CN107970258A - A kind of Chimeric antigen receptor T cell preparation - Google Patents
A kind of Chimeric antigen receptor T cell preparation Download PDFInfo
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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Abstract
The present invention discloses a kind of immunocyte preparation, and said preparation includes immunocyte and preserve without the immunocyte of dimethyl sulfoxide (DMSO) and Dextran 40 to feed back liquid；The immunocyte, which preserves feedback liquid, includes following each component：Isotonic electrolyte solution, human serum albumin injection, glucose injection with blood plasma.Wherein, electrolyte solution is any of sodium chloride injection, compound sodium chloride injection, Multiple electrolytes injection, Lactated Ringer'S Solution.Immunocyte preparation provided by the invention is adapted to Chinese population to concentrate the characteristics of intensive, uses at the immunocyte preparation center for closing on treatment mechanism, need to only be preserved at 4 15 DEG C.Using the immunocyte preparation of the present invention, particularly Chimeric antigen receptor T cell preparation, preparation flow can be simplified, logistics cost is substantially reduced, improves the security of preparation, shortens treatment cycle, so as to reduce the price of Chimeric antigen receptor T cell treatment, many patients are benefited.
Description
Technical field
The invention belongs to immunology and technical field of molecular biology, is related to a kind of immunocyte preparation, more particularly to one
Kind Chimeric antigen receptor T cell preparation.
Background technology
In medical domain, the treatment method such as traditional Radiotherapy chemotherapy, operation, hematopoietic stem cell transplantation extends part blood
The life span of System Malignant Tumor patient, but recur, refractory or even drug resistance phenomenon, it is still the huge challenge faced at present.
In recent years, it is more to become hematological system etc. because achieving breakthrough in the tumour of tumour for cellular immunotherapy
The important means of kind oncotherapy, the first place of ten big sciences breakthrough in 2013 is classified as by Science magazines.Wherein chimeric antigen by
Body T cell Advances of immunologic therapy is especially prominent.Chimeric antigen receptor T cell (CAR-T) immunotherapy techniques are to pass through in vitro
Technique for gene engineering, which modifies autologous patient or allogeneic T cells, becomes the Chimeric antigen receptor T cell of expression Chimeric antigen receptor.It is embedding
Closing antigen receptor molecule includes a single-chain antibody that can specifically identify cancer cell surface antigens albumen (scFv), inosculating antibody
ScFv in original receptor imparts the ability that Chimeric antigen receptor T cell specifically identifies cancer cell, when feeding back in patient body
Chimeric antigen receptor T cell just can specifically kill tumour cell afterwards.The immunization therapy of Chimeric antigen receptor T cell has special
Property it is strong, it is safe, can it is permanently effective control even completely cure tumour, be oncotherapy technology important breakthrough.CD19 is special
The opposite sex is expressed in pernicious and normal B cells and B cell precursor cell, and candidate stem cell and non-hematopoietic cell are then not present
CD19 is expressed, therefore can specifically kill expression CD19 malignant B cell tumours for the chimeric antigen Protein T cell of CD19.
The treatment of Chimeric antigen receptor T cell is from the outer of the mechanism collection autologous patient in hospital or the medical qualification of other tools
All blood monocytes start.It is independent using setting up in the production organizational mode of the Chimeric antigen receptor T cell of U.S.'s listing at present
The mode at preparation center, this is to adapt to the scarcely populated national conditions in the U.S. to a certain extent.In the whole America various regions medical institutions
The autologous peripheral blood monocyte of patient is gathered all by less than -120 DEG C cold chain transportations to this unique preparation center, is being made
Standby center is completed after preparing, and Chimeric antigen receptor T cell is transported to by less than -120 DEG C cold chains again after being detected by strict quality control
The whole America various regions medical institutions carry out adoptive therapy to patient.Cold chain transportation less than -120 DEG C is not only expensive, to inosculating antibody
Original receptor-T cell preparation prepares the requirement that it is also proposed higher, this all greatly encourages the price of Chimeric antigen receptor-T cell,
Longer treatment cycle.In addition, to preserve immunocytes, the dimethyl sulfoxide (DMSO) added in cell-preservation liquid less than -120 DEG C
(DMSO) and D-40 40 may cause serious allergic reaction to include anaphylactic shock in patient's body, thus
The treatment use of Chimeric antigen receptor T cell is limited under the conditions of some.
The content of the invention
For above-mentioned deficiency of the prior art, the present invention provides a kind of suitable Chinese population to concentrate the characteristics of intensive,
Close on treatment mechanism immunocyte prepare center use, only need to 4-15 DEG C preservation immunocyte preparation.Use this hair
Bright immunocyte preparation, particularly Chimeric antigen receptor T cell preparation, preparation flow can be simplified, substantially reduce logistics into
This, improves the security of preparation, shortens treatment cycle, so as to reduce the price of Chimeric antigen receptor T cell treatment, benefits vast
Patient.
To achieve the above object, present invention employs following technical scheme：
A kind of immunocyte preparation, said preparation include immunocyte and without dimethyl sulfoxide (DMSO) (DMSO) and Dextran 40s
Immunocyte preserve feed back liquid；
The immunocyte preserves feedback liquid includes following each component with volume percent：
The electrolyte solution 78%~93% isotonic with blood plasma；
Human serum albumin injection 5%~20%；
Glucose injection 1%~4%.
Preferably, the immunocyte in said preparation preserves feedback liquid includes following each component with volume percent：
The electrolyte solution 88% isotonic with blood plasma；
Human serum albumin injection (specification 50ml, 10g) 10%；
10% glucose injection 2%.
Further, the electrolyte solution is sodium chloride injection, compound sodium chloride injection, compound electrolyte injection
Any of liquid, Lactated Ringer'S Solution；Or the electrolyte solution is physiological saline, potassium chloride injection, gluconic acid
The mixed solution of calcium parenteral solution.
Further, the storage temperature of the immunocyte is 4 DEG C~15 DEG C.
Further, the density of the immunocyte is no more than 10,000,000/ml.
Preferably, the Chimeric antigen receptor T cell which modifies for the Chimeric antigen receptor of targeting CD19.
Preferably, the immunocyte in said preparation has the T cell of Chimeric antigen receptor for cell surface expression.
Further, the Chimeric antigen receptor includes the interleukin-22 signal peptide of sequential series, anti-CD19 single-chain antibodies,
The hinge region of CD8 protein moleculars, transmembrane region and intracellular signal domain, the intracellular signal transduction domain of CD3 ζ protein moleculars；
Its amino acid sequence such as SEQ ID NO:Shown in 9.
Further, Chimeric antigen receptor T cell outer surface, can tumor cell surface C D19 antigens be mouse source
Anti- CD19 single-chain antibodies (clone FMC63), the amino acid sequence such as SEQ of the heavy chain variable region of the anti-CD19 single-chain antibodies
ID NO:Shown in 2, the amino acid sequence such as SEQ ID NO of light chain variable region:Shown in 4, the amino of the bonding pad between light and weight chain
Acid sequence such as SEQ ID NO:Shown in 3；
Weight chain variable of the Chimeric antigen receptor at its N- end with the signal peptide of interleukin-22 with the single-chain antibody of anti-CD19
Area connects, the wherein amino acid sequence of interleukin-22 signal peptide such as SEQ ID NO:Shown in 1.
Further, the anti-CD19 single-chain antibodies in the Chimeric antigen receptor by the hinge areas of CD8 protein moleculars with
Transmembrane region is connected；The amino acid sequence of the hinge area such as SEQ ID NO:Shown in 5.
Further, the intracellular signal transduction domain (or signal costimulation area) comes from same signal with transmembrane region
Protein molecular, the signal protein molecule be selected from 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 special with CD83
The ligand that the opposite sex combines.
Further, the transmembrane region of the Chimeric antigen receptor with intracellular signal domain be selected from human tumour necrosis factor by
Body 9；The cross-film region amino acid sequence of 9 protein molecular of human tumor necrosis factor receptor such as SEQ ID NO:Shown in 6；The human tumour
The intracellular signal transduction domain amino acid sequence of 9 protein molecular of mecrosis factor receptors such as SEQ ID NO:Shown in 7.
Further, intracellular signal transduction domain (or the stimulus signal release area) of the CD3 ζ protein moleculars its ammonia
Base acid sequence such as SEQ ID NO:Shown in 8.
Above-mentioned technical proposal of the present invention devises a kind of artificial Chimeric antigen receptor albumen, and utilizes technique for gene engineering
The T cell of autologous patient is modified in vitro, the Chimeric antigen receptor protein expression of design is allowed on cell membrane
For Chimeric antigen receptor T cell；After Chimeric antigen receptor T cell feeds back to patient's body, Chimeric antigen receptor utilizes its position
Identified in the single chain antibody portion of cell surface and combine the specific antigen positioned at tumor cell surface, it is this specifically to combine
Change the conformation in the signal release area that Chimeric antigen receptor is located in T cell, signal release area will send stimulus signal to swash
Killing mechanism of the T cell living to tumour cell, so as to kill tumour cell；The area of Chimeric antigen receptor signal release at the same time is sent
Stimulus signal can also stimulate T cell to grow, and produce more Chimeric antigen receptor T cells, until killing all tumour cells.
The present invention can provide a kind of nucleic acid molecules, it encodes the Chimeric antigen receptor, and its nucleotide sequence such as SEQ
ID NO:Shown in 10.
The present invention may also provide a kind of carrier for including above-mentioned nucleic acid molecules；The carrier is to utilize the nucleic acid molecules pair
GFP genes in slow virus carrier pRRLSIN.cPPT.PGK-GFP.WPRE are substituted, and with people's elongation factor 1 alpha differential gene
Promoter substitute PGK promoters therein；Its nucleotide such as SEQ ID NO:Shown in 11.
Above-mentioned Chimeric antigen receptor T cell formulation application is in treatment hematologic malignancies；Wherein, the haematological malignant
Tumour includes the B-lineage Acute Lymphocyte Leukemia of the CD19 positives, diffusivity large B cell lymphoid tumor and non Hodgkin lymphom.
The beneficial effects of the present invention are：
1) feedback liquid is preserved used in invention formulation uses parenteral solution to configure, its component is simple, can directly use
In feedback；Coordinate dedicated gas permeability shipping container at the same time, can be extended the cell survival time, vigor reduces slow；Through examination
When testing using immunocyte preparation of the present invention progress immunocyte preservation, suitable storage and transport under the conditions of 4-15 DEG C, and
Cost of transportation is relatively low, need not again do and recover and cleaning operation before feedback, this greatly facilitates the clinical practice of cell preparation.
2) CD19 is specific expressed in pernicious and normal B cells and B cell precursor cell, and candidate stem cell and non-makes
Haemocyte can specifically kill expression CD19 evils then there is no CD19 expression for the Chimeric antigen receptor T cell of CD19
Property B cell tumour.The present invention in vitro modifies the T cell of autologous patient using technique for gene engineering according to the thinking,
Make the Chimeric antigen receptor protein expression of design on cell membrane, there is provided Chimeric antigen receptor T cell is as immunocyte；And
And test proves that, Chimeric antigen receptor T cell provided by the invention, which has, to be suppressed tumor cell proliferation, adjusts exempting from for body
The growth of epidemic disease defense mechanism limitation tumour, the advantage of specific killing CD19 positive tumor cells, can be applied to the treatment of tumour.
3) preparation provided by the invention can simplify the preparation flow of Chimeric antigen receptor T cell preparation, and the present invention
The preparation of offer is adapted to Chinese population to concentrate the characteristics of intensive, and preparing center in the immunocyte for closing on treatment mechanism uses, only
It need to can simplify preparation flow in 4-15 DEG C of preservation, substantially reduce logistics cost, shorten treatment cycle, so that chimeric reducing
On the premise of antigen receptor T cell treatment price, while the security of Chimeric antigen receptor T cell preparation is improved, benefited vast
Patient.
Brief description of the drawings
Fig. 1 is FCM analysis comparative result figure of the Chimeric antigen receptor T cell of the present invention with compareing T- cells.
Fig. 2 is Chimeric antigen receptor T cell of the present invention and the in vitro test result compares figure of normal T-cell.
Fig. 3 compares for Chimeric antigen receptor T cell of the present invention with the result of the test of the stimulated secretion INF- γ of normal T-cell
Figure.
Embodiment
The technical solution in the present invention is clearly and completely described below in conjunction with the embodiment of the present invention.Following reality
Apply example to be only used for clearly illustrating technical scheme, and be not intended to limit the protection scope of the present invention and limit the scope of the invention.
【Embodiment 1】Structure of the expression for the Chimeric antigen receptor slow virus carrier of the Chimeric antigen receptor gene of CD19
(1) all (including people extends 1 α genes of the factor to the artificial CAR genes of artificial synthesized encoding chimeric antigen acceptors
Promoter, and add EcoRV restriction enzyme sites at 5 '-end, in 3 '-end plus SalI restriction enzyme sites).
(2) EcoRV and SalI (NEB companies) double digestions are used from slow virus carrier pRRLSIN.cPPT.PGK-GFP.WPRE
Middle excision GFP genes and PGK promoters, the artificial synthesized encoding chimera for the promoter that 1 α genes of the factor are extended comprising people is resisted
The artificial CAR genes substitution GFP genes and PGK promoters of original receptor, thus obtain expressing the chimeric antigen for being directed to CD19 by
The Chimeric antigen receptor slow virus carrier of body gene.
【Embodiment 2】The artificial gene for including encoding chimeric antigen acceptor is packed in 293T cells using three carrier systems
Recombinant vector false lentiviral particle
293T is cultivated in the DMEM culture mediums containing 10vol%FBS, and consumptive material includes 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 10 lis
Rice Tissue Culture Dish (Fisher Scientific, 310109011).
The false lentiviral particle preparation flow of the recombinant vector of artificial gene of the packaging comprising encoding chimeric antigen acceptor is such as
Under：
0th day：Bed board
Before transfection 24 it is small when, with pancreatin digestion exponential phase 293FT cells, with seed culture medium (DMEM+10%
FBS+500 μ g/ml G418) adjustment cell density, it is re-seeded into 10cm Tissue Culture Dish, 37 DEG C, 5%CO2Constant temperature incubation
Case culture, cell confluency degree reaches 90%-95% after ensureing 24h, for use in transfection.
1st day：Transfection
Change liquid：Fresh DMEM is replaced when 2 is small before transfection；6ml is added in each 10cm culture dishes and is preheated to 37 DEG C fresh
DMEM。
Transfection cocktail prepares：According to the form below prepares transfection cocktail in 15ml sterile centrifugation tubes：
Transfection：Add 1ml transfection cocktails in each 10cm culture dishes, it is necessary to slowly, be uniformly added into；All around gently
Shake up, 37 DEG C, 5%CO2Cultivated in constant incubator.
2nd day：Change liquid
Culture supernatant is discarded, 37 DEG C of PBS cleaning is preheated to once with 10ml.Add the fresh bag that 6ml is preheated to 37 DEG C
Dress culture medium (DMEM+500 μ g/ml G418), 37 DEG C, 5%CO2Cultivated in constant incubator.
3rd day：Collect first viral supernatants
Collect the culture medium in Tissue Culture Dish, i.e. first viral supernatants, 4 DEG C of preservations.It is pre- that 6ml is added into culture dish
The fresh packaging culture medium of heat to 37 DEG C, 37 DEG C, 5%CO2Cultivated in constant incubator.
4th day：Collect second batch viral supernatants
Harvest second batch culture supernatant.Remaining incasing cells and Tissue Culture Dish are handled according to biologic garbage.
It is exactly the false slow of the recombinant vector of the artificial gene comprising encoding chimeric antigen acceptor in the viral supernatants being collected into
Virion, you can prepared for Chimeric antigen receptor T cell.
【Embodiment 3】Use the slow disease of vacation comprising coding for the recombinant vector of the artificial gene of CD19 Chimeric antigen receptors
The T cell (abbreviation Chimeric antigen receptor T cell) of malicious particle preparation expression Chimeric antigen receptor
(1) preparation of monokaryon lymphocyte (PBMC)
Agents useful for same consumptive material includes serum free medium, serum-free frozen stock solution, PBS, Ficoll lymphocyte separation mediums (GE
Healthcare#17-5442-03), the disposable sterilized pipette of 5ml, 10ml and 25ml (Thermo), sterile pipette tips (QSP),
Centrifuge tube (Thermo, 339652), cell freezing pipe (Thermo, 375418).
The autologous patient peripheral blood of collection is added in 50ml sterile centrifugation tubes, is put in horizontal centrifuge, 450g, 20
DEG C, centrifuge 8 minutes；Peripheral blood in centrifuge tube can be layered, upper strata is blood plasma, and lower floor is whole blood cells；Remove a layer whole blood cells
Isometric PBS is added to be diluted；Then in 50ml centrifuge tubes, often pipe presses 1:The lymphocyte separation medium of 2 ratio mixed room temperatures
With whole blood cells mixed liquor：It is slowly added into the diluted whole blood cells of 25ml pipette, extracts in lymphocyte separation medium upper strata；
Centrifuge tube after sample-adding is placed in horizontal centrifuge, 500g, 4 DEG C, centrifuged 25 minutes；Gentle aspiration leukocytic cream, is incorporated in
In 50ml centrifuge tubes, supplement pre-temperature PBS is 45ml to cumulative volume, screws tube cover, and centrifuge tube is turned upside down and is mixed 4-5 times,
500g, 4 DEG C, from 25 minutes；After observing cell precipitation, incline supernatant, supplements pre- warm saline to cumulative volume 40ml, gently hangs
Floating cell precipitation, screws tube cover, and centrifuge tube is turned upside down and is mixed 4-5 times, then, 450g, centrifuges 8 minutes by 20 DEG C；On inclining
Clearly, then with PBS whiten cell twice, every time 400g, 20 DEG C, centrifuge 8 minutes；Last leucocyte is resuspended in PBS, and is counted
Number, that is, the monokaryon lymphocyte (PBMC) prepared.
(2) the sorting purifying of positive (CD3+) the T lymphocytes of CD3
Selecting reagent and consumptive material includes 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 dyestuffs (Nexcelom, CS2-0105), cell counting count board (Nexcelom, #SD100) and LS
Column (Miltenyi, cat#130-042-401).Prepare 15ml buffer solutions (15ml PBS+75mg BSA+60 μ l 0.5M
EDTA), 4 DEG C of pre- cold standbies；The PBMC of 1.0E+07 is taken to be resuspended in PBS buffer, 300g, centrifuges 10 minutes, abandon by 4 DEG C
Clearly；Cell is resuspended with 40 μ l precoolings buffer solutions, adds 10 μ l Pan T Cell Biotin-Antibody Cocktail,
Human, is placed 10 minutes after mixing at 4 DEG C；Add 30 μ l buffer solutions and 20 μ l Pan T Cell MicroBead
Cocktail, human, are placed 15 minutes after mixing at 4 DEG C；LS columns are placed on MidiMACS Beads enrichment devices, are buffered with 3ml
Liquid rinse LS columns (attention avoids bubble foul columns)；Cell suspension is taken out from 4 DEG C, addition LS columns, collection efflux (for
CD3+T cells)；Add 3ml buffer solutions in pillar, collect efflux (cell containing CD3+)；Merge the outflow of the cell containing CD3+T
Liquid, cell count, that is, the CD3+T cells purified are carried out according to the standard practice instructions of cell count.
(3) Activated in Vitro of T cell and Chimeric antigen receptor vacation slow virus virus transfection
Selecting reagent and consumptive material includes serum free medium, IL-2, PBS (Hyclone, #SH30256.01),Human T-Activated CD3/CD28 (GIBCO, 11131D), AO/PI dyestuffs (Nexcelom, CS2-
0105)。
First day
Take the serum free medium without IL-2 to be put into 37 DEG C of incubators to preheat 30 minutes；The T cell of separator well is taken to use
The serum free medium without IL-2 of preheating washs 1 time, 300g, and room temperature centrifuges 10 minutes, carries out cell count, then with pre-
Culture medium dilution T cell to the concentration of heat is 0.5E+06 cells/ml；It is light in each culture hole central area of 6 porocyte culture plates
It is slow to add T cell, culture mediums of the 2ml containing T cell is added per hole, adds 1.0E+06 cell altogether, 20 μ l are added per holeHuman T-Activited CD3/CD28 are put into incubator, 37 DEG C, 5%CO2, culture；
Second day
Cultivated 24 it is small when T- cells in add IL-2 to IL-2 final concentration of 200U/ml, 37 DEG C, 5%CO2, carry out
Stimulate amplification cultivation；
3rd day
Chimeric antigen receptor vacation slow virus is added in the T cell of activation, then puts 37 DEG C, 5%CO2Trained in incubator
Support；
4th day
Change fresh culture (the final concentration of 200U/ml of IL-2).
Chimeric antigen receptor T cell was collected after the 6th day according to required cell concentration.
【Embodiment 4】With the Chimeric antigen receptor on FCM analysis method detection Chimeric antigen receptor T cell surface
First antibody used in Chimeric antigen receptor detection is rabbit anti-mouse igg (H+L) F (ab')2(Jackson companies).Operation
Step is as follows：The Chimeric antigen receptor T cell that 1.0E+6 is prepared is collected, is added in 1.5ml EP pipes, 300g, is centrifuged
5min, abandons supernatant；Add 200 μ l PBS and cell is resuspended in 1.5ml EP pipes, it is anti-to add first according to the requirement of the description of product
Body, after 4 DEG C are incubated 30 minutes, 500g is centrifuged 4 minutes, discards supernatant；With 200 μ l Staining Buffer be resuspended, 500g from
The heart 4 minutes, discards supernatant；Add 200 μ l PBS and cell is resuspended, add secondary antibody according still further to the description of product, 4 DEG C of lucifuges are incubated
30 minutes；500g is centrifuged 4 minutes again, abandons supernatant, adds 400 μ l Staining Buffer resuspensions；Upper machine testing.
Testing result is as shown in Figure 1, show：Compared with no processed normal control T cell, the present invention prepares gained
Chimeric antigen receptor T cell in Chimeric antigen receptor expression rate reach 29%, be higher by 6 times compared with control group.
【Embodiment 5】Detect the cytotoxicity that Chimeric antigen receptor-T cell is specifically directed to target cell
5.1 detection Chimeric antigen receptor-T cells are specifically directed to the killing ability of target cell
Detection with LDH-Cytotoxicity Colorimetric Assay Kit II kits (Biovision) and
RPMI-1640 culture mediums (Hyclone, cat#SH30809.01B).Cell needed for collection, one is washed with fresh culture 1640
It is secondary；First target cell (such as CD19 positive Raji cells) is added in 96 hole U-shaped boards with every hole 5.0E+04, at the same it is cloudy with CD19
Property K562 cells compare；Effector cell's (Chimeric antigen receptor-T cell and T cell) is separately added into corresponding target cell again
Kong Zhong, ensures per hole final volume at 100 microlitres；3 multiple holes are set per hole, while add the high limit control (cell of target cell+10%
The culture medium of lysate+1640) and lower bound control (culture medium of target cell+1640) and lower bound sample controls (Chimeric antigen receptor-
T cell or T cell+culture medium)；Mixed cell is put into 37 DEG C (in 96 orifice plates), 5%CO2It is incubated in incubator 4 small
When；It will be taken out in 96 orifice plate of cell mixture, be put into 600g in centrifuge, 4 DEG C, centrifuge 10 minutes, take 10 microlitres of supernatant, be put into
LDH detections are carried out in 96 new hole flat undersides and (in addition, taking 5 microlitres again, are put into progress gamma interferon inspection in another 96 orifice plate
Survey)；Add advance prepared 100 μ l of LDH reaction mixtures, room temperature avoid light place 30 minutes after mixing；Add 10% eventually
Only liquid terminates reaction, is put into microplate reader and reads OD450nm (reference 650nm)；Acquired results are analyzed with excel.
Testing result is as shown in Fig. 2, show：Chimeric antigen receptor-the T cell prepared by the present invention is with general T-cell
Compare, although to the no difference of killing of CD19 feminine gender K562 cells, CD19 positive tumor target cell Raji cells are killed
Overstrain is improved more than one times, while also demonstrates Chimeric antigen receptor-T cell for preparing of the present invention and have and be specifically directed to
The lethality of CD19 positive tumor cells.
Gamma interferon (IFN-γ) of the 5.2 detection Chimeric antigen receptor T cells by the special secretion inducing of target cell
Chimeric antigen receptor T cell by target cell stimulate secretion inducing IFN-γ amount can reflect indirectly chimeric antigen by
Specific killing ability of the body T cell to tumour target cell.ELISA detection kit (RAYBIO) is used in detection.Raji cells are
The B cell leukemia cell of the CD19 positives, K562 cells are the B cell lymphoma cells of CD19 feminine genders.Take and imitate in specific proportions
Answer cell and target cell mix be incubated after centrifuge each 5 μ l of supernatant are added in 96 orifice plates；First antibody is diluted with PBS,
Add 50 μ l antibody in 96 orifice plates per hole, 4 DEG C overnight；Per 250 μ l PBST board-washings of hole, wash altogether 3 times；5% skimmed milk power (PBST)
Closing, per 250 μ l PBST board-washings of hole, is washed 3 times altogether；Adding standard items and sample, (standard items are prepared：10 μ l are taken to contain in 640 μ l
In the PBS of 1%BSA, concentration 1000pg/ml, carries out 2 times as mother liquor and dilutes, respectively 500pg/ml, 250pg/ml,
125pg/ml, 62.5pg/ml, 31.3pg/ml, 15.6pg/ml), sample distribution is arranged according to oneself custom；Per 250 μ l of hole
PBST board-washings, are washed 3 times altogether；The secondary antibody that 50 μ l have diluted is added per hole, when 37 degree of incubations 1 are small；Per 250 μ l PBST of hole
Board-washing, is washed 3 times altogether；50 μ l streptavidin-HRP (taking 375 μ l in 15ml Reagent diluent) are added per hole,
Room temperature 30 minutes；Per 250 μ l PBST board-washings of hole, wash altogether 3 times；50 μ l TMB (Tiangeng #PA107-01), room temperature are added per hole
30 minutes；The 50 μ l 1M concentrated sulfuric acids are added per hole, terminate reaction；Microplate reader reads OD 450nm, Reference 620nm, calculates
Gamma interferon content in sample.
Gamma interferon is a kind of cell factor, has the immune defence mechanism limit for suppressing tumor cell proliferation, adjusting body
A variety of effects such as the growth of tumour processed.Testing result shows as shown in Figure 3：CD19 negative tumours K562 cells prepare the present invention
Chimeric antigen receptor-T cell and the gamma interferon secretory volume of general T-cell have not significant impact, but CD19 positive tumors
Chimeric antigen receptor-T cell that Raji cells but can specifically induce the present invention to prepare, which is largely secreted, suppresses tumour cell life
Long gamma interferon, is compared with CD19 negative tumours K562 cells, CD19 positive tumor Raji cells induce chimeric antigen by
The amount of body-T cell secretion gamma interferon improves at least octuple.
Proved by above-mentioned experiment, Chimeric antigen receptor T cell provided by the invention, which has, to be suppressed tumor cell proliferation, adjusts
Growth, the advantage of specific killing CD19 positive tumor cells of the immune defence mechanism limitation tumour of body are saved, can be applied to
The treatment of tumour.
【Embodiment 6】It is prepared by Chimeric antigen receptor-T cell preparation
It is spare that 6.1 preparations preserve feedback liquid
Following each component is mixed with volume percent：
The electrolyte solution 78%~93% isotonic with blood plasma；
Human serum albumin injection 5%~20%；
Glucose injection 1%~4%.
As a kind of preferable proportioning, the immunocyte in said preparation, which preserves, feeds back liquid with volume percent by following each
Content is mixed, and is preserved as spare Chimeric antigen receptor T cell and is fed back liquid；
The electrolyte solution 88% isotonic with blood plasma；
Human serum albumin injection 10%；
Glucose injection 2%.
Provided by the invention preserve feeds back liquid using parenteral solution configuration, its component is simple, meets the requirement of cytoactive, can
It is directly used in feedback.Above-mentioned immunocyte, which preserves feedback liquid, includes the electrolyte solution and albumin isotonic with blood plasma, glucose；
Albumin therein is the highest protein of content in blood plasma, there is following functions：Maintain the constant of colloid osmotic pressure；Albumin category
In non-specific transport protein, can reversibly be combined to form with the small organic molecule and inorganic ions of many slightly solubilities readily soluble
The compound of property, plays the role of removing toxic substances and transport；Albumin can guarantee that the exchange between intracellular fluid, extracellular fluid；Albumin
There is protective effect to the stability of cell membrane；Albumin is a kind of important nutriment；Albumin runs into heavy metal ion
When, it can be combined automatically with heavy metal ion, play the role of removing toxic substances.Glucose therein is the important energy matter of cell, is used
In generation ATP and partially synthetic metabolism.
Wherein, the electrolyte solution for sodium chloride injection, compound sodium chloride injection, Multiple electrolytes injection,
Any of Lactated Ringer'S Solution；Or the mixed of physiological saline+potassium chloride injection+calcium gluconate injection also can be selected
Close solution.These electrolyte solutions preserve the inorganic ions for providing basis for cell, maintain the current potential of cell membrane and other electricity raw
Manage function；Maintain osmotic pressure；The nutriments such as cotransport glucose, help excretion metabolism product etc., so as to maintain cell just
Normal energetic supersession, ensures survival rate of the cell during preservation.
Each parenteral solution is clinical special medicament, and formulation and concentration meet corresponding parenteral solution regulation, each parenteral solution tool
The component proportion of body is as shown in table 1.
1. immunocyte of table preserves the proportioning for feeding back each parenteral solution in liquid
6.2 prepare Chimeric antigen receptor-T cell preparation
By feedback requirement dosage, collect (4 DEG C, 1200rpm, centrifuge 7 minutes) and examine qualified Chimeric antigen receptor T cell,
After physiology salt washing three times, with 12ml physiological saline suspension cell in 15ml centrifuge tubes；12ml cells feedback liquid will be housed to hang
The 15ml centrifuge tubes of floating cell are in DynaMagTM5 minutes are stood in -15Magnet, supernatant is transferred in new 15ml centrifuge tubes,
It is repeated 3 times and removes activating cell magnetic bead, then supernatant is transferred in new 15ml centrifuge tubes, centrifuges (4 DEG C, 1200rpm, 7 points
Clock) cell is collected, suspension cell is in Chimeric antigen receptor T cells of the 50ml without dimethyl sulfoxide (DMSO) (DMSO) and Dextran 40
Preserve and feed back in liquid, ensure that the density of Chimeric antigen receptor T cell is no more than 10,000,000/ml, be filled into gas permeability immunocyte
Preserve and feed back in bag, be placed on 4 DEG C to 15 DEG C storage and transport.
Check experiment is as follows：1 experimental condition of group is that Chimeric antigen receptor T cell of the present invention is suspended in the present invention is free of
The Chimeric antigen receptor T cell of dimethyl sulfoxide (DMSO) (DMSO) and Dextran 40, which preserve, to be fed back in liquid, and Chimeric antigen receptor T is thin
Born of the same parents' suspension is stored in air-locked Normal Saline bag；The experiment of group 2, using at present in the U.S. by Novartis Co., Ltd
(Novartis) method of invention, is returned with the Chimeric antigen receptor T cell containing dimethyl sulfoxide (DMSO) (DMSO) and Dextran 40
Prepared by infusion, the Chimeric antigen receptor T cell preparation of the preservation at -80 DEG C；Group 3, group 4,5 experimental conditions of group are that the present invention is chimeric
Antigen receptor T cell is suspended in Chimeric antigen receptor T cell of the present invention without dimethyl sulfoxide (DMSO) (DMSO) and Dextran40 and protects
It is stored back in infusion, Chimeric antigen receptor T cell suspension is stored in ventilative cell preserves in feedback bag.Platform phenol indigo plant dyeing counting is thin
Born of the same parents' survival rate, the results are shown in Table 2.
Cell viability compares after preserving CAR-T cells under the different preservation conditions of table 2.
The result shows that：Preserved back with Chimeric antigen receptor T cell of the present invention without dimethyl sulfoxide (DMSO) and Dextran 40
Infusion gas permeability immunocyte preserve feed back bag in, in 4 DEG C, 10 DEG C, 15 DEG C preserve 24 it is small when, Chimeric antigen receptor T cell
Motility rate it is almost unchanged；If preserving Chimeric antigen receptor T cell with the method for Novartis Co., Ltd not only needs setting for additional expensive
Standby and reagent, preservation effect are also slightly worse than the present invention；It is and chimeric if preserving the present invention with common airtight normal saline bag
Antigen receptor T cell then can largely reduce the motility rate of Chimeric antigen receptor T cell.
So as to which the present invention is preserved by the Chimeric antigen receptor T cell without dimethyl sulfoxide (DMSO) (DMSO) and Dextran40
Feeding back liquid coordinates gas permeability immunocyte to preserve feedback bag use, is optimized the preservation condition of immunocyte preparation, and
And excellent preservation effect is obtained, it is easy to utilize.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, some improvement and deformation can also be made, these are improved and deformation
Also it should be regarded as protection scope of the present invention.
Sequence table
<110>Ying Pule inspires confidence in biotechnology（Shanghai）Co., Ltd
<120>A kind of 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 (10)
- A kind of 1. immunocyte preparation, it is characterised in that：Said preparation includes immunocyte and without dimethyl sulfoxide (DMSO) and dextrose The immunocyte of acid anhydride 40, which preserves, feeds back liquid；The immunocyte preserves feedback liquid includes following each component with volume percent：The electrolyte solution 78%~93% isotonic with blood plasma；Human serum albumin injection 5%~20%；Glucose injection 1%~4%.
- A kind of 2. immunocyte preparation according to claim 1, it is characterised in that：Immunocyte in said preparation preserves back Infusion includes following each component with volume percent：The electrolyte solution 88% isotonic with blood plasma；Human serum albumin injection 10%；Glucose injection 2%.
- A kind of 3. immunocyte preparation according to claim 1 or 2, it is characterised in that：The electrolyte solution is chlorination Any of sodium injection, compound sodium chloride injection, Multiple electrolytes injection, Lactated Ringer'S Solution；Or the electricity Electrolyte solution is physiological saline, potassium chloride injection, the mixed solution of calcium gluconate injection.
- A kind of 4. immunocyte preparation according to claim 1, it is characterised in that：Immunocyte in said preparation is cell Surface expression has the T cell of Chimeric antigen receptor.
- A kind of 5. immunocyte preparation according to claim 4, it is characterised in that：The Chimeric antigen receptor includes order The interleukin-22 signal peptide of series connection, anti-CD19 single-chain antibodies, the hinge region of CD8 protein moleculars, transmembrane region, intracellular signal domain With the intracellular signal transduction domain of CD3 ζ protein moleculars；Its amino acid sequence such as SEQ ID NO:Shown in 9.
- A kind of 6. immunocyte preparation according to claim 5, it is characterised in that：The anti-CD19 single-chain antibodies heavy chain can Become the amino acid sequence such as SEQ ID NO in area:Shown in 2, the amino acid sequence such as SEQ ID NO of light chain variable region:Shown in 4, gently The amino acid sequence of bonding pad between heavy chain such as SEQ ID NO:Shown in 3；The Chimeric antigen receptor is connected at its N- end with the signal peptide of interleukin-22 with the heavy chain variable region of anti-CD19 single-chain antibodies Connect, the amino acid sequence such as SEQ ID NO of interleukin-22 signal peptide:Shown in 1；The amino acid sequence of the CD8 protein moleculars hinge area Row such as SEQ ID NO:Shown in 5.
- A kind of 7. immunocyte preparation according to claim 5, it is characterised in that：The intracellular signal domain and cross-film Area comes from same signal protein molecule, the signal protein molecule be selected from CD27, CD28, human tumor necrosis factor receptor 9, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1, CD2, CD7, LIGHT, NKG2C, B7-H3 or With the ligand of CD83 specific bindings.
- A kind of 8. immunocyte preparation according to claim 7, it is characterised in that：The transmembrane region is with signal intracellular signal Domain is selected from human tumor necrosis factor receptor 9；The cross-film region amino acid sequence of 9 protein molecular of human tumor necrosis factor receptor Such as SEQ ID NO:Shown in 6；The signal structure domain amino acid sequence of 9 protein molecular of human tumor necrosis factor receptor such as SEQ ID NO:Shown in 7.
- A kind of 9. immunocyte preparation according to claim 5, it is characterised in that：The CD3 ζ protein molecular intracellular signals The amino acid sequence in conducting structure domain such as SEQ ID NO:Shown in 8.
- A kind of 10. immunocyte preparation according to claim 4, it is characterised in that：Encode the Chimeric antigen receptor Nucleotide sequence such as SEQ ID NO:Shown in 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 |
| CN112120012A (en) * | 2020-09-30 | 2020-12-25 | 广东康盾生物工程技术有限公司 | 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 |
| WO2021108389A1 (en) * | 2019-11-29 | 2021-06-03 | Nkmax Co., Ltd. | Method of producing natural killer cells and compositions thereof |
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