CN111876382A - Method for preparing universal immune cells and application thereof - Google Patents

Method for preparing universal immune cells and application thereof Download PDF

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CN111876382A
CN111876382A CN202010821035.7A CN202010821035A CN111876382A CN 111876382 A CN111876382 A CN 111876382A CN 202010821035 A CN202010821035 A CN 202010821035A CN 111876382 A CN111876382 A CN 111876382A
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cell
cells
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immune cell
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刘华
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Shanghai Xinghua Biomedical Technology Co ltd
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Abstract

The invention provides a simple and convenient method for preparing universal immune cells and application thereof, and the method cultures allogeneic immune cells, specific mitogens, cytokines and immunologic adjuvants in a liquid cell culture medium in a culture container together, thereby obtaining the universal immune cell culture with high immunocompetence. The method is simple and easy to operate, and adopts allogeneic immune cells, so that the quality of immune cell preparation can be improved, the cost of cell therapy can be reduced, and the safety of cell therapy can be improved. In addition, a large amount of practical application in clinical practice proves that the universal immune cell is safe, reliable, lasting and effective and has no rejection reaction. Therefore, the cell can be used as a parent cell of other types of universal immune cell preparations, such as CAR-T, TCR-T, and is used in the fields of adoptive cellular immunotherapy and the like. The invention solves the problems of high cost, poor safety, low efficiency and the like of the existing cellular immunotherapy, and provides a new way for the application and popularization of the cellular immunotherapy.

Description

Method for preparing universal immune cells and application thereof
Technical Field
The invention relates to the field of biomedicine, in particular to a method for preparing universal immune cells and application thereof.
Background
In recent decades, the field of tumor therapy has made great progress. Cell-based therapies, particularly against Tumor Associated Antigens (TAAs), have evolved rapidly. T cells were engineered to have the ability to attack tumor cells by generating CAR constructs consisting of genes encoding scFv, costimulatory domains (CD28 or TNFRSF9) and CD247 signaling domains for T cell proliferation and activation. In principle, CAR-T cells are activated by scFv recognition of TAAs on the surface of T cells, and then intracellular signaling domains linked by scFv are subsequently activated to induce downstream signaling pathways involved in T cell proliferation, activation and cytokine production.
Currently, the effective function of CAR-T cell therapy has been demonstrated in a variety of diseases including hematological malignancies, solid tumors, autoimmune diseases, and allergic diseases such as asthma. Furthermore, antigen-specific T regulatory cells (Tregs) and gene-edited T cells appear to be beneficial in controlling the inflammation of allergic asthma.
CAR T therapy differs from traditional drugs, but is primarily an autologous T cell therapy that incorporates the CAR gene. This therapy requires rapid completion of blood draw, isolation, activation, transfection, amplification, formulation, release, cryopreservation, transport and administration. CAR T cell therapy is therefore a rather expensive and personalized process, requiring expensive measures to collect the patient's cells, design them, and reinject them into the patient at each stage, with appropriate quality control. In addition, traditional Chimeric Antigen Receptors (CARs) have a fixed design, and one type of CAR T cell can only target one epitope. This rigid design limits clinical applications and results in extremely high manufacturing costs.
In 2017 the FDA approved two CAR-T cell therapies (yescata and kymeriah), which has become one of the most promising therapies for treating certain types of cancer. This success provides the opportunity to optimize the production of CAR T cells, making the patient more easily treated. However, currently two approved T cell products with Chimeric Antigen Receptors (CARs) are derived from autologous T cells. These CAR T cells approved for clinical use must be generated on a customized basis. Due to the expensive and lengthy production process, this autologous T cell production platform remains a significant limiting factor for large-scale clinical applications. Production failures also present an inherent risk. Personalized, customized autologous CAR T cell production processes have also impacted the wide range of applications for various tumor types.
In order to more broadly implement advanced cell therapy and reduce costs, it would be advantageous to use allogeneic "universal" cell therapy products that can be stored in cell banks in a manner similar to biopharmaceutical drug products and provided on demand. Thus, universal allogeneic T cells can be used to prepare universal CAR T cells, which can be used as "off-the-shelf" ready-to-use biotherapeutic agents for large-scale clinical applications. This would greatly reduce the cost and time required for traditional CAR T cell therapy. Currently, the development of universal CAR-T mainly involves the type of immune cells and their production, as well as the vector and structure of CAR-T. The main technical route is as follows: abrogating expression of endogenous α β T Cell Receptor (TCR) to prevent graft versus host responses; gene editing techniques; other types of immune cells; and (4) iPSC technology.
When allogenic CAR T cell infusion is considered, host versus graft and graft-versus-host rejection must be avoided to prevent rejection of adoptive transfer cells, host tissue damage and cause significant anti-tumor consequences. One modification currently employed is to knock out genes that prevent allogeneic therapy, such as endogenous T cell receptors, at the same time the CAR is introduced. Making it non-existent at its cell surface TCR α β receptors to prevent their alloreactivity. This technique clearly further increases the complexity of the manufacturing procedure and also has the potential for incomplete TCR α β receptor knock-out.
Since multiple gene targets can be edited simultaneously, some clinical trials employ the CRISPR/Cas9 system for CAR-T cell improvement. The combination of these powerful technologies is expected to provide a new generation of CAR-T cell-based immunotherapy for clinical application, i.e., knocking out endogenous TCR by gene editing technology and improving the anti-tumor activity and safety of T cells. However, this method also further increases the complexity of the manufacturing operation and also increases the production cost.
Natural Killer (NK) cells can be xenografted and are likely to be off-the-shelf products, making CAR-NK cell therapy a versatile product. These products may be safer than CAR-T cell therapy. Scientists reported a novel PCa treatment based on NK-92 cell engineering, CAR recognized human Prostate Specific Membrane Antigen (PSMA), which is overexpressed in prostate tumor cells. Following CAR transduction, NK-92/CAR cells acquired high and specific lytic activity in vitro against PSMA-expressing prostate cancer cells, and also degranulated and produced high levels of IFN- γ in response to antigen recognition. However, this technique requires lethal irradiation of the effector, a safety measure required for clinical applications targeting NK-92 cells, which can completely eliminate replication, but does not affect phenotype and short-term function.
Another investigator explored a technical route to the development of ready-to-use cell therapies based on Induced Pluripotent Stem Cell (iPSC) -derived Natural Killer (NK) cells. Strategies to design iPSC-derived NK (iPSC-NK) cells to enhance immune cell function, prolong persistence and promote cell homing are proposed. However, to date, there has been no report of iPSC-derived CTL (iPSC-CTL).
The invention discloses a method for preparing antigen-specific immunocyte medicaments (CN101626781B, CN102847143B and CN102847144B) applied in the prior art, which is clinically applied in the last 10 yearsIn use, approximately 800 patients with tumors were treated, ranging in age from 18 months to 93 years, for a maximum of 500 uses over a maximum of ten years. The total number of peripheral blood leukocytes is in the range of 0.9-30.0 × 109And L. Proved by clinical multi-center application: the safety is good, and there are no graft-versus-host rejection (GVHD) and autoimmune diseases, and about 50-60% of patients treated by traditional DLI have GVHD.
Therefore, the applicant intends to design a method for preparing universal allogeneic T cells based on the basis of the prior patents, and provide maternal cells, such as CAR-T, TCR-T or other immune cell types, for other types of universal immune cell preparations for the fields of adoptive immune cell therapy and the like, in order to improve the quality of immune cells, reduce the cost of cell therapy and improve the safety thereof.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a simple and convenient method for preparing universal immune cells and application thereof. The prepared universal immune cell is safe, reliable, durable and effective, and has no graft-versus-host rejection reaction.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for preparing universal immune cells, comprising the steps of:
step 1, allogeneic immune cells (such as mononuclear cells) and mitogen, cytokines and immune adjuvants are placed in a culture container in a liquid cell culture medium for co-culture, so that a universal immune cell culture is obtained;
step 2, separating a cell population from the culture obtained in step 1;
wherein the cytokine is derived from one or more of lymphokines, monokines, inflammatory cytokines and hematopoietic cytokines produced by lymphocytes, monocytes and other cells.
Further, the cytokine is selected from any one or more of interleukins, interferons, tumor necrosis factors, colony stimulating factors, chemotactic cytokines and transforming growth factors.
Furthermore, the above cytokine is one or more of interleukin-2, interleukin-6, interleukin-15, and interferon.
Further, the concentration of the cytokine is 20 to 500 ten thousand units/L.
Further, the concentration of the cytokine is 50 to 200 ten thousand units/L.
Further, the mitogen is selected from one or more of canavalin, phytohemagglutinin, pokeweed, lipopolysaccharide, and dextran.
Further, the concentration of the mitogen is 10 to 1000 ten thousand units/L.
Further, the concentration of the mitogen is 0.1mg to 10 mg/L.
Furthermore, the immunological adjuvant is selected from one or more of biological adjuvant, inorganic adjuvant, organic adjuvant, synthetic adjuvant, oil agent and Freund's adjuvant, and the concentration is 0.01ml-1 ml/L.
Further, the immune cells are derived from peripheral blood or umbilical cord blood.
Further, the immune cells may be allogeneic or autologous cells.
Further, the concentration of the above immunocytes is 1X 103-1×1011One per ml.
Further, the cell population includes one or more of Cytotoxic T Lymphocytes (CTL), Tumor Infiltrating Lymphocytes (TIL), cytokine-activated killer Cells (CIK), lymphokine-activated killer cells (LAK), natural killer cells (NK), tumor-associated macrophages (TAM), Activated Killer Monocytes (AKM), and Dendritic Cells (DC).
Further, the above-mentioned cell population having immunological activity is lymphocytes such as CTL, NK, CIK and B cells, etc., and most preferably cytotoxic T lymphocytes.
Further, the co-cultivation time in the step 1 is 3-180 days.
Further, the culture vessel is a three-dimensional large-volume high-density cell culture vessel.
Further, the method comprises a cloning step: cloning the culture obtained in the step 1 or the cell population with immunocompetence obtained in the step 2 in a liquid cell culture medium to obtain a cell strain with immunocompetence. The cloning adopts an intermittent cycle stimulation method or a continuous stimulation method.
In a second aspect of the present invention, there is provided a universal immune cell prepared by the above method, wherein the universal immune cell comprises one or more of Cytotoxic T Lymphocyte (CTL), Tumor Infiltrating Lymphocyte (TIL), cytokine-activated killer Cell (CIK), lymphokine-activated killer cell (LAK), natural killer cell (NK), tumor-associated macrophage (TAM), Activated Killer Monocyte (AKM), and Dendritic Cell (DC).
The third aspect of the present invention provides the use of the universal immune cells of the second aspect in the preparation of an immune cell preparation, wherein the maternal cells of the immune cell preparation are universal immune cells.
Further, the immune cell preparation comprises CAR-T cells or TCR-T cells.
In a fourth aspect of the present invention, an immune cell preparation is provided, wherein the immune cell preparation is prepared by using the universal immune cell of the second aspect as a maternal cell.
In a fifth aspect, the present invention provides a biologic drug for adoptive cellular immunotherapy, comprising a container and the immune cell preparation of the fourth aspect disposed in the container.
Furthermore, the biological medicine also comprises normal saline, albumin and other stabilizing agents.
A sixth aspect of the invention provides the use of an immune cell preparation according to the fourth aspect of the invention in the manufacture of a medicament for the treatment and prevention of disorders associated with abnormal function and number of lymphocytes.
Further, the administration route of the drug is intravenous, thoracic, intraperitoneal, intraspinal, intradermal, subcutaneous, or intratumoral injection, etc.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the method for preparing the universal immune cells is simple and convenient, is easy to operate, and can improve the quality of immune cell preparations, reduce the cost of cell therapy and improve the safety of the cell therapy by adopting allogeneic immune cells. In addition, a large amount of practical application in clinical practice proves that the universal immune cell is safe, reliable, lasting and effective and has no rejection reaction. Therefore, the cell can be used as a parent cell of other types of universal immune cell preparations, such as CAR-T, TCR-T or other immune cell types, for the fields of adoptive cellular immunotherapy and the like, the problems of high cost, poor safety, low efficiency and the like of the existing cellular immunotherapy are solved, and a new way is provided for the application and popularization of the cellular immunotherapy.
In addition, the method for preparing the medicament of the invention changes the action mode of the traditional vaccine, the traditional vaccine is made of pathogenic organisms or antigenic substances thereof, after being input into a human body, the immune system generates protective substances (such as antibodies), and the immune system manufactures more protective substances to prevent pathogenic bacteria from being damaged according to the original memory.
Drawings
FIG. 1 is a graph showing the distribution of the significant difference gene Pathway of Cell1 based on Pathway analysis in an effective example of the present invention;
FIG. 2 is a diagram illustrating the interaction between Cell1 and Cell2 signal paths for activation in an advantageous embodiment of the present invention; the circle with the inner shadow is a signal path which is not changed, the circle with the inner oblique line is a signal path activated in the Cell1, and the blank circle is a signal path activated in the Cell 2;
FIG. 3 is a graph showing the distribution of the significantly different gene pathways of Cell2 based on Pathway analysis in an effective example of the present invention.
Detailed Description
After extensive and intensive studies, the inventors have found through extensive experiments and experiments that allogeneic mononuclear cells can be cultured together with mitogens, cytokines and immunoadjuvants to produce safe, reliable, durable, effective, general-purpose immunocytes (particularly CTLs) without rejection, and provide low-cost, safe and reliable maternal cells for other types of general-purpose immunocyte preparations, such as CAR-T, TCR-T or other immunocyte type cellular immunotherapies.
The first aspect of the invention is to provide a simple method for preparing universal immune cells, which cultures allogeneic mononuclear cells together with mitogens, cytokines and immunoadjuvants to obtain a large amount of universal immune cells. The method specifically comprises the following steps:
step 1, allogeneic immune cells (such as mononuclear cells) and mitogens, cytokines and immune adjuvants are placed in a culture container in a liquid cell culture medium to be cultured together, so that a universal immune cell culture with high immune activity is obtained;
step 2, separating the cell population with immunocompetence from the culture obtained in the step 1;
wherein the cytokine is derived from one or more of lymphokines, monokines, inflammatory cytokines and hematopoietic cytokines produced by lymphocytes, monocytes and other cells.
In a preferred embodiment of the present invention, the cytokine is derived from any one or more of interleukins, interferons, tumor necrosis factors, colony stimulating factors, chemotactic cytokines and transforming growth factors; in a preferred embodiment of the present invention, the cytokine is selected from one or more of interleukin-2, interleukin-6, interleukin-15, and interferon.
In a preferred embodiment of the present invention, the cytokine is interleukin-2 and/or interferon. During co-culture, allogeneic mononuclear cells can also secrete IL-2 and other cytokines autonomously due to stimulation by mitogens, cytokines and immunoadjuvants, and thus have low dependence on IL-2, and even do not require IL-2. Of course, exogenous IL-2 may be added simultaneously during the culture, thereby further promoting the mass expansion of activated T lymphocytes.
In a preferred embodiment of the present invention, the concentration of the cytokine is 20 to 500 ten thousand units/L in the culture in order to obtain a culture of the immunocompetent cell population. In a preferred embodiment of the present invention, the concentration of the cytokine is 50 to 200 ten thousand units/L.
In a preferred embodiment of the present invention, the mitogen is selected from one or more of concanavalin, phytohemagglutinin, pokeweed, lipopolysaccharide and dextran.
In a preferred embodiment of the invention, the concentration of said mitogen is 10 to 1000 ten thousand units/L when cultured in order to obtain a culture of an immunocompetent cell population. In a preferred embodiment of the invention, the concentration of said mitogen is between 50 and 500 ten thousand units/L.
In a preferred embodiment of the invention, the concentration of said mitogen is 0.1mg to 10mg/L during the cultivation in order to obtain a culture of an immunocompetent cell population. In a preferred embodiment of the invention, the concentration of said mitogen is between 0.5mg and 5 mg/L.
In a preferred embodiment of the present invention, the immunological adjuvant is selected from one or more of a biological adjuvant, an inorganic adjuvant, an organic adjuvant, a synthetic adjuvant, an oil agent and a freund adjuvant; in order to obtain a culture of the immunocompetent cell population, the concentration of the immunoadjuvant is 0.01ml to 1ml/L when the culture is performed. In a preferred embodiment of the present invention, the concentration of the immunoadjuvant is 0.1ml to 0.5 ml/L.
In a preferred embodiment of the invention, the immune cells are derived from peripheral blood or umbilical cord blood. In a preferred embodiment of the invention, the immune cells are derived from peripheral blood.
In a preferred embodiment of the invention, the immune cells are allogeneic or autologous cells. In a preferred embodiment of the invention, the immune cells are allogeneic cells.
In a preferred embodiment of the present invention, in order to obtain a culture of an activated immune cell population, the concentration of the immune cells is 1X 10 at the time of culturing3-1×1011One per ml. In a preferred embodiment of the present invention, the concentration of the immune cells is 1 × 105-1×109One per ml.
However, the ratio of different types of immune cells (i.e., allogeneic monocytes) to mitogens, cytokines and immune adjuvants may be different, and one skilled in the art can determine the appropriate ratio by experimentation or the nature of the immune cells.
In a preferred embodiment of the present invention, the immunocompetent cell population includes Cytotoxic T Lymphocytes (CTL), Tumor Infiltrating Lymphocytes (TIL), cytokine-activated killer Cells (CIK), lymphokine-activated killer cells (LAK), natural killer cells (NK), tumor-associated macrophages (TAM), Activated Killer Monocytes (AKM), and Dendritic Cells (DC).
In a preferred embodiment of the present invention, the above-mentioned cell population having immune activity is lymphocytes, such as CTL, NK, CIK and B cells; in a more preferred embodiment, the population of immunocompetent cells is cytotoxic T lymphocytes.
In a preferred embodiment of the present invention, the co-cultivation time in step 1 is 3-180 days; in a preferred embodiment of the present invention, the co-cultivation time in step 1 is 3 to 60 days; in a preferred embodiment of the present invention, the co-cultivation time in step 1 is 7-28 days; in a more preferred embodiment, the co-cultivation time in step 1 is 14-21 days.
In a preferred embodiment of the present invention, the culture vessel is a three-dimensional large-volume high-density cell culture vessel, but may be any other known culture vessel including a bioreactor.
In a preferred embodiment of the present invention, the method further comprises a cloning step: cloning the culture obtained in the step 1 or the cells with immunocompetence obtained in the step 2 in a liquid cell culture medium to obtain a cell strain with immunocompetence. The cloning adopts an intermittent cycle stimulation method or a continuous stimulation method.
In a second aspect of the present invention, there is provided a universal immune cell prepared by the above method, wherein the universal immune cell comprises one or more of Cytotoxic T Lymphocyte (CTL), Tumor Infiltrating Lymphocyte (TIL), cytokine-activated killer Cell (CIK), lymphokine-activated killer cell (LAK), natural killer cell (NK), tumor-associated macrophage (TAM), Activated Killer Monocyte (AKM), and Dendritic Cell (DC).
In a preferred embodiment of the present invention, the universal immune cells are lymphocytes, such as CTL, NK, CIK and B cells. In a more preferred embodiment, the universal immune cell is a cytotoxic T lymphocyte.
In a third aspect of the present invention, there is provided a use of the universal immune cell of the second aspect as a parent cell of an immune cell preparation in preparing an immune cell preparation.
In a preferred embodiment of the present invention, the immune cell preparation comprises CAR-T cells or TCR-T cells; the immune cell preparation can be used for adoptive cell immunotherapy and can be clinically used as a cell biological medicament for treating and preventing various blood system diseases and various solid tumors.
In a preferred embodiment of the present invention, the hematological disease includes: 1) red blood cell diseases such as aplastic anemia, Paroxysmal Nocturnal Hemoglobinuria (PNH), etc.; 2) leukocyte diseases, such as various leukemias, malignant lymphomas, malignant lympho-reticulocytosis, plasmacytosis, histiocytic proliferative diseases, myelodysplastic syndromes, myeloproliferative diseases (polycythemia vera, essential thrombocythemia, myelofibrosis) and the like.
In a preferred embodiment of the present invention, the solid tumor may be selected from: nasopharyngeal carcinoma, esophageal carcinoma, gastric carcinoma, liver cancer, breast cancer, colorectal cancer, prostate cancer, lung cancer, cervical cancer, leukemia, oral cancer, salivary gland tumor, malignant tumor of nasal cavity and paranasal sinus, laryngeal carcinoma, ear tumor, eye tumor, thyroid tumor, mediastinal tumor, chest wall, pleural tumor, small intestine tumor, biliary tract tumor, pancreatic cancer and periampulla tumor, mesenteric and retroperitoneal tumor, kidney tumor, adrenal tumor, bladder tumor, prostate cancer, testicular tumor, penile cancer, endometrial cancer, ovarian malignant tumor, malignant trophoblastic tumor, vulval cancer and vaginal cancer, malignant lymphoma, multiple myeloma, soft tissue tumor, bone tumor, skin and accessory tumor, malignant melanoma, nervous system tumor, various pediatric tumors, and the like.
In a fourth aspect of the present invention, an immune cell preparation is provided, wherein the immune cell preparation is prepared by using the universal immune cell of the second aspect as a maternal cell.
In a fifth aspect, the present invention provides a biologic drug for adoptive cellular immunotherapy, comprising a container and the immune cell preparation of the fourth aspect disposed in the container.
In a preferred embodiment of the present invention, the biological drug further comprises physiological saline, albumin, and other stabilizers.
The sixth aspect of the present invention provides the use of the immune cell preparation of the fourth aspect of the present invention in the preparation of a medicament for treating and preventing diseases associated with abnormal function and number of lymphocytes, including but not limited to the following fields: tumor immunity, transplantation immunity, hypersensitivity immunity, autoimmune disease, immunoproliferative disease, immunodeficiency disease, infection and immunity, aging and immunity, reproduction and immunity, reproductive system and immunity, immunohematologic disease, respiratory system disease and immunity, renal disease and immunity, digestive system disease and immunity, endocrine disease and immunity, cardiovascular system disease and immunity, connective tissue disease, immunodermatology, trauma and immunity, parasitic disease and immunity. For example, for the treatment and prevention of diseases such as tumors, AIDS and viral hepatitis, coronavirus or other types of viruses, or for delaying aging).
The medicine can be a vaccine for improving the immune function of the body, or a medicine for treating diseases by improving the immune function of the body. The vaccine and the medicament can be prepared into any available dosage form.
In a preferred embodiment of the present invention, the administration route of the drug is intravenous, intrathoracic, intraperitoneal, intraspinal, intradermal, subcutaneous, or intratumoral injection, etc.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1
This example provides a simple method for preparing universal immune cells, comprising the following steps:
allogenic mononuclear cells derived from normal human peripheral blood are cultured together with mitogen, cytokines and immunologic adjuvants in a liquid culture medium, wherein the consumption of the mitogen concanavalin in the culture medium is 10 ten thousand units/L; the dosage of the cytokine interleukin-2 in the culture medium is 50 ten thousand units/L; the dosage of the immunologic adjuvant (5% Tween-80) is 0.5 mL/L.
The allogeneic mononuclear cells are cultured together with mitogen, cytokines and immunologic adjuvants for 30 days to obtain immune cell culture with immunocompetence, mainly cytotoxic T lymphocytes.
Example 2
This example provides a simple method for preparing universal immune cells, comprising the following steps:
mononuclear cells derived from allogeneic peripheral blood, co-cultured with a mitogen, a cytokine and an immunoadjuvant in a liquid culture medium comprising: mitogen phytohemagglutinin 0.5 mg/L; the cytokine interferon is 500 ten thousand units/L; the dosage of the immunologic adjuvant (5% Tween-80) is 0.1 mL/L.
The allogeneic mononuclear cells are cultured together with mitogen, cytokines and immunologic adjuvants for 30 days to obtain immune cell culture with immunocompetence, mainly cytotoxic T lymphocytes.
Example 3
This example provides a simple method for preparing universal immune cells, comprising the following steps:
allogenic mononuclear cells derived from normal human umbilical cord blood are co-cultured with mitogen, cytokines and immunologic adjuvants in a liquid culture medium, wherein the dosage of the mitogen concanavalin in the culture medium is 100 ten thousand units/L; the dosage of the cytokine interleukin-2 in the culture medium is 100 ten thousand units/L; the dosage of the immunologic adjuvant (5% Tween-80) is 0.1 mL/L.
Allogeneic mononuclear cells are co-cultured with mitogens, cytokines and immunoadjuvants for 45 days to obtain an immune cell culture with activated immunocompetence, mainly cytotoxic T lymphocytes.
Example 4
This example provides a simple method for preparing universal immune cells, comprising the following steps:
mononuclear cells derived from allogeneic cord blood, co-cultured with mitogens, cytokines and immunoadjuvants in a liquid culture medium comprising: 1mg/L of cytomitogen phytohemagglutinin; 300 ten thousand units/L of cytokine interferon; the dosage of the immunologic adjuvant (5% Tween-80) is 0.5 mL/L.
The allogeneic mononuclear cells are cultured together with mitogen, cytokines and immunologic adjuvants for 60 days to obtain immune cell culture with immunocompetence, mainly cytotoxic T lymphocytes.
Effect example 1
Using T lymphocytes as an example, a large number of activated and expanded immune cells can be obtained by the above method. The single cell whole gene expression sequencing method and the whole gene expression sequencing method are used. Characteristic gene screening analysis 2 different groups of immune effector cells: cell2 is cultured by immune cells, mitogens, cytokines and immunologic adjuvants for 8 days; cell1 is cultured by immune cells together with mitogen, cytokine and immunoadjuvant for 26 days. The following results are the immune cell whole gene signature found together using both assays (Table 1).
TABLE 1 Whole Gene signature information for immune cells
Gene log2FoldChange Cell type Description of the molecules
CFTR 4.91 Cell 1 Cystic fibrosis transmembrane conductance regulator
CFH 4.73 Cell 1 Complement factor H
FGR 1.95 Cell 1 FGR protooncogene, Src family tyrosine kinase
TMEM176A -1.92 Cell 2 Transmembrane protein 176A
KLHL13 -5.56 Cell 2 Kelch-like protein 13
CD38 -6.28 Cell 2 CD38 molecule
When two groups of samples are different, the difference gene is taken as a research object to be further researched. Comparing the two groups by using DESeq2 to obtain the Fold-change of Marker gene difference between samples; in this analysis, two groups were compared and differential gene screening was performed under the conditions that the Fold change was log-transformed into log2Fold change, and genes with log2Fold change >1.9 or < -1.9 were selected as marker genes, and as can be seen from table 1, the differential expression characteristics of these genes were evident.
Effect example 2
Taking T lymphocyte as an example, the method can obtain a large amount of activated and expanded immune cells. Cell1 is cultured by immune cells, mitogen, cytokine and immunologic adjuvant for 26 days; then Pathway analysis was performed.
The system analyzes the relationship among genes (and coding products thereof), gene functions and a database of genome information, and is helpful for researchers to research the genes and expression information as a whole network. The integrated metabolic pathways (pathways) provided by KEGG, including the metabolism of carbohydrates, nucleosides, amino acids, etc., and the biodegradation of organic matter, not only provide all possible metabolic pathways, but also make comprehensive annotations on the enzymes that catalyze the reactions of the various steps. KEGG is a powerful tool for in vivo metabolic analysis and metabolic network research. Currently, the KEGG Pathway is divided into 8 categories, which are eight major categories of overall network, metabolic process, genetic information transfer, environmental information transfer, intracellular biological process, biological system, human disease and drug development.
The Pathway analysis is based on a KEGG database, Fisher accurate test and chi-square test are utilized for differential genes, Pathway in which target genes participate is subjected to significance analysis, and the Pathway is screened according to p value <0.05 to obtain significant Pathway. Based on the significance level (p value) of each of the differential gene pathways with up-and down-regulated significance and the number of differential genes included in the analysis results, a significant differential gene pathway profile can be constructed. In the figure, the ordinate is the name of the differential gene pathway, and the abscissa is the negative logarithm of p value (-LgP), and a larger value indicates that p value is smaller, that is, the significance level of the differential gene pathway is higher, and the size of the bubble represents how much the pathway contains the number of the differential gene. For the convenience of the researcher, we performed a bubble map of the significantly different gene functions of up-and down-regulation of the fractions (fractions with p value less than 0.05; if more than 0.05, only the first 30 smaller p value are shown). Taking the significance pathway diagram as an example, the significance part results are shown in fig. 1.
As can be seen from FIG. 1, there are two signal pathways enriched with marker genes, which are: the hematopoetic Cell line (Hematopoietic Cell line) signal pathway and the Cytokine-Cytokine receptor interaction (Cytokine interaction) signal pathway, which are the main characteristic pathways for Cell1 activation.
Effect example 3
Taking T lymphocyte as an example, the method can obtain a large amount of activated and expanded immune cells. Cell1 is cultured by immune cells together with mitogen, cytokine and immunologic adjuvant for 26 days, and Cell2 is cultured by immune cells together with mitogen, cytokine and immunologic adjuvant for 8 days; the Cell1 and Cell2 signal pathways were then analyzed for activation interaction, and the results are shown in fig. 2.
In fig. 2, these effects lead to the formation of signaling pathways that activate the transmission network, thereby cascade inducing an immune cell phenotype. After analyzing all immune signal pathways, we found that the hematopoietic lineage signal pathway is a marker pathway of Cell1, receives stimulation from an upstream signal pathway, is a very important effector signal pathway, and a cytokine-cytokine receptor interaction signal pathway is a core effector target stimulated by a plurality of upstream signal pathways.
Effect example 4
Taking T lymphocyte as an example, the method can obtain a large amount of activated and expanded immune cells. Cell2 is cultured by immune cells together with mitogen, cytokine and immune adjuvant for 8 days, then Pathway analysis is carried out (as shown in figure 3), and similarly, we analyze that the main signal path activated by Cell2 is the ECM receptor interaction signal path, and the enrichment degree of the marker gene is most obvious (-LgP max): based on the significance level (p value) of each of the differential gene pathways with up-and down-regulated significance and the number of differential genes included in the analysis results, a significant differential gene pathway profile can be constructed. The ordinate of the graph is the name of the differential gene pathway, and the abscissa is the negative logarithm of p value (-LgP), and a larger value indicates that p value is smaller, i.e., the level of significance of the differential gene pathway is higher. Wherein the shade of the color represents the degree of up-and-down regulation and the additive effect of the genes in the signal pathway, and the size of the bubble represents the number of the pathways containing the different genes.
Through the signaling pathway activation network, it was found that the ECM receptor interaction (receptor interaction) signaling pathway activates and re-strengthens the local adhesion signaling pathway in Cell2 in reverse.
Effect example 5
Using T lymphocyte as example, the invention can obtain a large amount of activated and expanded immune cells by the method
Figure BDA0002634415930000131
The cell concentration and activity rate of the PBMC samples is detected by a Rigel fluorescence analyzer and an AOPI dye method, and the specific results are shown in the following table 2:
table 2 activated immune cells: (
Figure BDA0002634415930000132
Rigel fluorescence Analyzer, AOPI method)
Figure BDA0002634415930000141
Flow cytometry analysis of immune cell subsets showed CD8 for samples 1, 2, and 3+T lymphocytes were 95.34%, 96.9% and 96.2%, respectively. These results are similar to those we have previously obtained using similar methods: CD8+T lymphocytes are increased by over 95%, mainly CD45RO positive cells, about 93%, strongly suggesting that the obtained immune effector cells belong to Cytotoxic T Lymphocytes (CTL).
Effect example 6
The universal immune cells prepared by the method provided by the invention are infused into a subject to verify the safety and effectiveness of the subject.
Subject information: the allogeneic universal immune cells prepared by the method are intravenously infused into Liu Jie, male, 65 years old and last two years every three months for one treatment course. Each treatment course comprises three times of venous return, the return is performed once every other day, and the number range of the returned cells is 5 multiplied by 108～1×1010。
The effect is as follows: the whole process is well tolerated without any clinical symptoms and signs related to rejection reaction of organs or systems such as skin, liver and intestinal tract, such as rash, jaundice and diarrhea. Moreover, after the allogeneic universal immune cells prepared by the method are input, ten great benefits are shown: (1) senile plaque is relieved; (2) improvement in skin texture; (3) disappearance of chronic eczema; (4) white hair is reduced; (5) enhancing the memory; (6) mild reduction in blood lipid; (7) the glycosylated hemoglobin is increased from lightness to normal; (8) the oral mucosa ulcer does not occur; (9) joint damage is reduced; (10) the vigor is vigorous.
Effect example 7
The universal immune cells prepared by the method provided by the invention are infused into a subject to verify the safety and effectiveness of the subject.
Subject information: xue Yi (a kind of Xue), male, 56 years old, liver cancer patients. The allogeneic universal immune cells prepared by the method are infused intravenously for nearly four years. The method is characterized in that the venous transfusion is performed for one treatment course every month at first, each treatment course is divided into five venous return transfusions, after two years, the venous transfusion is performed for one treatment course every other month, each treatment course is still five venous return transfusions, and the venous return transfusions are performed once every other day. The number of cells per return was in the range of 5X 108～1×109。
The effect is as follows: the tolerance is good in the whole process, and other adverse reactions are not observed except for occasional transient fever. The repeated examination shows that no special abnormal findings exist in the routine blood, liver function and kidney function. Various examinations, including imaging examinations, indicated that the tumor was in complete remission.
Effect example 8
The universal immune cells prepared by the method provided by the invention are infused into a subject to verify the safety and effectiveness of the subject.
Subject information: wangzhi, female, 75 years old, colon cancer patient. The allogeneic universal immune cells prepared by the method are infused intravenously once a month for nearly ten years, and the number of the cells infused back into the vein is 5 multiplied by 109。
The effect is as follows: the tolerance is good in the whole process, and no adverse reaction is observed. Clinical symptoms and signs of organs or systems such as skin, liver and intestinal tract, such as rash, jaundice, diarrhea, etc., do not appear. The repeated examination shows that no special abnormal findings exist in the routine blood, liver function and kidney function. Clinically there were no special symptoms and signs, and the various hematological examinations and tumor markers were normal. No special abnormal findings are found in the enteroscopy. Patient complains of common cold obviously reduced.
Effect example 9
The universal immune cells prepared by the method provided by the invention are infused into a subject to verify the safety and effectiveness of the subject.
Subject information: li A certain, male, age 70. Patients with malignant melanoma. The allogeneic universal immune cells prepared by the method are injected in various ways including intravenous infusion, intraperitoneal infusion and subcutaneous (intratumoral) injection within four years. Initially one treatment period every two weeks, later on, a transition is made to one treatment period every month, every three months or every six months. Each treatment course comprises four times of reinfusion, which is infused once every other day, and the number of cells reinfused each time ranges from 5X 108～1×109。
The effect is as follows: the whole process has good tolerance, pain is caused when the injection is injected subcutaneously, and no other adverse reaction is observed. Repeatedly checking that blood routine and liver and kidney functions have no special abnormal findings; clinical symptoms and signs of organs or systems such as skin, liver and intestinal tract, such as rash, jaundice, diarrhea, etc., do not appear. The patients are advanced malignant melanoma, relapse and metastasis after the operation and ineffective chemotherapy. The allogeneic universal immune cells prepared by the method are used for single treatment, and the tumor control is good.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications or alterations to this practice will occur to those skilled in the art and are intended to be within the scope of this invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (23)

1. A method for preparing a universal immune cell, comprising the steps of:
step 1, putting allogeneic immune cells, mitogens of cells, cytokines and immune adjuvants in a liquid cell culture medium and putting the liquid cell culture medium in a culture container for co-culture, thereby obtaining a universal immune cell culture;
step 2, separating a cell population from the culture obtained in step 1;
wherein the cell factor is derived from any one or more of lymphokines, monokines, cytokines for activating inflammation and cytokines for stimulating hematopoiesis, which are produced by lymphocytes, monocytes and other cells.
2. The method of claim 1, wherein the cytokine is derived from any one or more of interleukins, interferons, tumor necrosis factors, colony stimulating factors, chemotactic cytokines, and transforming growth factors.
3. The method of claim 1, wherein the cytokine is one or more of interleukin-2, interleukin-6, interleukin-15, and interferon.
4. The method of claim 1, wherein the cytokine is present at a concentration of 20 to 500 ten thousand units/L.
5. The method of claim 1, wherein the cytokine is present at a concentration of 50 to 200 ten thousand units/L.
6. The method according to claim 1, wherein the mitogen is selected from any one or more of concanavalin, phytohemagglutinin, pokeweed, lipopolysaccharide and dextran.
7. The method of claim 6, wherein the concentration of mitogen is from 10 to 1000 ten thousand units/L.
8. The method of claim 6, wherein the mitogen concentration is 0.1mg to 10 mg/L.
9. The method according to claim 1, wherein the immunoadjuvant is selected from any one or more of a biological adjuvant, an inorganic adjuvant, an organic adjuvant, a synthetic adjuvant, an oil agent and Freund's adjuvant, and the concentration is 0.01 ml/L to 1 ml/L.
10. The method of claim 1, wherein the immune cells are allogeneic or autologous cells derived from peripheral blood or umbilical cord blood.
11. The method of claim 1, wherein the immune cells are at a concentration of 1 x 103-1×1011One per ml.
12. The method of claim 1, wherein the population of cells comprises one or more of cytotoxic T lymphocytes, tumor infiltrating lymphocytes, cytokine-activated killer cells, lymphokine-activated killer cells, natural killer cells, tumor-associated macrophages, activated killer monocytes, and dendritic cells.
13. The method according to claim 1, wherein the co-cultivation time in step 1 is 3 to 180 days.
14. The method of claim 1, wherein the culture vessel is a three-dimensional high-volume high-density cell culture vessel.
15. The method of claim 1, further comprising the cloning step of: cloning in a liquid cell culture medium to obtain a cell strain by taking the culture obtained in the step 1 or the cell population obtained in the step 2 as a raw material;
the cloning adopts an intermittent cycle stimulation method or a continuous stimulation method.
16. A universal immune cell prepared according to any one of claims 1 to 15 comprising one or more of cytotoxic T lymphocytes, tumor infiltrating lymphocytes, cytokine-activated killer cells, lymphokine-activated killer cells, natural killer cells, tumor-associated macrophages, activated killer monocytes, dendritic cells.
17. Use of the universal immune cell of claim 16 in the preparation of an immune cell preparation, wherein the parent cell of the immune cell preparation is the universal immune cell.
18. The use of claim 17, wherein the immune cell preparation comprises CAR-T cells or TCR-T cells.
19. An immune cell preparation prepared from the universal immune cell of claim 16 as a precursor cell.
20. A biologic drug for use in adoptive cellular immunotherapy comprising a container and the immune cell preparation of claim 11 disposed in said container.
21. The biologic drug of claim 20, further comprising saline, albumin, and other stabilizers.
22. Use of an immune cell preparation according to claim 19 in the manufacture of a medicament for the treatment and prevention of disorders associated with abnormal lymphocyte function and number.
23. The use of claim 22, wherein the drug is administered by intravenous, intrathoracic, intraperitoneal, intraspinal, intradermal, subcutaneous, or intratumoral injection.
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