CN107970258B - Chimeric antigen receptor T cell preparation - Google Patents
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Abstract
The invention discloses an immune cell preparation, which comprises immune cells and immune cell preservation transfusion liquid without dimethyl sulfoxide and dextran 40; the immune cell preservation infusion solution comprises the following components: electrolyte solution isotonic with blood plasma, human serum albumin injection, and glucose injection. Wherein the electrolyte solution is any one of sodium chloride injection, compound electrolyte injection and lactated ringer's solution. The immune cell preparation provided by the invention is suitable for the characteristic of concentrated population in China, is used in an immune cell preparation center close to a treatment institution, and only needs to be stored at 4-15 ℃. The immune cell preparation, especially the chimeric antigen receptor T cell preparation, can simplify the preparation process, greatly reduce the logistics cost, improve the safety of the preparation and shorten the treatment period, thereby reducing the price of the chimeric antigen receptor T cell treatment and benefiting the majority of patients.
Description
Technical Field
The invention belongs to the technical field of immunology and molecular biology, relates to an immune cell preparation, and particularly relates to a chimeric antigen receptor T cell preparation.
Background
In the medical field, the traditional treatment methods such as radiotherapy, chemotherapy, surgery, hematopoietic stem cell transplantation and the like prolong the survival time of partial malignant tumor patients in the blood system, but the phenomena of relapse, difficult treatment and even drug resistance still face huge challenges at present.
In recent years, cell immunotherapy has been a breakthrough in tumor, and has become an important means for treating various tumors such as blood systems, and is listed as the first ten scientific breakthroughs in 2013 by the Science journal. The chimeric antigen receptor T cell immunotherapy is particularly advanced. The chimeric antigen receptor T cell (CAR-T) immunotherapy technology is to modify autologous or allogeneic T cells of a patient into chimeric antigen receptor T cells expressing chimeric antigen receptors in vitro by genetic engineering techniques. The chimeric antigen receptor molecule includes a single chain antibody (scFv) that specifically recognizes a cancer cell surface antigen protein, wherein the scFv of the chimeric antigen receptor confers upon the chimeric antigen receptor T cell the ability to specifically recognize cancer cells, and wherein the chimeric antigen receptor T cell specifically kills tumor cells when returned to the patient. The chimeric antigen receptor T cell immunotherapy has strong specificity and high safety, can effectively control and even completely cure tumors for a long time, and is a great breakthrough of tumor therapy technology. CD19 is specifically expressed in malignant and normal B cells and B cell precursor cells, but hematopoietic stem cells and non-hematopoietic cells do not have CD19 expression, so the chimeric antigen protein T cells aiming at CD19 can kill CD19 malignant B cell tumor specifically.
Chimeric antigen receptor T cell therapy begins with the collection of patient autologous peripheral blood mononuclear cells in a hospital or other medically qualified facility. The production organization of chimeric antigen receptor T cells currently marketed in the United states uses a method of setting up a separate preparation center, which is in part intended to accommodate the widely prevalent national conditions in the United states. The autologous peripheral blood mononuclear cells collected by medical institutions in various places in the whole world are transported to the unique preparation center through a cold chain at the temperature lower than-120 ℃, and after the preparation is completed in the preparation center, the chimeric antigen receptor T cells are transported to the medical institutions in various places in the whole world through the cold chain at the temperature lower than-120 ℃ after strict quality control detection so as to carry out reinfusion treatment on the patients. Cold chain transport at temperatures below-120 ℃ is not only expensive, but also places higher demands on the preparation of chimeric antigen receptor-T cell preparations, which greatly contributes to the price of chimeric antigen receptor-T cells and a longer treatment period. In addition, to preserve immune cells below-120 ℃, the addition of dimethyl sulfoxide (DMSO) and low molecular dextran40 to the cell preservation solution may cause severe allergic reactions including anaphylactic shock in patients, thus limiting the therapeutic application of chimeric antigen receptor T cells under certain conditions.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the immune cell preparation which is suitable for the characteristic of concentrated population in China, is used in the immune cell preparation center near a treatment institution and only needs to be stored at 4-15 ℃. The immune cell preparation, especially the chimeric antigen receptor T cell preparation, can simplify the preparation process, greatly reduce the logistics cost, improve the safety of the preparation and shorten the treatment period, thereby reducing the price of the chimeric antigen receptor T cell treatment and benefiting the majority of patients.
In order to achieve the purpose, the invention adopts the following technical scheme:
an immune cell preparation comprising immune cells and a dimethyl sulfoxide (DMSO) and dextran40 free immune cell preservation infusate;
the immune cell preservation infusion fluid comprises the following components in percentage by volume:
78% -93% of electrolyte solution which is isotonic with blood plasma;
5-20% of human serum albumin injection;
1 to 4 percent of glucose injection.
Preferably, the immune cell preservation infusion solution in the preparation comprises the following components in percentage by volume:
88% of electrolyte solution isotonic with plasma;
10% of human serum albumin injection (specification 50ml, 10 g);
2% of 10% glucose injection.
Further, the electrolyte solution is any one of sodium chloride injection, compound electrolyte injection and lactated ringer's solution; or the electrolyte solution is a mixed solution of normal saline, potassium chloride injection and calcium gluconate injection.
Further, the preservation temperature of the immune cells is 4-15 ℃.
Further, the density of the immune cells does not exceed 1000 ten thousand per ml.
Preferably, the immune cell is a chimeric antigen receptor T cell modified by a chimeric antigen receptor targeted to CD 19.
Preferably, the immune cells in the preparation are T cells expressing a chimeric antigen receptor on the cell surface.
Further, the chimeric antigen receptor comprises an interleukin 2 signal peptide, an anti-CD 19 single-chain antibody, a hinge region, a transmembrane region and an intracellular signal domain of a CD8 protein molecule, and an intracellular signal conduction domain of a CD3 zeta protein molecule which are sequentially connected in series; the amino acid sequence is shown in SEQ ID NO. 9.
Furthermore, on the outer surface of the chimeric antigen receptor T cell, the anti-CD 19 single-chain antibody (clone number FMC63) which can recognize CD19 antigen on the surface of tumor cells is murine, the amino acid sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is shown as SEQ ID NO:2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO:4, and the amino acid sequence of the connecting region between the light chain and the heavy chain is shown as SEQ ID NO: 3;
the chimeric antigen receptor is connected with a heavy chain variable region of a single-chain antibody of anti-CD 19 at the N-terminal thereof by a signal peptide of interleukin 2, wherein the amino acid sequence of the signal peptide of interleukin 2 is shown as SEQ ID NO. 1.
Further, the anti-CD 19 single-chain antibody in the chimeric antigen receptor is connected with the transmembrane region through the hinge region of the CD8 protein molecule; the amino acid sequence of the hinge region is shown as SEQ ID NO. 5.
Further, the intracellular signaling domain (or signaling costimulatory region) and the transmembrane region are derived from the same signaling protein molecule selected from the group consisting of CD27, CD28, human tumor necrosis factor receptor 9, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand that specifically binds to CD 83.
Further, the transmembrane region and intracellular signaling domain of the chimeric antigen receptor are selected from the group consisting of human tumor necrosis factor receptor 9; the transmembrane region amino acid sequence of the human tumor necrosis factor receptor 9 protein molecule is shown as SEQ ID NO 6; the intracellular signal transduction structural domain amino acid sequence of the human tumor necrosis factor receptor 9 protein molecule is shown as SEQ ID NO. 7.
Further, the amino acid sequence of the intracellular signaling domain (or the stimulation signal release region) of the CD3 zeta protein molecule is shown as SEQ ID NO. 8.
The technical scheme of the invention designs an artificial chimeric antigen receptor protein, and modifies autologous T cells of a patient in vitro by utilizing a genetic engineering technology, so that the designed chimeric antigen receptor protein is expressed on a cell membrane to become chimeric antigen receptor T cells; when the chimeric antigen receptor T cell is returned to the patient, the chimeric antigen receptor uses the single-chain antibody part on the cell surface to recognize and combine with the specific antigen on the surface of the tumor cell, the specific combination changes the conformation of the signal release area of the chimeric antigen receptor in the T cell, and the signal release area can send out a stimulation signal to activate the killing mechanism of the T cell to the tumor cell, thereby killing the tumor cell; meanwhile, the stimulation signal emitted by the signal release area of the chimeric antigen receptor can stimulate the growth of T cells and generate more chimeric antigen receptor T cells until all tumor cells are killed.
The invention can provide a nucleic acid molecule which encodes the chimeric antigen receptor and the nucleotide sequence of which is shown as SEQ ID NO. 10.
The present invention also provides a vector comprising the above-described nucleic acid molecule; the vector is used for replacing a GFP gene in a lentiviral vector pRRLSIN.cPPT.PGK-GFP.WPRE by using the nucleic acid molecule, and replacing a PGK promoter in the lentiviral vector pRRLSIN.cPPT.PGK-GFP.WPRE by a promoter of a human elongation factor 1 alpha gene; the nucleotide is shown in SEQ ID NO. 11.
The chimeric antigen receptor T cell preparation is applied to treating hematological malignancy; wherein said hematologic malignancy comprises acute B-lymphocytic leukemia positive for CD19, diffuse large B-cell lymphoma and non-Hodgkin's lymphoma.
The invention has the beneficial effects that:
1) the preparation of the invention uses the injection for storing the infusion solution, has simple components and can be directly used for the infusion solution; meanwhile, the special air-permeable transport container is matched, so that the survival time of the cells can be prolonged, and the activity is slowly reduced; the experiment shows that the immune cell preparation is suitable for being stored and transported at 4-15 ℃ when being used for preserving the immune cells, the transportation cost is low, and resuscitation and cleaning operations are not needed before the cell preparation is returned, so that the clinical application of the cell preparation is greatly facilitated.
2) CD19 is specifically expressed in malignant and normal B cells and B cell precursors, whereas hematopoietic stem cells and non-hematopoietic cells are absent CD19 expression, so chimeric antigen receptor T cells against CD19 can specifically kill CD19 expressing malignant B cell tumors. According to the thought, the invention modifies autologous T cells of a patient in vitro by utilizing a genetic engineering technology, so that the designed chimeric antigen receptor protein is expressed on a cell membrane, and the chimeric antigen receptor T cells are provided as immune cells; and tests prove that the chimeric antigen receptor T cell provided by the invention has the advantages of inhibiting tumor cell proliferation, regulating the immune defense mechanism of an organism to limit the growth of tumors and specifically killing CD19 positive tumor cells, and can be applied to the treatment of tumors.
3) The preparation provided by the invention can simplify the preparation process of the chimeric antigen receptor T cell preparation, is suitable for the characteristic of concentrated and dense population in China, is used in an immune cell preparation center close to a treatment institution, only needs to be stored at 4-15 ℃, can simplify the preparation process, greatly reduces the logistics cost, shortens the treatment period, and simultaneously improves the safety of the chimeric antigen receptor T cell preparation on the premise of reducing the treatment price of the chimeric antigen receptor T cells, thereby being beneficial to a large number of patients.
Drawings
FIG. 1 is a graph comparing the flow cytometric assay results of chimeric antigen receptor T cells of the present invention and control T-cells.
FIG. 2 is a graph showing the results of in vitro experiments on chimeric antigen receptor T cells of the present invention and normal T cells.
FIG. 3 is a graph showing the results of the experiment in which the chimeric antigen receptor T cells of the present invention and normal T cells are stimulated to secrete INF-gamma.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1 construction of chimeric antigen receptor Lentiviral vectors expressing chimeric antigen receptor genes against CD19
(1) All synthetic artificial CAR genes encoding chimeric antigen receptors (including the promoter of the human elongation factor 1 alpha gene with an EcoRV endonuclease site at the 5 '-end and a SalI endonuclease site at the 3' -end).
(2) GFP gene and PGK promoter were excised from lentiviral vector prrlsin. cppt. PGK-GFP. wpre by double digestion with EcoRV and SalI (NEB), and the GFP gene and PGK promoter were replaced with an artificial synthetic CAR gene encoding a chimeric antigen receptor containing the promoter of human elongation factor 1 α gene to obtain a chimeric antigen receptor lentiviral vector capable of expressing the chimeric antigen receptor gene for CD 19.
Example 2 packaging of pseudolentivirus particles comprising recombinant vectors encoding artificial genes for chimeric antigen receptors in 293T cells Using the three vector System
293T were cultured in DMEM medium containing 10 vol% FBS, and consumables included DMEM (Gibco, C11995500BT), FBS (Gibco, 10099-141), pancreatin (Gibco, 25200-056), Dulbecco's PBS (Hyclone, cat # SH30256.01), PEI (1mg/ml, Life Sciences, 23966-2), Opti-MEM (Gibco, 51985-034) and 10cm cell culture dish (Fisher Scientific, 310109011).
The procedure for preparing pseudolentiviral particles packaging recombinant vectors comprising an artificial gene encoding a chimeric antigen receptor is as follows:
day 0: floor board
24 hours before transfection, 293FT cells in logarithmic growth phase were trypsinized, cell density was adjusted with seed medium (DMEM + 10% FBS + 500. mu.g/ml G418), and reseeded in 10cm cell culture dishes at 37 ℃ with 5% CO2And (5) culturing in a constant-temperature incubator, and ensuring that the cell confluency reaches 90-95% after 24 hours so as to be used for transfection.
Day 1: transfection
Liquid changing: fresh DMEM was replaced 2 hours before transfection; 6ml of fresh DMEM pre-heated to 37 ℃ was added to each 10cm dish.
Preparation of transfection mixture: transfection mixtures were prepared in 15ml sterile centrifuge tubes according to the following table:
transfection: adding 1ml of transfection mixed solution into each 10cm culture dish, and slowly and uniformly adding the transfection mixed solution; lightly shaking the whole body at 37 deg.C and 5% CO2Culturing in a constant temperature incubator.
Day 2: liquid changing device
The culture supernatant was discarded and washed once with 10ml of PBS pre-warmed to 37 ℃. 6ml fresh packaging medium (DMEM + 500. mu.g/ml G418), preheated to 37 ℃ 5% CO was added2Culturing in a constant temperature incubator.
Day 3: collecting the first viral supernatant
The medium, i.e., the first viral supernatant, was collected from the cell culture dish and stored at 4 ℃. 6ml of fresh packaging medium preheated to 37 ℃ at 37 ℃ 5% CO was added to the petri dish2Culturing in a constant temperature incubator.
Day 4: collecting the second viral supernatant
The second culture supernatant was harvested. The remaining packaging cells and cell culture dishes are processed according to the biological waste.
The collected virus supernatant is the pseudolentiviral particles containing the recombinant vector encoding the artificial gene for the chimeric antigen receptor, and can be used for the preparation of chimeric antigen receptor T cells.
Example 3 preparation of chimeric antigen receptor-expressing T cells (chimeric antigen receptor T cells for short) Using pseudolentiviral particles comprising a recombinant vector encoding an artificial gene for the chimeric antigen receptor CD19
(1) Preparation of mononuclear lymphocytes (PBMC)
The reagent consumables used include serum-free medium, serum-free frozen stock solution, PBS, Ficoll lymphocyte separation medium (GE Healthcare #17-5442-03), disposable sterile pipettes (Thermo) of 5ml, 10ml and 25ml, sterile pipette tips (QSP), centrifuge tubes (Thermo, 339652), and cell freezing tubes (Thermo, 375418).
Adding collected peripheral blood of a patient into a 50ml sterile centrifuge tube, placing the tube in a horizontal centrifuge, centrifuging the tube for 8 minutes at 450g and 20 ℃; the peripheral blood in the centrifugal tube can be layered, the upper layer is plasma, and the lower layer is whole blood cells; taking the lower layer whole blood cells and adding PBS with the same volume for dilution; then, the room-temperature lymphocyte separation solution and the whole blood cell mixed solution were mixed in a 50ml centrifuge tube at a ratio of 1: 2: sucking diluted whole blood cells by using a 25ml pipette, and slowly adding the whole blood cells into the upper layer of the lymphocyte separation solution; placing the centrifuge tube after sample adding in a horizontal centrifuge, centrifuging for 25 minutes at 500g and 4 ℃; gently sucking leukocyte layers, mixing in a 50ml centrifuge tube, supplementing pre-warmed PBS until the total volume is 45ml, screwing a tube cover, turning the centrifuge tube upside down, mixing uniformly for 4-5 times, 500g, 4 ℃, and separating for 25 minutes; observing cell precipitation, pouring off supernatant, supplementing pre-warmed physiological saline to the total volume of 40ml, lightly suspending the cell precipitation, screwing a tube cover, turning a centrifugal tube upside down, uniformly mixing for 4-5 times, and then centrifuging for 8 minutes at 450g and 20 ℃; the supernatant was decanted and the leukocytes were washed twice with PBS, 400g each time, centrifuged at 20 ℃ for 8 minutes; finally, the white blood cells are resuspended in PBS and counted, thus obtaining the prepared mononuclear lymphocytes (PBMC).
(2) Sorting and purifying CD3 positive (CD3+) T lymphocyte
The reagents and consumables used include Dulbecco's PBS (Hyclone, cat # SH30256.01), BSA (Sigma, cat # V900933), EDTA, 0.5M (VWR, cat #45001-122), Pan T-Cell Isolation Kit (Miltenyi, cat #130-096-535), AO/PI dye (Nexcell, CS2-0105), Cell counting plate (Nexcell, # SD100) and LS column (Miltenyi, cat # 130-042-401). Preparing 15ml buffer (15ml PBS +75mg BSA + 60. mu.l 0.5M EDTA), and precooling at 4 ℃ for later use; taking 1.0E +07 PBMC to be resuspended in PBS buffer solution, centrifuging for 10 minutes at 300g and 4 ℃, and discarding supernatant; resuspending the cells in 40. mu.l of pre-chilled buffer, adding 10. mu.l of Pan T Cell Biotin-Antibody Cocktail, human, mixing well and then standing at 4 ℃ for 10 minutes; then adding 30 μ l of buffer solution and 20 μ l of Pan T Cell MicroBead Cocktail and human, mixing uniformly, and standing at 4 ℃ for 15 minutes; place the LS column on a mitimacs magnetic bead separator and rinse the LS column with 3ml buffer (care was taken to avoid air bubble clogging the column); taking out the cell suspension from 4 ℃, adding the cell suspension into an LS column, and collecting effluent (CD3+ T cells); 3ml of buffer was added to the column and the effluent (containing CD3+ cells) was collected; the CD3+ T cell containing effluents were pooled and cell counts were performed according to standard protocols for cell counting, resulting in purified CD3+ T cells.
(3) In vitro activation of T cells and chimeric antigen receptor pseudolentivirus viral transfection
The selected reagents and consumables include serum-free medium, IL-2, PBS (Hyclone, # SH30256.01),
human T-Activated CD3/CD28(GIBCO, 11131D), AO/PI dye (Nexcelom, CS 2-0105).
Day one
Taking a serum-free culture medium without IL-2, and putting the serum-free culture medium into a 37 ℃ incubator for preheating for 30 minutes; washing the separated T cells for 1 time by using preheated serum-free culture medium without IL-2, centrifuging for 10 minutes at room temperature, counting the cells, and then diluting the T cells to the concentration of 0.5E +06 cells/ml by using the preheated culture medium; gently adding T cells into the central region of each culture well of 6-well cell culture plate, adding 2ml of T cell-containing culture medium into each well, adding 1.0E +06 cells, and adding 20 μ l of T cell-containing culture medium into each well
Placing the Human T-activated CD3/CD28 into an incubator at 37 ℃ and 5% CO2Culturing;
the next day
The T-cells which have been cultured for 24 hours are added with IL-2 to a final IL-2 concentration of 200U/ml, 37 ℃ and 5% CO2Performing stimulation amplification culture;
the third day
The chimeric antigen receptor pseudolentivirus was added to activated T cells and then incubated at 37 ℃ with 5% CO2Culturing in an incubator;
the fourth day
Fresh medium was changed (final concentration of IL-2 was 200U/ml).
Chimeric antigen receptor T cells can be harvested after the sixth day depending on the desired cell mass.
Example 4 detection of chimeric antigen receptor on the surface of chimeric antigen receptor T cells by flow cytometry
The primary antibody used in the chimeric antigen receptor assay was rabbit anti-mouse IgG (H + L) F (ab')2(Jackson Corp.). The operation steps are as follows: collecting the chimeric antigen receptor T cells prepared by 1.0E +6, adding the chimeric antigen receptor T cells into a 1.5ml EP tube, centrifuging for 5min at 300g, and discarding the supernatant; adding 200 μ l PBS to resuspend the cells in a 1.5ml EP tube, adding the primary antibody according to the requirements of the product specification, incubating for 30 minutes at 4 ℃, centrifuging for 4 minutes at 500g, and discarding the supernatant; resuspend with 200. mu.l of stabilizing Buffer, centrifuge at 500g for 4 min, discard the supernatant; adding 200. mu.l PBS to resuspend the cells, adding the secondary antibody according to the product instructions, and incubating for 30 minutes at 4 ℃ in the dark; centrifuging at 500g for 4 min, discarding the supernatant, and adding 400 μ l of stabilizing Buffer for resuspension; and (6) performing detection on the machine.
The detection results are shown in fig. 1, and show that: compared with the untreated normal control T cell, the chimeric antigen receptor T cell prepared by the invention has 29 percent of chimeric antigen receptor expression rate and 6 times higher than that of a control group.
Example 5 detection of cytotoxicity of chimeric antigen receptor-T cells specific for target cells
5.1 detection of the killing Capacity of chimeric antigen receptor-T cells specific to target cells
Detection was carried out using the LDH-cytoxicity Colorimetric Assay Kit II Kit (Biovision) and RPMI-1640 medium (Hyclone, cat # SH30809.01B). The desired cells were collected and washed once with fresh medium 1640; target cells (such as CD19 positive Raji cells) are added into a 96-well U-shaped plate at 5.0E +04 per well, and CD19 negative K562 cells are used as controls; then adding effector cells (chimeric antigen receptor-T cells and T cells) into corresponding target cell wells respectively to ensure that the final volume of each well is 100 microliters; each well is provided with 3 multiple wells, and simultaneously, a high-limit control (target cells + 10% cell lysate +1640 culture medium), a low-limit control (target cells +1640 culture medium) and a low-limit sample control (chimeric antigen receptor-T cells or T cells + culture medium) are added; the mixed cells (in 96-well plates) were placed at 37 ℃ in 5% CO2Incubating for 4 hours in an incubator; taking out the cell mixed solution 96-well plate, putting the cell mixed solution into a centrifuge at 600g and 4 ℃, centrifuging the cell mixed solution for 10 minutes, taking 10 microliter of supernatant, and putting the supernatant into a new 96-well flat-bottom plate for LDH detection (in addition, taking 5 microliter of supernatant, putting the supernatant into another 96-well plate for gamma-interferon detection); then adding 100 mul of prepared LDH reaction mixed solution, and placing for 30 minutes at room temperature in a dark place after mixing; adding 10% stop solution to terminate the reaction, and placing the mixture into an enzyme-linked immunosorbent assay to read OD450nm (reference 650 nm); the results were analyzed in excel.
The detection results are shown in fig. 2, which shows that: compared with the common T-cell, the chimeric antigen receptor-T cell prepared by the invention has no difference in killing to the CD19 negative K562 cell, but has over one time of killing to the Raji cell of the CD19 positive tumor target cell, and simultaneously proves that the chimeric antigen receptor-T cell prepared by the invention has the killing power specifically aiming at the CD19 positive tumor cell.
5.2 detection of interferon-gamma (IFN-. gamma.) secreted by chimeric antigen receptor T cells specifically induced by target cells
The amount of IFN-gamma secreted by the chimeric antigen receptor T cells induced by the stimulation of target cells can indirectly reflect the specific killing capacity of the chimeric antigen receptor T cells on tumor target cells. ELISA test kit (RAYBIO) for detection. Raji cells are CD19 positive B cell leukemia cells, K562 cells are CD19 negative B cell lymphoma cells. Mixing effector cells and target cells according to a specific proportion, incubating, centrifuging and separating to obtain supernatant, and adding 5 mu l of each supernatant into a 96-well plate; the primary antibody was diluted with PBS, 50. mu.l of antibody was added to each well in a 96-well plate, overnight at 4 ℃; wash plate with 250 μ l PBST per well for 3 times; 5% skimmed milk Powder (PBST) was blocked, and each well was washed 3 times with 250. mu.l PBST; adding a standard substance and a sample (the standard substance is prepared by taking 10 mu l of the standard substance in 640 mu l of PBS containing 1% BSA with the concentration of 1000pg/ml, and taking the concentration as a mother solution to carry out 2-fold dilution, wherein the dilution is respectively 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ml and 15.6pg/ml), and the distribution of the sample is arranged according to the habit of a user; wash plate with 250 μ l PBST per well for 3 times; add 50. mu.l of diluted secondary antibody per well and incubate for 1 hour at 37 ℃; wash plate with 250 μ l PBST per well for 3 times; add 50. mu.l streptavidin-HRP (375. mu.l in 15ml Reagent) per well for 30 min at RT; wash plate with 250 μ l PBST per well for 3 times; add 50. mu.l TMB (Tiangen # PA107-01) per well for 30 min at room temperature; adding 50 mul of 1M concentrated sulfuric acid into each hole, and stopping the reaction; and reading OD450nm and Reference 620nm by using a microplate reader, and calculating the content of gamma-interferon in the sample.
Gamma-interferon is a cytokine with multiple functions of inhibiting tumor cell proliferation, regulating immune defense mechanism of body, limiting tumor growth, etc. The detection results are shown in fig. 3: the CD19 negative tumor K562 cells have no obvious influence on the gamma-interferon secretion amount of the chimeric antigen receptor-T cells and the common T-cells prepared by the invention, but the CD19 positive tumor Raji cells can specifically induce the chimeric antigen receptor-T cells prepared by the invention to secrete a large amount of gamma-interferon for inhibiting the growth of tumor cells, and compared with the CD19 negative tumor K562 cells, the amount of the gamma-interferon secreted by the chimeric antigen receptor-T cells induced by the CD19 positive tumor Raji cells is increased by at least eight times.
The tests prove that the chimeric antigen receptor T cell provided by the invention has the advantages of inhibiting the proliferation of tumor cells, regulating the immune defense mechanism of an organism to limit the growth of tumors and specifically killing CD19 positive tumor cells, and can be applied to the treatment of tumors.
[ example 6 ] preparation of chimeric antigen receptor-T cell preparation
6.1 preparation and preservation of the infusion solution for later use
The following components are mixed in percentage by volume:
78% -93% of electrolyte solution which is isotonic with blood plasma;
5-20% of human serum albumin injection;
1 to 4 percent of glucose injection.
As a preferable proportion, the immune cell preservation infusion fluid in the preparation is mixed according to the following contents in percentage by volume and is used as a standby chimeric antigen receptor T cell preservation infusion fluid;
88% of electrolyte solution isotonic with plasma;
10% of human serum albumin injection;
2 percent of glucose injection.
The storage transfusion liquid provided by the invention is prepared from the injection, has simple components, meets the requirement of cell activity, and can be directly used for transfusion. The immune cell preservation infusion fluid comprises an electrolyte solution which is isotonic with blood plasma, albumin and glucose; the albumin is the protein with the highest content in blood plasma and has the following functions: maintaining the colloid osmotic pressure constant; the albumin belongs to non-specific transport protein, can be reversibly combined with a plurality of insoluble small molecular organic matters and inorganic ions to form a soluble compound, and plays roles in detoxification and transportation; the albumin can ensure the communication between the intracellular fluid and the extracellular fluid; albumin has a protective effect on the stability of cell membranes; albumin is an important nutrient; when meeting heavy metal ions, the albumin can be automatically combined with the heavy metal ions to play a role in detoxification. Glucose is an important energy substance of cells, and is used for generating ATP and partially anabolism.
Wherein the electrolyte solution is any one of sodium chloride injection, compound electrolyte injection and lactated ringer's solution; or a mixed solution of normal saline, potassium chloride injection and calcium gluconate injection can be selected. These electrolyte solutions provide the basic inorganic ions for cell preservation, maintaining the potential and other electrophysiological functions of cell membranes; maintaining osmotic pressure; glucose and other nutrients are transported in a coordinated manner, and metabolic products and the like are excreted, so that normal energy metabolism of cells is maintained, and the survival rate of the cells in the preservation process is ensured.
The injection is a special clinical medicine, the dosage form and the concentration of the injection accord with the corresponding injection specification, and the specific component proportion of each injection is shown in table 1.
TABLE 1 ratio of injection solutions in immune cell preservation and infusion solution
6.2 preparation of chimeric antigen receptor-T cell preparations
Collecting qualified chimeric antigen receptor T cells (4 ℃, 1200rpm, 7 minutes of centrifugation) according to the required dosage of the feedback, washing the chimeric antigen receptor T cells with physiological saline for three times, and suspending the cells in a 15ml centrifuge tube by using 12ml of physiological saline; placing a 15ml centrifuge tube containing 12ml of cell transfusion suspension cells in DynaMagTM-15Magnet for 5 minutes, transferring the supernatant to a new 15ml centrifuge tube, repeating 3 times to remove magnetic beads for activated cells, transferring the supernatant to a new 15ml centrifuge tube, centrifuging (4 ℃, 1200rpm, 7 minutes) to collect cells, suspending the cells in 50ml chimeric antigen receptor T cell storage reinfusion fluid without dimethyl sulfoxide (DMSO) and Dextran40 to ensure that the density of chimeric antigen receptor T cells does not exceed 1000 ten thousand/ml, perfusing the chimeric antigen receptor T cells into a gas permeable immune cell storage reinfusion bag, and placing the air permeable immune cell storage reinfusion bag at 4 ℃ to 15 ℃ for storage and transportation.
The control test was as follows: the experimental conditions of group 1 were that the chimeric antigen receptor T cells of the present invention were suspended in the chimeric antigen receptor T cells of the present invention, which did not contain dimethyl sulfoxide (DMSO) and Dextran40, and stored in an air-impermeable normal saline bag; group 2 assay, chimeric antigen receptor T cell preparation prepared with a chimeric antigen receptor T cell reinfusion containing dimethyl sulfoxide (DMSO) and Dextran40, stored at-80 ℃, using the method currently invented in the united states by Novartis (Novartis); the test conditions of groups 3, 4 and 5 were that the chimeric antigen receptor T cells of the present invention were suspended in the chimeric antigen receptor T cell preservation reinfusion solution of the present invention which did not contain dimethyl sulfoxide (DMSO) and Dextran40, and the chimeric antigen receptor T cell suspension was stored in the air-permeable cell preservation reinfusion bag. The cell viability was counted by talarol blue staining, and the results are shown in table 2.
TABLE 2 comparison of cell viability after storage of CAR-T cells under different storage conditions
The results show that: the chimeric antigen receptor T cell preservation feedback fluid without dimethyl sulfoxide and Dextran40 is preserved in a breathable immune cell preservation feedback bag at 4 ℃, 10 ℃ and 15 ℃ for 24 hours, and the survival rate of the chimeric antigen receptor T cell is almost unchanged; if the method of Nowa company is used for preserving the chimeric antigen receptor T cells, not only additional expensive equipment and reagents are needed, but also the preservation effect is slightly inferior to that of the invention; however, if the chimeric antigen receptor T cells of the present invention are stored in a normal air-impermeable physiological saline bag, the survival rate of the chimeric antigen receptor T cells is greatly reduced.
Therefore, the chimeric antigen receptor T cell preservation reinfusion fluid without dimethyl sulfoxide (DMSO) or Dextran40 is matched with the air-permeable immune cell preservation reinfusion bag for use, so that the preservation condition of the immune cell preparation is optimized, an excellent preservation effect is obtained, and the popularization and the application are facilitated.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Enpulfuu Biotechnology (Shanghai) Co., Ltd
<120> a chimeric antigen receptor T cell preparation
<130> 2017
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Thr Ala Met Gly Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Ala Ser Ala
20
<210> 2
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Gly Val Leu Leu Gly Gly Ser Gly Pro Gly Leu Val Ala Pro Ser Gly
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Ala Thr
20 25 30
Gly Val Ser Thr Ile Ala Gly Pro Pro Ala Leu Gly Leu Gly Thr Leu
35 40 45
Gly Val Ile Thr Gly Ser Gly Thr Thr Thr Thr Ala Ser Ala Leu Leu
50 55 60
Ser Ala Leu Thr Ile Ile Leu Ala Ala Ser Leu Ser Gly Val Pro Leu
65 70 75 80
Leu Met Ala Ser Leu Gly Thr Ala Ala Thr Ala Ile Thr Thr Cys Ala
85 90 95
Leu His Thr Thr Thr Gly Gly Ser Thr Ala Met Ala Thr Thr Gly Gly
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 3
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
1 5 10 15
<210> 4
<211> 106
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Ala Ile Gly Met Thr Gly Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Ala Ala Val Thr Ile Ser Cys Ala Ala Ser Gly Ala Ile Ser Leu Thr
20 25 30
Leu Ala Thr Thr Gly Gly Leu Pro Ala Gly Thr Val Leu Leu Leu Ile
35 40 45
Thr His Thr Ser Ala Leu His Ser Gly Val Pro Ser Ala Pro Ser Gly
50 55 60
Ser Gly Ser Gly Thr Ala Thr Ser Leu Thr Ile Ser Ala Leu Gly Gly
65 70 75 80
Gly Ala Ile Ala Thr Thr Pro Cys Gly Gly Gly Ala Thr Leu Pro Thr
85 90 95
Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile
100 105
<210> 5
<211> 47
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala Cys Ala Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Ala Gly Leu Ala Pro Ala Cys Ala Ile Thr
35 40 45
<210> 6
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Ile Ile Ser Pro Pro Leu Ala Leu Thr Ser Thr Ala Leu Leu Pro Leu
1 5 10 15
Leu Pro Pro Leu Thr Leu Ala Pro Ser Val Val
20 25
<210> 7
<211> 42
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Leu Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met
1 5 10 15
Ala Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro
20 25 30
Pro Gly Gly Gly Gly Gly Gly Cys Gly Leu
35 40
<210> 8
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Ala Val Leu Pro Ser Ala Ser Ala Ala Ala Pro Ala Thr Leu Gly Gly
1 5 10 15
Gly Ala Gly Leu Thr Ala Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr
20 25 30
Ala Val Leu Ala Leu Ala Ala Gly Ala Ala Pro Gly Met Gly Gly Leu
35 40 45
Pro Ala Ala Leu Ala Pro Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu
50 55 60
Ala Leu Met Ala Gly Ala Thr Ser Gly Ile Gly Met Leu Gly Gly Ala
65 70 75 80
Ala Ala Gly Leu Gly His Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala
85 90 95
Thr Leu Ala Thr Thr Ala Ala Leu His Met Gly Ala Leu Pro Pro Ala
100 105 110
<210> 9
<211> 490
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Met Thr Ala Met Gly Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Ala Ser Ala Gly Val Leu Leu Gly Gly Ser Gly Pro Gly Leu
20 25 30
Val Ala Pro Ser Gly Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val
35 40 45
Ser Leu Pro Ala Thr Gly Val Ser Thr Ile Ala Gly Pro Pro Ala Leu
50 55 60
Gly Leu Gly Thr Leu Gly Val Ile Thr Gly Ser Gly Thr Thr Thr Thr
65 70 75 80
Ala Ser Ala Leu Leu Ser Ala Leu Thr Ile Ile Leu Ala Ala Ser Leu
85 90 95
Ser Gly Val Pro Leu Leu Met Ala Ser Leu Gly Thr Ala Ala Thr Ala
100 105 110
Ile Thr Thr Cys Ala Leu His Thr Thr Thr Gly Gly Ser Thr Ala Met
115 120 125
Ala Thr Thr Gly Gly Gly Thr Ser Val Thr Val Ser Ser Thr Gly Gly
130 135 140
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Ile Gly Met
145 150 155 160
Thr Gly Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Ala Ala Val Thr
165 170 175
Ile Ser Cys Ala Ala Ser Gly Ala Ile Ser Leu Thr Leu Ala Thr Thr
180 185 190
Gly Gly Leu Pro Ala Gly Thr Val Leu Leu Leu Ile Thr His Thr Ser
195 200 205
Ala Leu His Ser Gly Val Pro Ser Ala Pro Ser Gly Ser Gly Ser Gly
210 215 220
Thr Ala Thr Ser Leu Thr Ile Ser Ala Leu Gly Gly Gly Ala Ile Ala
225 230 235 240
Thr Thr Pro Cys Gly Gly Gly Ala Thr Leu Pro Thr Thr Pro Gly Gly
245 250 255
Gly Thr Leu Leu Gly Ile Thr Thr Thr Pro Ala Pro Ala Pro Pro Thr
260 265 270
Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala Pro Gly Ala
275 280 285
Cys Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly Leu Ala Pro
290 295 300
Ala Cys Ala Ile Thr Ile Ile Ser Pro Pro Leu Ala Leu Thr Ser Thr
305 310 315 320
Ala Leu Leu Pro Leu Leu Pro Pro Leu Thr Leu Ala Pro Ser Val Val
325 330 335
Leu Ala Gly Ala Leu Leu Leu Leu Thr Ile Pro Leu Gly Pro Pro Met
340 345 350
Ala Pro Val Gly Thr Thr Gly Gly Gly Ala Gly Cys Ser Cys Ala Pro
355 360 365
Pro Gly Gly Gly Gly Gly Gly Cys Gly Leu Ala Val Leu Pro Ser Ala
370 375 380
Ser Ala Ala Ala Pro Ala Thr Leu Gly Gly Gly Ala Gly Leu Thr Ala
385 390 395 400
Gly Leu Ala Leu Gly Ala Ala Gly Gly Thr Ala Val Leu Ala Leu Ala
405 410 415
Ala Gly Ala Ala Pro Gly Met Gly Gly Leu Pro Ala Ala Leu Ala Pro
420 425 430
Gly Gly Gly Leu Thr Ala Gly Leu Gly Leu Ala Leu Met Ala Gly Ala
435 440 445
Thr Ser Gly Ile Gly Met Leu Gly Gly Ala Ala Ala Gly Leu Gly His
450 455 460
Ala Gly Leu Thr Gly Gly Leu Ser Thr Ala Thr Leu Ala Thr Thr Ala
465 470 475 480
Ala Leu His Met Gly Ala Leu Pro Pro Ala
485 490
<210> 10
<211> 1473
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atgtatcgca tgcagctgct gagctgcatc gccctgtccc tggccctggt gaccaacagc 60
gccgaggtga agctgcagga gtccggacca ggactggtgg caccttccca gtctctgagc 120
gtgacatgta ccgtgtccgg cgtgtctctg cctgactacg gcgtgtcctg gatcaggcag 180
ccacctagga agggactgga gtggctgggc gtgatctggg gctctgagac cacatactat 240
aatagcgccc tgaagtcccg gctgacaatc atcaaggata actccaagtc tcaggtgttc 300
ctgaagatga atagcctgca gacagacgat accgccatct actattgcgc caagcactac 360
tattacggcg gctcttatgc catggactac tggggccagg gcacaagcgt gaccgtgagc 420
tccaccggcg gcggcggctc tggaggagga ggaagcggag gaggaggcga tatccagatg 480
acacagacca catctagcct gagcgcctcc ctgggcgaca gagtgaccat ctcttgtagg 540
gccagccagg atatctccaa gtatctgaac tggtaccagc agaagcctga cggcacagtg 600
aagctgctga tctatcacac ctctcgcctg cacagcggag tgccatcccg gttctctgga 660
agcggatccg gaacagacta ctctctgacc atcagcaacc tggagcagga ggatatcgcc 720
acatatttct gccagcaggg caatacactg ccatacacct ttggcggcgg caccaagctg 780
gagatcacca caaccccagc acctaggcca ccaacaccag caccaaccat cgcatcccag 840
ccactgtctc tgagaccaga ggcatgcagg cctgcagcag gaggagccgt gcacacacgg 900
ggcctggact ttgcctgtga tatctatatc atctccttct ttctggccct gacatctacc 960
gccctgctgt tcctgctgtt ctttctgacc ctgaggttta gcgtggtgaa gagaggcagg 1020
aagaagctgc tgtacatctt caagcagcct tttatgagac cagtgcagac aacccaggag 1080
gaggacggct gctcctgtag gttcccagaa gaggaggagg gaggatgtga gctgcgcgtg 1140
aagttttctc ggagcgccga tgcccctgcc tataagcagg gccagaatca gctgtacaac 1200
gagctgaatc tgggccggag agaggagtac gacgtgctgg ataagaggag gggaagagat 1260
ccagagatgg gaggcaagcc acggagaaag aacccccagg agggcctgta taatgagctg 1320
cagaaggaca agatggccga ggcctacagc gagatcggca tgaagggaga gaggcgccgg 1380
ggcaagggac acgatggcct gtatcagggc ctgtccacag ccaccaagga cacctacgat 1440
gccctgcaca tgcaggccct gcctccaagg tga 1473
<210> 11
<211> 2695
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ttgatatcgt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg 60
gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag 120
tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc 180
agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc 240
cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat 300
tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg 360
ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg 420
gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc 480
tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg 540
gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc 600
cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg 660
agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc 720
tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc 780
agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag 840
gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc 900
gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga 960
ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat 1020
ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta 1080
attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac 1140
agtggttcaa agtttttttc ttccatttca ggtgtcgtga ggaatttcga catttaaatt 1200
taattaagcc accatgtacc gcatgcagct gctgagctgc atcgccctga gcctggccct 1260
ggtgaccaac agcgccgagg tgaagctgca ggagagcggc cccggcctgg tggcccccag 1320
ccagagcctg agcgtgacct gcaccgtgag cggcgtgagc ctgcccgact acggcgtgag 1380
ctggatccgc cagccccccc gcaagggcct ggagtggctg ggcgtgatct ggggcagcga 1440
gaccacctac tacaacagcg ccctgaagag ccgcctgacc atcatcaagg acaacagcaa 1500
gagccaggtg ttcctgaaga tgaacagcct gcagaccgac gacaccgcca tctactactg 1560
cgccaagcac tactactacg gcggcagcta cgccatggac tactggggcc agggcaccag 1620
cgtgaccgtg agcagcaccg gcggcggcgg cagcggcggc ggcggcagcg gcggcggcgg 1680
cgacatccag atgacccaga ccaccagcag cctgagcgcc agcctgggcg accgcgtgac 1740
catcagctgc cgcgccagcc aggacatcag caagtacctg aactggtacc agcagaagcc 1800
cgacggcacc gtgaagctgc tgatctacca caccagccgc ctgcacagcg gcgtgcccag 1860
ccgcttcagc ggcagcggca gcggcaccga ctacagcctg accatcagca acctggagca 1920
ggaggacatc gccacctact tctgccagca gggcaacacc ctgccctaca ccttcggcgg 1980
cggcaccaag ctggagatca ccaccacccc cgccccccgc ccccccaccc ccgcccccac 2040
catcgccagc cagcccctga gcctgcgccc cgaggcctgc cgccccgccg ccggcggcgc 2100
cgtgcacacc cgcggcctgg acttcgcctg cgacatctac atcatcagct tcttcctggc 2160
cctgaccagc accgccctgc tgttcctgct gttcttcctg accctgcgct tcagcgtggt 2220
gaagcgcggc cgcaagaagc tgctgtacat cttcaagcag cccttcatgc gccccgtgca 2280
gaccacccag gaggaggacg gctgcagctg ccgcttcccc gaggaggagg agggcggctg 2340
cgagctgcgc gtgaagttca gccgcagcgc cgacgccccc gcctacaagc agggccagaa 2400
ccagctgtac aacgagctga acctgggccg ccgcgaggag tacgacgtgc tggacaagcg 2460
ccgcggccgc gaccccgaga tgggcggcaa gccccgccgc aagaaccccc aggagggcct 2520
gtacaacgag ctgcagaagg acaagatggc cgaggcctac agcgagatcg gcatgaaggg 2580
cgagcgccgc cgcggcaagg gccacgacgg cctgtaccag ggcctgagca ccgccaccaa 2640
ggacacctac gacgccctgc acatgcaggc cctgcccccc cgctgagtcg acaat 2695
Claims (4)
1. An immune cell preparation, comprising: the preparation comprises immune cells and immune cell preservation transfusion liquid without dimethyl sulfoxide and dextran 40;
the immune cell preservation infusion fluid comprises the following components in percentage by volume:
78% -93% of electrolyte solution which is isotonic with blood plasma;
5-20% of human serum albumin injection;
1% -4% of glucose injection;
the immune cells in the preparation are T cells with chimeric antigen receptors expressed on the cell surfaces; the chimeric antigen receptor comprises an interleukin 2 signal peptide, an anti-CD 19 single-chain antibody, a hinge region of a CD8 protein molecule, a transmembrane region, an intracellular signaling domain and an intracellular signaling domain of a CD3 zeta protein molecule which are sequentially connected in series; the amino acid sequence is shown as SEQ ID NO. 9;
the amino acid sequence of the heavy chain variable region of the anti-CD 19 single-chain antibody is shown as SEQ ID NO. 2, the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4, and the amino acid sequence of the connecting region between the light chain and the heavy chain is shown as SEQ ID NO. 3.
2. An immune cell preparation according to claim 1, wherein: the immune cell preservation infusion solution in the preparation comprises the following components in percentage by volume:
88% of electrolyte solution isotonic with plasma;
10% of human serum albumin injection;
2 percent of glucose injection.
3. An immune cell preparation according to claim 1 or 2, wherein: the electrolyte solution is any one of sodium chloride injection, compound electrolyte injection and lactated ringer's solution; or the electrolyte solution is a mixed solution of normal saline, potassium chloride injection and calcium gluconate injection.
4. An immune cell preparation according to claim 1, wherein: the nucleotide sequence for coding the chimeric antigen receptor is shown as SEQ ID NO. 10.
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| CN108378021A (en) * | 2018-03-19 | 2018-08-10 | 英普乐孚生物技术(上海)有限公司 | A kind of stored refrigerated system of lymphocyte |
| CN110468098A (en) * | 2019-07-05 | 2019-11-19 | 北京中瑞联合生物科技有限公司 | A kind of dental pulp stem cell preparation method |
| WO2021108389A1 (en) * | 2019-11-29 | 2021-06-03 | Nkmax Co., Ltd. | Method of producing natural killer cells and compositions thereof |
| CN112120012B (en) * | 2020-09-30 | 2022-03-22 | 广东康盾创新产业集团股份公司 | CAR-T cell cryopreservation method |
| CN112806354A (en) * | 2021-01-18 | 2021-05-18 | 圣至同合(北京)生物科技有限公司 | Immune cell cryopreservation liquid as well as preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102948413A (en) * | 2011-08-29 | 2013-03-06 | 北京清美联创干细胞科技有限公司 | Hepatic stem cell preserving solution and applications of hepatic stem cell preserving solution |
| CN103404509A (en) * | 2013-08-07 | 2013-11-27 | 彭乐 | Cell preserving fluid as well as preparation method and application of cell preserving fluid |
| CN106665560A (en) * | 2017-01-16 | 2017-05-17 | 哈尔滨医科大学 | Direct venous re-transfusion immune cell cryopreservation medium and application thereof |
| CN106701827A (en) * | 2016-12-05 | 2017-05-24 | 刘晓明 | Transformation, multiplication culture and preservation method for T cell for expressing CD19 and CD20 antibody gene CAR (chimeric antigen receptor) |
| CN106922648A (en) * | 2017-02-22 | 2017-07-07 | 海南新生命干细胞医疗有限公司 | A kind of mescenchymal stem cell cryopreservation solution and preparation method thereof |
| CN107047536A (en) * | 2016-11-22 | 2017-08-18 | 浙江三誉生物科技有限公司 | A kind of cell-preservation liquid and its application |
| CN107148967A (en) * | 2016-11-09 | 2017-09-12 | 深圳宾德生物技术有限公司 | A kind of antigenspecific T lymphocyte frozen stock solution and its preparation method and application |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102584999B (en) * | 2011-01-13 | 2014-10-15 | 四川大学 | Human VEGFR-1 (Vascular Endothelial Growth Factor Receptor-1) targeting genetically engineered lymphocyte as well as preparation method and application thereof |
| SG11201509609SA (en) * | 2013-05-24 | 2015-12-30 | Univ Texas | Chimeric antigen receptor-targeting monoclonal antibodies |
| CN105418765B (en) * | 2014-08-26 | 2019-09-06 | 西比曼生物科技（上海）有限公司 | The Chimeric antigen receptor and NKT cell and its preparation method of CD19 targeting and application |
| CN104542578B (en) * | 2015-02-05 | 2017-01-18 | 广州赛莱拉干细胞科技股份有限公司 | Cell preservation solution and preparation method and applications thereof |
-
2017
- 2017-11-20 CN CN201711156576.7A patent/CN107970258B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102948413A (en) * | 2011-08-29 | 2013-03-06 | 北京清美联创干细胞科技有限公司 | Hepatic stem cell preserving solution and applications of hepatic stem cell preserving solution |
| CN103404509A (en) * | 2013-08-07 | 2013-11-27 | 彭乐 | Cell preserving fluid as well as preparation method and application of cell preserving fluid |
| CN107148967A (en) * | 2016-11-09 | 2017-09-12 | 深圳宾德生物技术有限公司 | A kind of antigenspecific T lymphocyte frozen stock solution and its preparation method and application |
| CN107047536A (en) * | 2016-11-22 | 2017-08-18 | 浙江三誉生物科技有限公司 | A kind of cell-preservation liquid and its application |
| CN106701827A (en) * | 2016-12-05 | 2017-05-24 | 刘晓明 | Transformation, multiplication culture and preservation method for T cell for expressing CD19 and CD20 antibody gene CAR (chimeric antigen receptor) |
| CN106665560A (en) * | 2017-01-16 | 2017-05-17 | 哈尔滨医科大学 | Direct venous re-transfusion immune cell cryopreservation medium and application thereof |
| CN106922648A (en) * | 2017-02-22 | 2017-07-07 | 海南新生命干细胞医疗有限公司 | A kind of mescenchymal stem cell cryopreservation solution and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 不同保存介质对脐带间充质干细胞活性的影响;周楠 等;《中国组织工程研究》;20140402;第18卷(第14期);2153-2160 * |
| 临床治疗用人骨髓间充质干细胞保存条件的研究;陈京 等;《国际检验医学杂志》;20141231;第35卷(第12期);1542-1544 * |
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