma and inactivating
The blood was put into a sterile 50ml centrifuge tube, lifted 9 and lowered 7, and centrifuged at 750g for 15 min. Collecting supernatant plasma in 50ml centrifuge tube, incubating at 56 deg.C for 30min, standing at 4 deg.C for 30min, centrifuging at 1000g for 10min, collecting supernatant, and storing at 4 deg.C for use.
(2) Isolation of leukocytes
The centrifuged blood cell sediment is resuspended and mixed evenly by using 2-3 times of volume (volume of blood) of normal saline, and the diluted blood is slowly added into the upper layer of the human mononuclear cell separating medium (purchased from Oriental Huahui, goods number: 25810), and the volume ratio of the diluted blood to the human mononuclear cell separating medium is 2: 1. Then, the speed is increased and decreased by 4 degrees, 2000rpm is carried out, centrifugation is carried out for 20min, leucocyte layer cells are collected, and the leucocyte layer cells are washed twice by normal saline.
(3) Erythrocyte lysis
Adding 1 Xerythrocyte lysate (purchased from BD company, cat # 555899) for lysis for 10min, centrifuging at 2000rpm for 5min, discarding the supernatant, adding physiological saline for resuspension and washing, centrifuging at 2000rpm for 5min, and discarding the supernatant. The mononuclear cells are obtained, the cells are resuspended by using 5ml of physiological saline, counted, sampled and tested for the proportion of CD3, CD4 and CD 8.
(4) CD4-/CD 8-cell sorting
a. Preparing 0.5% HAS sorting buffer solution with the formula of 1.25ml HSA +48.75ml DPBS;
b. centrifuging the mononuclear cell suspension at 2000rpm/min for 5min, discarding the supernatant, and adding the supernatant at 1 × 107Adding 80 μ l of sorting buffer, 20 μ l of CD4 MicroBeads human and 20 μ l of CD8 MicroBeads human into each cell, mixing uniformly, incubating for 15min at 4 ℃, and shaking uniformly every 5 min;
c. each 1 × 107Adding 1-2 ml of sorting buffer solution into each cell to clean the cell, centrifuging at 2000rpm/min for 5min, and removing supernatant;
d. the number of cells is less than 1 × 108Adding 500 μ l of sorting buffer to the sample, the amount is more than 1 × 108Proportionally increasing sorting buffer solution, and resuspending cells;
e. preparing LD magnetic sorting column, rinsing the column with 2ml buffer solution for 1 time, adding 500 μ l cell suspension to sort cells, and then adding 1ml buffer solution to rinse the column for 2 times, wherein the negative cell is CD4-/CD 8-cell.
2. Antibody coating
Coating with a secondary antibody; AffiniPure F (ab') 2Fragment Goat Anti-mouse lgG, Fcy Fragment specificity (concentration 1.3. mu.g/. mu.l) working concentration 10. mu.g/ml, T25 cell culture flask each 3ml antibody diluent, 37 ℃ incubation for 1h, abandoning the secondary antibody, adding DPBS to wash T25 cell culture flask once.
Primary anti-coating: LEAFTMThe working concentration of the Purified anti-human TCR gamma/delta (concentration of 1mg/ml) is 100ng/ml, 3ml of antibody diluent is added into each bottle of a T25 cell culture bottle, the mixture is incubated for 1h at room temperature, primary antibody is removed, and DPBS is added to soak the T25 cell culture bottle for later use.
3. Cell culture
The sorted CD4-CD 8-cells were inoculated into antibody-coated T25 bottles, and placed at 37 ℃ in 5% CO2Culturing in an incubator. The cell culture medium used in the first 6-8 days of the cell culture process was activated medium (SLAM): KBM581+ OK432(100ng/ml) + IFN-gamma (1000IU/ml) + IL-2(1000IU/ml) + IL-15(100ng/ml) + IL-21(100ng/ml) + 10% autologous plasma (inactivated); gradually changing the culture medium to an amplification culture medium by means of half-liquid change from 7 to 9 days of culture: KBM581+ IL-2(1000IU/ml) + 5% autologous plasma (inactivated).
4. Results
1) Bottled cell density affects late cell growth
Different numbers of CD4-CD 8-cells (1.5X 10)6、3×106、6×106、9×106) The cell expansion curves for different cell plating densities when seeded into antibody-coated T25 flasks are shown in FIG. 1. 6X 106The cells grew fastest in the/T25 flask.
2) Vial-plated cell density effects proportional changes in NK and γ δ T cells
Different numbers of CD4-CD 8-cells (1.5X 10)6、3×106、6×106、9×106) The ratio of NK and γ δ T cells among the number of cells cultured at different cell plating densities inoculated into antibody-coated T25 flasks was varied as shown in fig. 2, with the higher the ratio of γ δ T cells as the number of cells per T25 flask increased.
3) Effect of anti-human TCR gamma/delta Secondary stimulation on cell growth
Sorting the obtained CD4-CD 8-cells (6X 10)6) Inoculating into T25 flask coated with antibody, using activated medium (SLAM), placing at 37 deg.C and 5% CO2Culturing in an incubator. Cells cultured up to day 6 to 8 were inoculated into flasks re-coated with anti-human TCR γ/δ antibody, and the cell expansion curves are shown in fig. 3. The results in fig. 3 show that secondary stimulation with the antibody can significantly improve the expansion efficiency and growth rate of cells, and increase the number of cells.
4) Growth of cells, phenotypic changes
Sorting the obtained CD4-CD 8-cells (6X 10)6) Inoculating into T25 flask coated with antibody, using activated medium (SLAM), placing at 37 deg.C and 5% CO2Culturing in an incubator. Inoculating the cells cultured to the 6 th to 8 th days into a culture flask re-coated with anti-human TCR gamma/delta antibody for secondary stimulation, culturing the cells to the 25 th to 30 th days for harvesting, and carrying out in vitro function detection on the harvested cells. The amplification curve of MILS cells is shown in FIG. 4, and the MILS cells were amplified from 6E6 to 1.84E10 within 30 days of cultureIncreased by 3000 times. The ratio of each cell in MILS changes in the culture process is shown in figure 5, the MILS cell culture is initially mainly composed of NK cells, CD3-CD 56-cells and a small amount of gamma delta T cells, and the gamma delta T cells and the NK cells are continuously expanded along with the gradual reduction and disappearance of cells for culturing CD3-CD 56-. The proportion of the sub-population of γ δ T cells is shown in FIG. 6, and the γ δ T of cord blood-derived MILS (CB-MILS) is mainly γ δ T of v δ 1 type and a fraction of v δ 1-vδ2-Type gamma delta T, v delta 1-vδ2-And the v delta 2 proportion is 63.12 +/-8.5%, 34.44 +/-7.53% and 2.44 +/-1.63% respectively. The gamma delta T of peripheral blood derived MILS (PB-MILS) is mainly gamma delta T of the v delta 2 type, v delta 2, v delta 1-vδ2-And the v delta 1 ratios are 75.87 +/-4.37%, 14.43 +/-2.14% and 9.59 +/-5.56%, respectively.
5) Has killing effect on AML primary tumor cells
PBMC are separated from peripheral blood of Acute Myelocytic Leukemia (AML), CFSE stained PBMC is used as target cells, MILS cells are added into the target cells according to different effect target ratios (2:1, 10: 1, 50: 1), after mixing, the cells are incubated for 4 hours, and the cell killing is detected by flow cytometry. MILS had a killing effect on AML primary cells (fig. 7), with a potency to target ratio of 2: the killing rate of 1 was 8.2%, 10: the killing rate of 1 was 28.13%, 50: the killing rate of 1 was 65.36%.
6) Killing effect on tumor cell line
K562 cells stained with CFSE as target cells (2X 106Staining was performed at a rate of 100. mu.l of 2. mu.M CFSE working solution added to each cell). At a rate of 1X 10 per hole5Cells were added to 96-well plates per 100. mu. l K562. Then adding a proper amount of MILS effector cells into the target cells according to the effective target ratio of 1:1, mixing, incubating for 4 hours, and detecting the cell killing ratio by flow cytometry. MILS had a killing effect on K562 cells (FIG. 8), with a killing rate of 80.0%.
While specific embodiments of the invention have been described in detail, those skilled in the art will understand that: various modifications and changes in detail can be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. A full appreciation of the invention is gained by taking the entire specification as a whole in the light of the appended claims and any equivalents thereof.

Claims (10)

1. An activation medium comprising the following cytokine combinations: OK432, IFN-gamma, IL-2, IL-15, IL-21; preferably, the final concentration of OK432 is 100ng/ml, the final concentration of IFN-gamma is 1000IU/ml, the final concentration of IL-2 is 1000IU/ml, the final concentration of IL-15 is 100ng/ml, and the final concentration of IL-21 is 100 ng/ml;
preferably, the activation medium further comprises: KMB581, autologous plasma or human AB serum; preferably, the ratio of autologous plasma or human AB serum is 10%.
2. A method for producing mixed innate lymphocytes, the method comprising the steps of:
1) CD4-/CD8-T cell sorting;
2) mixed innate lymphocyte induction: culturing CD4-/CD8-T cells using the activated medium of claim 1;
3) mixed innate lymphocyte expansion: culturing the cells obtained in step 2) using an amplification medium;
preferably, the amplification medium comprises KMB581, IL-2, autologous plasma or human AB serum; preferably, the final IL-2 concentration is 1000IU/ml, and the ratio of autologous plasma or human AB serum is 5%.
3. The method for preparing according to claim 2, wherein the step 1) comprises the steps of:
a) separating the white membrane layer cells;
b) red blood cell lysis;
c) CD4-/CD8-T cell sorting;
preferably, the buffy coat cells are isolated from blood; more preferably, the blood comprises umbilical cord blood or peripheral blood;
preferably, the step of isolating the cells of the buffy coat comprises: centrifuging the blood to obtain autologous plasma; resuspending the centrifuged blood cell sediment with normal saline to obtain diluted blood, adding a mononuclear cell separation solution, wherein the volume ratio of the diluted blood to the mononuclear cell separation solution is 2:1, then centrifuging to collect leucocyte layer cells, and washing twice with normal saline;
preferably, the step of lysing the red blood cells comprises: adding a red blood cell lysate to lyse the white membrane cells obtained in the step a), discarding the supernatant, adding physiological saline to carry out heavy suspension cleaning, centrifuging and discarding the supernatant to obtain mononuclear cells, and carrying out heavy suspension on the cells by using the physiological saline;
preferably, the step of sorting CD4-/CD8-T cells comprises:
i) preparing a sorting buffer solution;
ii) centrifuging the single nuclear cell suspension, removing the supernatant, adding a sorting buffer solution, CD4 MicroBeads human and CD8 MicroBeads human, uniformly mixing, and incubating at low temperature;
iii) adding a sorting buffer solution to wash the cells, centrifuging and removing a supernatant;
iiii) resuspending the cells by adding sorting buffer;
iiii) preparing LD magnetic sorting column, rinsing the column with buffer solution, adding the cell suspension obtained by iiii) to sort cells, and then adding buffer solution to clean the column, wherein the negative cell sorted out is CD4-/CD 8-cell.
4. The method of claim 2, wherein the step of inducing the mixed innate lymphocytes in the step 2) further comprises an antibody coating comprising: secondary antibody coating and primary antibody coating;
preferably, the primary antibody is coated with anti-human TCR gamma/delta;
preferably, the secondary antibody is coated by Goat Anti-mouse lgG;
preferably, a secondary antibody coating; affinipure F (ab') 2Fragment Goat Anti-mouse lgG, the working concentration of Fcy Fragment specificity is 10 mug/ml, each bottle of T25 cell culture bottle is 3ml of antibody diluent, the incubation is carried out for 1h at 37 ℃, secondary antibody is discarded, and DPBS is added to wash the T25 cell culture bottle for one time;
preferably, the primary antibody coating: LEAFTMThe working concentration of the Purified anti-human TCR gamma/delta is 100ng/ml, each bottle of T25 cell culture bottle is 3ml of antibody diluent, the incubation is carried out for 1h at room temperature, primary antibody is removed, DPBS is added to soak the T25 cell culture bottle for standby。
5. The method of claim 2, wherein the mixed innate lymphocytes induce: culturing CD4-/CD8-T cells for 6-8 days using the activated medium of claim 1.
6. The method of claim 2, wherein the mixed innate lymphocyte proliferation step is as follows: gradually changing the activation culture medium into an amplification culture medium by means of half liquid change from 7-9 days of culture;
preferably, starting from day 7 to day 9 of culture, cells cultured for 6 to 8 days are inoculated into culture flasks which are not coated or are coated with anti-human TCR gamma/delta antibody, and the activation medium is gradually changed to the amplification medium by means of half-liquid change.
7. Mixed innate lymphocytes obtained by the production method according to any one of claims 1 to 6;
preferably, the mixed innate lymphocytes are mixed lymphocytes predominantly composed of γ δ T cells and NK cells, wherein a subpopulation of γ δ T cells is predominantly composed of v δ 1, v δ 2 and v δ 1-vδ2-Three subgroups;
more preferably, the ratio of v δ 1 is highest among mixed innate lymphocytes obtained from cord blood, followed by v δ 1-vδ2-V δ 2 is the lowest; preferably, v δ 1-vδ2-The v delta 2 proportion is respectively 60 +/-10%, 35 +/-10% and 3 +/-2%;
more preferably, the ratio of v δ 2 is highest among mixed innate lymphocytes obtained from peripheral blood, followed by v δ 1-vδ2-V δ 1 is the lowest; preferably, v δ 2, v δ 1-vδ2-And v.delta.1 ratios of 75 + -10%, 15 + -5% and 10 + -5%, respectively.
8. A kit or medicament comprising the mixed innate lymphocyte of claim 7.
9. A method for preventing and/or treating a tumor or an autoimmune disease in a subject, comprising administering to a subject in need thereof an effective amount of the mixed innate lymphocyte of claim 7, the medicament of claim 8.
10. Use of the mixed innate lymphocyte of claim 7, characterized in that the use comprises any of:
1) the application in preparing the medicine for preventing and/or treating tumor or autoimmune disease;
2) the application of the chimeric antigen receptor in preparing immune cells expressing the chimeric antigen receptor;
3) use in the manufacture of a kit according to claim 8.
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Application publication date: 20210709