CN105950561A

CN105950561A - Products and preparation method of double chimeric antigen receptor gene modified T lymphocyte targeting breast cancer stem cells

Info

Publication number: CN105950561A
Application number: CN201610353118.1A
Authority: CN
Inventors: 王锦杰; 宋现让
Original assignee: Jie Sheng Bio Tech Ltd Jiangsu
Current assignee: Taizhou several biological technology Co., Ltd.
Priority date: 2016-05-26
Filing date: 2016-05-26
Publication date: 2016-09-21

Abstract

The invention provides a preparation method of a double chimeric antigen receptor gene modified T lymphocyte targeting breast cancer stem cells. The preparation method is characterized in that integrin-associated protein (CD47) and transcriptional coactivator (TAZ) are both highly expressed in breast cancer tissues, especially in the breast cancer stem cells; by established CD47 and TAZ over-expressed breast cancer cells, higher proliferation and metastasis capacity and cancer stem cell features are shown. Extracellular domain of the two breast cancer stem cell antigens CD47 and TAZ are targeted to generate monoclonal strains in specific binding under immunity action, and single-chain antibodies in specific binding with the CD47 and TAZ by genetic recombination are obtained to construct a human CD47 and TAZ containing double chimeric antigen receptor gene recombined to a virus vector to transfect human T lymphocyte. By high expression of the double chimeric antigen receptor gene in specific binding with human CD47 and TAZ expressing breast cancer stem cells, a first signal and a costimulatory signal can be activated to trigger anti-breast-cancer cytotoxicity, and high cytotoxicity in in-vivo and in-vitro anti-cancer experiments is achieved.

Description

The bispecific chimeric antigen receptor genetic modification of a kind of targeting breast carcinoma stem cell Preparation method of T lymphocyte and products thereof

Technical field

The present invention relates to the preparation of the T lymphocyte of the Chimeric antigen receptor genetic modification of a kind of targeting breast carcinoma stem cell Method, including following feature: breast cancer tissue and breast carcinoma stem cell specificity overexpression integrin associated protein (CD47) and transcribe the co-activation factor (TAZ), CD47 and the TAZ process LAN breast cancer cell of structure show higher propagation, Transfer ability and tumor stem cell feature；Targeting breast carcinoma stem cell antigen CD4 7 and TAZ extracellular domain of the present invention, it is thus achieved that with two The single-chain antibody of person's specific bond, is built into the double distinctive embedment antigen receptor gene (double containing anti-human CD47 and TAZ Chimeric antigen receptor, Di-CAR), recombinate on viral vector and transfect human T lymphocyte, can special tie Closing the breast carcinoma stem cell expressing people CD47 and TAZ, in vitro tests activates immunity signal and causes powerful anti-mammary gland in vivo Cancer toxicity；Present invention also offers a kind of test kit prepared containing the T autologous lymphocytes obtained by said method, available In the T lymphocyte preparing the Chimeric antigen receptor genetic modification that targeting breast carcinoma kills.

Background technology

Lymphocyte immunity technology at home and abroad has been used to clinical treatment malignant tumor, and obtains some clinical efficacies, Mainly include Cytokine-induced killer cells, cytotoxic T lymphocyte and infiltrating T lymphocytes etc., but it is non-spy Specific immunological effect, owing to the immunologic escape of tumor cell own and antigen are disguised, interior tumor cell is removed the most very by it Limited.

The clinical anticancer that appears as of Chimeric antigen receptor genetic modification lymphocyte technology provides new direction, its Technology can activate self immune system in vivo, and routinely targets neoplastic cells kills, and is finally reached fully erased evil Property tumor cell purpose, thus this technology to cancer clinical treatment provide more preferable application platform.But due at solid tumor Aspect tumor antigen finds limited, and a lot of tumor antigen is except expressing at tumor cell itself, table the most in the normal tissue Reaching, thus tumor cell cannot produce good specificity, killing tumor cell has also damaged normal structure and organ simultaneously, Find and there is the key that specific tumor antigen is the application of this technology.

The invention provides at breast cancer tissue and breast carcinoma stem cell specificity overexpression integrin associated protein (CD47) and transcribe the co-activation factor (TAZ), CD47 and TAZ process LAN breast cancer cell shows higher propagation, transfer energy Power and tumor stem cell feature, to combine double distinctive embedment antigen gene weights of CD47 and TAZ of tumor cell specifically expressing Group on viral vector and transfect human T lymphocyte, this bispecific chimeric antigen receptor gene of high expressed, can specific bond table Reach the breast cancer cell of CD47 and TAZ, particularly breast carcinoma stem cell；Activate the first signal and costimulatory signal and vivo and vitro Cause anti-breast cancer cellular cytoxicity activity, by malignant cell in purged body effectively and prolongation survival of patients phase.

Summary of the invention

The present invention is directed to existing clinical practice T lymphocyte technology targets neoplastic cells and kill poor specificity；And at body Interior activation patient's autoimmunization antitumor is not enough, especially to the mammary gland with high drug-resistance, self renewal and Tumor formation etc. Cancer stem cell, without lethal effect, causes most of malignant tumor clinical treatment limited.The present invention finds in previous research work Two tumor antigen CD47 and TAZ of the equal specifically expressing of breast cancer tissue, breast cancer cell and breast carcinoma stem cell.Simultaneously we By CD47 and the TAZ process LAN slow virus carrier built, transfect breast cancer cell after being packaged into virus, find that it has higher Propagation and transfer ability, and show tumor stem cell phenotype, with internal become tumor experiment there is tumor stem cell feature.

The present invention obtains special with both according to the extracellular region of these two tumor antigen CD47 and TAZ by hybridoma technology The monoclonal cell strain of property association reaction and monoclonal antibody, protein sequencing determines the heavy chain of monoclonal antibody and the variable of light chain District, utilizes gene PCR technology that by link (linker), heavy chain and light chain are connected generation single-chain antibody (single chain Antibody fragment, scFv), these two scFv of euzymelinked immunosorbent assay (ELISA) and cell flow cytometer detection can specific bond CD47 and Two tumor antigens of TAZ, the two scFv can be as the antibody combining two tumor antigens of CD47 and TAZ.

Present inventor utilizes DNA modification enzyme-1011 transposition methylcystein dioxygenase 2 (teneleven Translocation methylcytosine dioxygenase 2, TET2) interleukin 6 (interleukin 6, IL-can be suppressed 6) transcriptional expression, it has been found that TET2 high expressed in T lymphocyte can be suppressed IL-6 gene expression and secretion, thus stop T lymphocytic emiocytosis IL-6 and produce " immune factor storm ".Thus we are according to these scientific research results, it is built into containing spy Double distinctive embedment antigen receptor genes of different combination CD47 and TAZ, and introduce TET2 and CD28, CD137 and CD3 ζ intracellular The genetic fragment of signaling zone, recombinates on viral vector and transfects human T lymphocyte, and this bispecific chimeric antigen of high expressed is subject to Body gene, vivo and vitro test antitumor has and kills the most by force toxicity.

The present invention relates to the preparation of the T lymphocyte of the Chimeric antigen receptor genetic modification of a kind of targeting breast carcinoma stem cell Method, it is characterised in that CD47 and TAZ both of which high expressed in breast cancer tissue, especially expresses in breast carcinoma stem cell Special rising；By set up CD47 and TAZ process LAN breast cancer cell, its show higher propagation and transfer ability and Tumor stem cell feature；The rwo breast carcinoma stem cell antigen CD4 7 and TAZ extracellular domain of targeting of the present invention, immunity produces specificity In conjunction with monoclonal strain, obtained and the single-chain antibody of both specific bond by gene recombinaton, thus be built into containing anti-human CD47 Double distinctive embedment antigen receptor gene recombinaton with TAZ are on viral vector and transfect human T lymphocyte, and high expressed this pair is special Sex-mosaicism antigen receptor gene, can specific bond express people CD47 and TAZ breast carcinoma stem cell, activate the first signal and common thorn Energizing signal and cause anti-breast cancer toxic activity, in vitro tests antitumor has the strongest killing activity in vivo.

The cDNA of described bispecific chimeric antigen receptor (Di-CAR) gene builds in viral vector, and transfects T pouring Bar cell, then T lymphocyte is cultivated to the 14th day, it is thus achieved that Di-CAR-T cell；Detected by fluorescent quantitative PCR technique, turn Its expression Di-CAR gene of T lymphocyte having contaminated Di-CAR viral vector is more than 50 times higher than the T lymphocyte of untransfected；

The T lymphocyte obtained, this bispecific chimeric antigen receptor gene of high expressed, specific bond can express CD47 With the breast carcinoma stem cell of TAZ, activate the first signal and costimulatory signal simultaneously and cause antitumor cell toxic activity, internal In vitro tests antitumor has and kills the most by force toxicity；Its high expressed its specific markers CD3+/CD8+, positive rate higher than 90% with On, and Autoimmune disease CD4+/CD25+/Foxp3+ positive rate is less than less than 5%.

CD47 and TAZ gene of the present invention and protein high expressed in breast cancer tissue, pass through quantitative fluorescent PCR Detection, compared with normal galactophore tissue, both are respectively 89.00 and 134.55 times in expression in breast；Western blot Analyzing detection, both are respectively 23.49 and 35.67 times of normal galactophore tissue in expression in breast.

Preferably, described CD47 and TAZ gene and protein high expressed in breast carcinoma stem cell, In vitro culture obtains CD44+CD24-breast carcinoma stem cell fluorescence quantitative PCR detection, compared with breast cancer cell, both are table in breast cancer tissue Reach respectively 23.45 and 54.33 times；Western blot analysis detects, and both express respectively breast carcinoma in breast carcinoma stem cell 11.56 and 25.61 times of cell.

The present invention, also by building two slow virus carriers of CD47 and TAZ, transfects breast cancer cell respectively after packaging, it is high Express CD47 and TAZ gene and albumen；Body outer cell proliferation culture assays, the breast carcinoma of these two kinds of CD47 and TAZ virus transfections Cell is respectively 9.44 and 12.56 times of the Cells Proliferation of Human Breast Cancer of untransfected at the 7th day proliferation times；Transwell cell Migrating in experiment, these two kinds of cells there occurs significantly migration, and the breast cancer cell of untransfected migrates inconspicuous.And CD47 CD44+CD24-phenotypes are expressed by flow cytomery, both discoveries with the breast cancer cell of two slow-virus transfections of TAZ CD44+CD24-positive rate apparently higher than the breast cancer cell of untransfected, i.e. these two kinds of breast cancer cells is respectively 67.56% With 78.90%, and the breast cancer cell of untransfected is only 1.45%.

Become in tumor experiment in nude mouse, when the breast cancer cell of only 100 CD47 and TAZ virus transfections is inoculated, at the 7th day It is formed for tumor, and the breast cancer cell of untransfected inoculates 10⁶Square one-tenth tumor during the order of magnitude, show high expressed CD47 and The breast cancer cell of TAZ has phenotype and the characteristic of tumor stem cell.

Distinctive embedment antigen receptor gene containing specific bond CD47 of the present invention, including the list of specific bond CD47 Chain antibody connected two costimulatory molecules intracellular territories (CD28 and CD137) and the genetic fragment in CD3 ζ intracellular signal district, can Activate the first signal and costimulatory signal, i.e. activate immune response and cause antitumor cell toxic activity and tumor death Effect.

The distinctive embedment antigen receptor gene of TAZ of the present invention, including the single-chain antibody of TAZ and linked suppression The TET2 genetic fragment of IL-6 genetic transcription, suppresses T lymphocytic emiocytosis IL-6 to prevent " immune factor storm ".

Having linked furin cleavage site between the most described bispecific chimeric antigen receptor gene, it is at cell The Chimeric antigen receptor of two independent functions can be produced after interior protein translation.

In the method for the invention, T lymphocyte derives from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc. Mononuclearcell.Preferably, derive from cancer patient perform the operation one month after, the fresh peripheral blood that gathers after one month of chemicotherapy or Bone marrow.

Of the present invention by people's IFN γ-signal peptide-scFv (CD47)-CD8a hinge region-CD28-CD137-CD3 ζ intracellular District, and scFv (TAZ)-IgD hinge region-TET2 linked together by Furin cleavage site and catenation sequence, forms restructuring Gene order, gene order is obtained by chemosynthesis, forms the cDNA of complete bispecific chimeric antigen receptor gene, i.e. Di-CAR。

Di-CAR is built in viral vector, and transfects autologous patient T lymphocyte, then T lymphocyte is cultivated 14th day, it is thus achieved that Di-CAR-T lymphocyte.Being detected by fluorescent quantitative PCR technique, the T having transfected Di-CAR viral vector drenches Its expression Di-CAR gene of bar cell is more than 50 times higher than the T lymphocyte of untransfected.Western blotting technique detects its Di- CAR expressing fusion protein level, can express the fusion egg of scFv (CD47)-CAR 53kDa and scFv (TAZ)-CAR 42kDa In vain.

50ml PERIPHERAL BLOOD MONONUCLEAR CELL inducing culture of originating in the method for the invention obtains T lymphocyte, propagation times Number reaches more than 1000 times, cultivates and reaches 3 × 10 to 14 days T lymphocyte numbers⁹Above.

The method of the invention is cultivated the T lymphocyte of acquisition, high expressed its specific markers CD3+/CD8+, the positive Rate is higher than more than 90%, and Autoimmune disease CD4+/CD25+/Foxp3+ positive rate is less than less than 5%, and vivo and vitro tries Test and there is very powerful antitumor killing toxicity.

The T lymphocyte activator incubator that the present invention uses is coated for using mouse anti human CD3 monoclonal antibody (OKT3) Incubator, including Tissue Culture Plate, culture dish and culture bottle etc., it is therefore preferable to Tissue Culture Dish.

The cell culture medium that the present invention uses is that lymphocyte serum adds containing 1-500ng/ml1-500ng/ Ml IL-1 α, 10-2000 active unit (IU)/ml IL-2 and 5% (volume ratio) autoserum or hyclone, such as CellGro The commercial prods such as SCGM, AIM-V, X-VIVO and GT-T551.

The T lymph that present invention also offers a kind of Chimeric antigen receptor genetic modification preparing targeting breast carcinoma stem cell is thin The test kit of born of the same parents, it is characterised in that described test kit includes:

(1) viral vector stably expressing Di-CAR as above is obtained；

(2) T lymphocyte factor, including IL-1 α and IL-2；

(3) OKT3 is coated 75cm²Culture bottle；

(4) lymphocytes culture medium, including serum-free medium；

(5) lymphocyte separation medium；

(6) normal saline；

(7) operation instructions；

Wherein, described operation instructions include method as above.

Present invention also offers said method and test kit prepares T lymphocyte, can be used for the clinical immunization of breast carcinoma Treatment.

Accompanying drawing explanation

Fig. 1 is expressed as CD47 and TAZ gene expression in breast cancer tissue and normal galactophore tissue

Fig. 2 is expressed as western blotting technique detection CD47 and TAZ albumen table in patient with breast cancer and normal galactophore tissue Reach level

Fig. 3 is expressed as CD47 and TAZ gene expression in breast carcinoma stem cell and breast cancer cell

Fig. 4 is expressed as western blotting technique detection CD47 and TAZ albumen table in breast carcinoma stem cell and breast cancer cell Reach level

Fig. 5 is expressed as in mtt assay Detection results example three CD47-MCF7, TAZ-MCF7 and MCF-7 cell proliferation of preparation and lives Linearity curve

Fig. 6 is expressed as in effect example three CD47-MCF7, TAZ-MCF7 and MCF-7 cell FCM analysis of preparation

Fig. 7 is expressed as the expression after Di-CAR slow-virus transfection T lymphocyte after cultivating

Fig. 8 is expressed as western blotting technique detection Di-CAR fusion protein to be come Di-CAR slow-virus transfection patient with breast cancer The T Expressions In Lymphocytes level in source

Fig. 9 is expressed as patient with breast cancer's Di-CAR slow-virus transfection T lymphocyte fluidic cell knot of embodiment one preparation Fruit figure

Figure 10 be expressed as patient with breast cancer originate Di-CAR slow-virus transfection T lymphocyte (Di-CAR-T) to MCF-7 and The killing activity curve chart of BSC

Figure 11 is expressed as the tumor size change curve of Di-CART cell therapy breast carcinoma mouse model

Detailed description of the invention

Present invention discover that two tumor antigens of the equal specifically expressing of breast cancer tissue, breast cancer cell and breast carcinoma stem cell CD47 and TAZ.We are by CD47 and the TAZ process LAN slow virus carrier built simultaneously, transfect breast carcinoma after being packaged into virus Cell, finds that it has higher propagation and transfer ability, and shows tumor stem cell phenotype, with internal become tumor experiment have Tumor stem cell feature.By double distinctive embedment antigen receptor gene recombinaton of these two specific bond CD47 and TAZ to virus On carrier and transfect human T lymphocyte, this bispecific chimeric antigen receptor gene of high expressed, can specific bond express CD47 with And the breast cancer cell of TAZ and breast carcinoma stem cell, activate the first signal and costimulatory signal and vivo and vitro test causes Antitumor cell toxic activity.

By gathering patients with breast cancer tissue (put on record by Ethics Committee and sign Informed Consent Form with patient), CD47 and TAZ gene and protein carrying out in breast cancer tissue detection analyze, fluorescence quantitative PCR detection finds, and normally Mammary gland tissue is compared, and both are respectively 89.00 and 134.55 times in expression in breast；Western blot analysis detects, and two Person is respectively 23.49 and 35.67 times of normal galactophore tissue in expression in breast.

The CD44+CD24-breast carcinoma stem cell that In vitro culture of the present invention obtains, fluorescence quantitative PCR detection, CD47 and TAZ Gene and protein high expressed in breast carcinoma stem cell, compared with breast cancer cell, both divide in expression in breast It is not 23.45 and 54.33 times；Western blot analysis detects, and both express respectively breast cancer cell in breast carcinoma stem cell 11.56 and 25.61 times.

Additionally by building two slow virus carriers of CD47 and TAZ, after packaging, transfect breast cancer cell, its high expressed respectively CD47 and TAZ gene and albumen；Body outer cell proliferation culture assays, the breast cancer cell of these two kinds of CD47 and TAZ virus transfections It is respectively 9.44 and 12.56 times of the Cells Proliferation of Human Breast Cancer of untransfected at the 7th day proliferation times；Transwell cell migration In experiment, these two kinds of cells there occurs significantly migration, and the breast cancer cell of untransfected migrates inconspicuous.

The breast cancer cell of two slow-virus transfections of CD47 and TAZ passes through flow cytomery, finds that both express CD44+CD24-phenotype is apparently higher than the CD44+CD24-positive rate of the breast cancer cell of untransfected, i.e. these two kinds of breast cancer cells It is respectively 67.56% and 78.90%, and the breast cancer cell of untransfected is only 1.45%.Become in tumor experiment in nude mouse, only When the breast cancer cell of 100 CD47 and TAZ virus transfections is inoculated, it was formed for tumor at the 7th day, and the breast carcinoma of untransfected is thin Born of the same parents inoculate 10⁶Square one-tenth tumor during the order of magnitude, shows that the breast cancer cell of high expressed CD47 and TAZ has tumor stem cell Phenotype and characteristic.

To the T lymphocyte of the Chimeric antigen receptor genetic modification of a kind of targeting breast carcinoma stem cell that the present invention relates to Preparation method is specifically described.

The present invention relates to the preparation of the T lymphocyte of the Chimeric antigen receptor genetic modification of a kind of targeting breast carcinoma stem cell Method, it is characterised in that be built into the double distinctive embedment antigen receptor gene recombinaton containing specific bond CD47 and TAZ to virus On carrier and transfect human T lymphocyte, this bispecific chimeric antigen receptor gene of high expressed, can specific bond express CD47 with And the breast cancer cell of TAZ and breast carcinoma stem cell, activate the first signal and costimulatory signal and vivo and vitro test causes Antitumor cell toxic activity.

The distinctive embedment antigen receptor gene of TAZ of the present invention, including the single-chain antibody of TAZ and linked suppression The TET2 genetic fragment of IL-6 gene transcript expression, suppresses T lymphocytic emiocytosis IL-6 to prevent " immune factor wind in treatment Generation cruelly ".

Of the present invention by people's IFN γ-signal peptide-scFv (CD47)-CD8a hinge region-CD28-CD137-CD3 ζ intracellular District, and scFv (TAZ)-IgD hinge region-TET2 passes through Furin cleavage site and catenation sequence (Linker) links together, Forming recombination sequence, gene order is obtained by chemosynthesis, forms complete bispecific chimeric antigen receptor gene CDNA, i.e. Di-CAR.

By two specific chimeric antigen receptor genes, i.e. people's IFN γ-signal peptide-scFv (CD47)-CD8a hinge region- CD28-CD137-CD3 ζ intracellular region, and scFv (TAZ)-IgD hinge region-TET2 is by Furin cleavage site and catenation sequence Linking together, form recombination sequence, sequence is obtained by chemosynthesis, forms complete bispecific chimeric antigen and is subject to The cDNA of body gene, i.e. Di-CAR；

After the target DNA fragment of Di-CAR and pUC229 carrier Hind III/BamHI double digestion, at T4DNA ligase Under effect, by both in 4 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, after conversion competent escherichia coli cell DH5a Carry out positive colony PCR qualification and order-checking is identified；PCR primer detected through gel electrophoresis and order-checking qualification meet Di-CAR size and sequence After row, by checking order, correct bacterium solution is transferred in 10ml contains the LB fluid medium of corresponding antibiotic, and 37 DEG C of overnight incubation, with north Sky, capital root biology carries middle amount test kit carry out plasmid extraction without endotoxin plasmid is little, extracts qualified recombiant plasmid and places-80 DEG C The medium-term and long-term preservation of ultra cold storage freezer；

After the target DNA fragment of Di-CAR and pFLAG-CMV-2 carrier Hind III/BamH I double digestion, at T4DNA Under ligase effect, by both in 37 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, conversion competent escherichia coli cell Carry out positive colony PCR qualification after DH5a and order-checking is identified；

Take cell state good, be in the 293FT cell of exponential phase, after cell counting, according to the training of each 10cm Support ware 6 × 10⁶Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO₂Incubator in overnight incubation；Transfection reach in second day Remove culture fluid, change 5ml Opti-MEM culture fluid；Take 9 μ g packaging mixed liquors and 3 μ g slow virus expression plasmids add 1.5ml In Opti-MEM, mix gently, take 36 μ l lipofectamine2000 and add in 1.5ml Opti-MEM, mix gently, room Temperature places 5min；Mixing plasmid solution and lipofectamine 2000 diluent, put room temperature 20min；Mixed liquor is slowly added dropwise To the culture fluid of 293FT cell, mixing, in 37 DEG C, 5%CO₂Cell culture incubator is cultivated；Discard containing transfection after cultivating 6h The culture medium of mixture, the PBS liquid adding 10ml cleans once, after softly rocking the culture dish transfection mixture with washing remnants Abandon；It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO₂Continue in incubator to cultivate 48- 72h；

According to cell state, collect the 293FT cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged 10min, removes Cell debris；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Trim sample respectively, will be with viral supernatants The ultracentrifugation pipe of liquid is put into one by one to Beckman supercentrifuge, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature controls at 4 DEG C；After centrifugal end, supernatant discarded, remove as far as possible and remain in the liquid on tube wall, add virus Preserve liquid, the most repeatedly blow and beat resuspended；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence method Measuring titre, virus, according to 50 μ l 2E+8TU/ml subpackages, is stored in-80 DEG C of ultra cold storage freezers.

Collection derives from patient with breast cancer autologous peripheral blood 50-100ml, obtains list through lymphocyte separation medium is isolated and purified After individual nucleus, resuspended with serum-free medium and be diluted to 2-5 × 10⁶Cell/ml, and supplementary 1-500ng/ml IL-1 α, 10-2000IU/ml IL-2 and 5% autoserum, take 10ml and join mouse-anti people CD3 (OKT3) coated 75cm²Culture bottle In, in 37 DEG C, containing 5%CO₂Incubator cultivates the 3rd day；3rd day harvested by centrifugation T lymphocyte, and expect blue counting by tongue.

The T lymphocyte serum-free medium of the 3rd day results is resuspended for 5-10 × 10⁶Cell/ml, takes 6ml and joins The coated 75cm of OKT3²In culture bottle, and adding 100 μ l 1E+7TU/ml restructuring Di-CAR slow viruss, system is 6ml, mixing, 37 DEG C, 5%CO₂Incubator in hatch 8-12 hour after, change serum-free medium 10ml, described complete culture solution is preferred Containing 1-500ng/ml IL-1 α, 10-2000IU/ml IL-2 and 5% autoserum；When T lymphocyte growth density reach 10 × 10⁶During cell/ml, proceed to new 75cm²Culture bottle grows, and supplements complete serum-free medium to 50ml cultivating system；In 37 DEG C, containing 5%CO₂Cultivating by the 14th day in incubator, period changes supplementary containing according to T lymphocyte density and culture medium color 10-2000IU/ml IL-2 and the fresh complete serum-free medium of 5% autoserum, or expand bottle to 175cm²In culture bottle or turn Move on to 1-3L culture bag grows；

The T lymphocyte cultivated the 14th day is detected by fluorescent quantitative PCR technique, has transfected Di-CAR viral vector Its expression Di-CAR gene of T lymphocyte is more than 50 times higher than the T lymphocyte of untransfected；Western blotting technique detects it Di-CAR expressing fusion protein level, can express the fusion of scFv (CD47)-CAR 53kDa and scFv (TAZ)-CAR 42kDa Albumen.

The method of the invention is cultivated the T lymphocyte of acquisition, high expressed its specific markers CD3+/CD8+, the positive Rate is higher than more than 90%, and Autoimmune disease CD4+/CD25+/Foxp3+ positive rate is less than less than 5%, and described T drenches In bar cyton, in vitro tests has very powerful antitumor killing toxicity.

(1) viral vector stably expressing Di-CAR as above is obtained；

(2) T lymphocyte factor, including IL-1 α and IL-2；

(3) OKT3 is coated 75cm²Culture bottle；

(4) lymphocytes culture medium, including serum-free medium；

(5) lymphocyte separation medium；

(6) normal saline；

(7) operation instructions；

Wherein, described operation instructions include method as above.

The Tissue Culture Plate of use, Tissue Culture Dish, culture bottle, cell in cultivating as T lymphopoiesis of the present invention Culture bag, can be exemplified by 75cm²Tissue Culture Flask, 175cm²Tissue Culture Flask, 225cm²Tissue Culture Flask, 1L or 2L cell are trained Support the cells such as bag to cultivate with equipment (container), be used equally to the present invention, preferred cell culture bag.

T lymphocyte is carried out frozen by the manufacture method of the present invention, to frozen stock solution without particular restriction, but the most for example, 50% calf serum, 40% cell culture fluid and 10% dimethyl sulfoxide, wherein cell culture fluid is more preferably the training of T lymphocyte Nutrient solution.

Serum can be added in the medium or blood plasma is cultivated.They additions in the medium are not by special limit System, such as larger than 0 capacity % to 20 capacity %, and the consumption of serum or blood plasma can be changed according to different cultivation stages, excellent Elect 5% (volume ratio) as.For example, it is possible to interim minimizing serum or plasma concentration use.It addition, as serum or blood plasma Source, can be oneself (meaning identical with the cell derived cultivated) or oneself (cell meaning Yu being cultivated non- Source difference) in any one, set out from a security point, preferably oneself source serum or blood plasma.Alternatively, it is also possible to add Add the serum composition of purification as separated in human serum albumin etc.

The preparation of the T autologous lymphocytes propagation of the present invention uses above-mentioned various compositions and culture medium to implement.The present invention The cultivation condition of culture of middle use is also not particularly limited, it is possible to use the condition that common cell uses in cultivating.Such as, may be used At 37 DEG C, 5%CO₂Cultivate under the conditions of Deng.Can also implement such as inferior operation: interval reasonable time adds fresh culture Diluting cells culture fluid, or change culture medium, or change cell cultivation equipment etc..

The present invention also provides for the anti-breast cancer treatment in clinical practice repeatedly of T lymphocyte, the most in vivo Play anti-tumor immune response, reach good therapeutic effect.Additionally, above-mentioned T lymphocyte also has the advantage that, repeatedly T Lymphocyte treatment will preferably cause T lymphocyte to kill tumor activity, activate body autoimmune response, produce efficient, long-acting Ground antineoplastic immune effect, is therefore especially advantageous for improving and the prolongation of life cycle of patient with breast cancer's curative effect.

Hereinafter, the present invention is done in conjunction with the embodiments and more specifically describe, but the invention is not restricted to this.

Effect example one integrin associated protein (CD47) and transcribe the co-activation factor (TAZ) high expressed in breast cancer tissue

Gather 100 example patients with breast cancer tissues and 50 example volunteer normal galactophore tissues are (standby by Ethics Committee Case and sign Informed Consent Form with patient), take 1g and be organized in liquid nitrogen grinding, rear extract total serum IgE by Trizol one-step method, take 2 μ G reverse transcription synthesis template cDNA.Take 2 μ l reverse transcription product and carry out PCR reaction to detect the mrna expression level of target gene.By U.S. Invitrogen company of state PCR kit for fluorescence quantitative description, sets up the PCR reaction system that final volume is 20 μ l, and 2 μ l reverse Record product, 10 μ l SYBR Green Mix, each 0.5 μ l of upstream and downstream primer (10 μm ol/L).PCR thermal circulation parameters is: 95 DEG C 5min, then 94 DEG C of degeneration 30s, 60 DEG C of annealing 30s, carry out 45 circulations.Primer sequence is as follows:

TAZ positive-sense strand: 5 '-GTGTCCAATCACCAGTCCTG-3 ',

Antisense strand: 5 '-GCTGAAGAAGTGGGAGTGTAGC-3 '；

CD47 positive-sense strand: 5 '-GTGGTTGTTGGAGCCATCCT-3 ',

Antisense strand: 5 '-TGCCATGATGCAGAGACACA-3 '；

House-keeping gene NADPH positive-sense strand: 5 '-GACCCCTTCATTGACCTCAAC-3 ',

Antisense strand: 5 '-CGCTCCTGGGAAGATGGTGAT-3 '.

Machine testing on American AB I 7500 quantitative real time PCR Instrument, result uses 2^-ΔΔCtMethod calculates.Fig. 1 is CD47 and TAZ gene is expression in breast cancer tissue and normal galactophore tissue, and result shows that both are in breast cancer tissue Express average and be respectively 75.1 and 92.34 times of normal galactophore tissue；Show CD47 and TAZ gene high table in breast cancer tissue Reach.

Patient with breast cancer and normal healthy people 1g tissue carry out proteins gel electrophoresis after homogenate cracking.According to destination protein Molecular weight, prepare 10% separation gel, concentrating gum concentration is 5%.Protein sample applied sample amount to be detected: 20 μ g/ holes.Electrophoresis strip Part: concentration glue constant voltage 90V, about 20 minutes；By standard molecular weight pre-dyed albumen, separation gel constant voltage 120V, determines that electrophoresis stops Time.Wet robin, transferring film condition: 300mA constant current；0.45 μm aperture pvdf membrane, 80 minutes transferring film time.Transferring film is the beautiful spring after completing Film is dyeed by red colouring reagent, observes transferring film effect.Close: film is totally submerged in 5% (mg/mL) bovine serum albumin (BSA), in transferring film buffer (containing volume ratio 3% tween 20, TBST), under room temperature, horizontal shaker hatches 1 hour.One anti-incubates Educate: 5% (mg/mL) BSA-TBST dilution mouse anti human CD47 and TAZ monoclonal antibody 1: 2000, purchased from Santa company of the U.S.；4 DEG C of water Yawing bed overnight incubation.Next day, wash film: TBST and wash 3 times, each 10 minutes.The two of dropping horseradish peroxidase labelling resist film Hatching 1h, chemiluminescence develops the color.

Film scanner scanning is the TIF format picture more than 300DPI, uses Quantity One to analyze software (Bio-Rad company) carries out gray analysis, Fig. 2 be western blotting technique detection CD47 and TAZ albumen patient with breast cancer and just Often expression in mammary gland tissue, i.e. western blotting gray scale result figure, both are in expression in breast gray scale Average is respectively 1.96 and 2.88, and normal galactophore tissue is respectively 0.11 and 0.10；CD47 and TAZ albumen is in breast cancer tissue In be expressed as 17.36 and 29.65 times of normal galactophore tissue, show that CD47 and TAZ albumen both of which is high in breast cancer tissue Express.

Special high expressed in effect example two CD47 and TAZ breast carcinoma stem cell.

Also from above-mentioned 100 example patient with breast cancer's fresh tumor tissue, the tumor single cell suspension obtained through digestion makes With containing 20ng/ml recombinant human epidermal growth factor, 20ng/ml recombinant human fibroblast somatomedin, 10ng/ml insulin human and 1 DMEM/F12 (1: the 1) culture medium of × serum-free culture additive B27 is cultivated, and concrete grammar refers to Piscitelli E etc. " the becoming cultivation and the feature of the breast carcinoma stem cell of mammary gland ball " delivered in " molecular biology method " magazine for 2015, i.e. Piscitelli E et al.Culture and characterization of mammary cancer stem cells in mammospheres.Methods Mol Biol.2015；1235:243-62. the breast carcinoma stem cell obtained is thin through streaming It is 69.45% that born of the same parents' instrument detection CD44+CD24-leads, and is obtained the breast carcinoma stem cell of CD44+CD24-by selected by flow cytometry apoptosis. Breast cancer cell and normal breast cell are originated fresh tumor tissue and normal galactophore tissue respectively, by containing 10% hyclone DMEM (high sugar) culture medium culturing obtain.

Use fluorescence quantitative PCR detection CD47 and TAZ gene expression in breast carcinoma stem cell and breast cancer cell (concrete grammar take effect fruit example one), in Fig. 3, result show that both divide at expression average in breast carcinoma stem cell and breast cancer cell Wei 112.09 and 159.76,23.89 and 45.66 times of normal breast cell；Show that CD47 and TAZ gene is dry thin in breast carcinoma In born of the same parents, expression ratio breast cancer cell is higher.

CD47 and TAZ albumen expression in breast carcinoma stem cell and breast cancer cell is detected by western blotting technique And measure gray scale (concrete grammar take effect fruit example one), in Fig. 4 result show both in breast carcinoma stem cell and breast cancer cell Express gray average and be respectively 2.44 and 3.3,1.56 and 1.88, and normal breast cell is respectively 0.09 and 0.08；CD47 and TAZ albumen is expressed as 26.32 and 42.74 times of normal breast cell, 16.79 Hes in breast carcinoma stem cell and breast cancer cell 24.38 times, show CD47 and TAZ albumen both of which high expressed in breast carcinoma stem cell and breast cancer cell, and in breast carcinoma Stem cell is expressed higher than breast cancer cell.

Propagation that effect example three CD47 and TAZ process LAN breast cancer cell is higher and transfer ability

People CD47 and TAZ genetic fragment are designed the I/Hind III primer Han EcoR, by PCR respectively from human peripheral Middle amplification obtains, after pET-11c carrier EcoR I/Hind III double digestion, under T4DNA ligase effect, by CD47 and TAZ PCR primer respectively with the pET-11c after enzyme action in 4 DEG C, coupled reaction in 12 hours preparation clone is connected liquid, conversion escherichia coli sense By carrying out positive colony PCR qualification after state cell DH5a and order-checking is identified；PCR primer detected through gel electrophoresis and order-checking qualification meet After CD47 and TAZ size and sequence, the correct bacterium solution that checks order is transferred in 10ml contains the LB fluid medium of corresponding antibiotic, 37 DEG C of overnight incubation, carry middle amount test kit with sky, Beijing root biology carry out plasmid extraction without endotoxin plasmid are little, and it is qualified to extract Recombiant plasmid places-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer；Described CD47 and TAZ size is respectively 887bp and 1208bp；

After the target DNA fragment of CD47 and TAZ and GV358 carrier Pst I/Pst I double digestion, at T4DNA ligase Under effect, by both in 37 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, after conversion competent escherichia coli cell DH5a Carry out positive colony PCR qualification and order-checking is identified；

Take cell state good, be in the 293T cell of exponential phase, after cell counting, according to the cultivation of each 10cm Ware 6 × 10⁶Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO₂Incubator in overnight incubation；Remove before transfection in second day Culture fluid, changes 5ml Opti-MEM culture fluid；Take 9 μ g packaging mixed liquors and 3 μ g slow virus expression plasmids add 1.5ml Opti- In MEM, mixing gently, take 36 μ l lipofectamine2000 and add in 1.5ml Opti-MEM, mix gently, room temperature is placed 5min；Mixing plasmid solution and lipofectamine 2000 diluent, put room temperature 20min；Mixed liquor is slowly added dropwise to 293T In the culture fluid of cell, mixing, in 37 DEG C, 5%CO₂Cell culture incubator is cultivated；Discard after cultivating 6h containing transfection mixture Culture medium, the PBS liquid adding 10ml cleans once, softly rocks culture dish to abandon after the remaining transfection mixture of washing； It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO₂Continue in incubator to cultivate 72h.

According to cell state, collect the 293T cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged 10min, removes Cell debris；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Trim sample respectively, will be with viral supernatants The ultracentrifugation pipe of liquid is put into one by one to Beckman supercentrifuge, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature controls at 4 DEG C；After centrifugal end, supernatant discarded, remove as far as possible and remain in the liquid on tube wall, add virus Preserve liquid, the most repeatedly blow and beat resuspended；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence method Measuring titre, virus, according to 50 μ l 2E+8TU/ml subpackages, is stored in-80 DEG C of ultra cold storage freezers, has i.e. been prepared as CD47 and TAZ Two kinds of slow viruss.

With 3 × 10⁶Cell/ml breast cancer cell MCF-7 joins six well culture plates, and every hole is that 2ml RPMI 1640 trains Support base (containing 10% hyclone), in 37 DEG C, containing 5%CO₂Overnight incubation in incubator.6th day by 20 μ l 1E+7TU/ml weights In six well culture plates that group CD47 and TAZ slow virus is added separately to, system is 1ml, mixing, 37 DEG C, 5%CO₂Incubator In hatch 8-12 hour after, change complete culture solution RPMI 1640 2ml；When MCF-7 growth fusion reaches 90%, will MCF-7 cell transfers to 75cm²Culture bottle, supplements complete culture solution RPMI 1640 to 10ml cultivating system and carries out amplification cultivation. Detected according to operation instructions (American I nvitrogen company) by PCR kit for fluorescence quantitative, cultivating to the 10th generation MCF-7 cell is detected by fluorescent quantitative PCR technique, and it expresses purpose base to have transfected CD47 and TAZ virus transfection MCF-7 cell The MCF-7 of cause respectively higher than untransfected is 289.9 and 320.87 times.CD47-MCF7 lymph is the MCF-7 of CD47 slow-virus transfection Cell, TAZ-MCF7 is the MCF-7 cell of TAZ slow-virus transfection.

CD47-MCF7, TAZ-MCF7 and MCF-7 cell is with 5 × 10⁷/ L density is inoculated in 96 orifice plates, and often group sets 6 again Hole.Each group is terminated, in experiment, the MTT 20 μ l that front 4h adds 5g/L, cultivates 4h and discards culture fluid, and each hole adds 150 μ l dimethyl Sulfoxide (DMSO), shaken at room temperature 15min, microplate reader is surveyed absorbance (A) value at wavelength 490nm and is represented cytoactive.Above reality Test and be repeated 3 times.Mtt assay detection Cells Proliferation of Human Breast Cancer activity curve in Fig. 5, CD47-MCF7 and TAZ-MCF7 breeds speed Rate is apparently higher than MCF-7, and has statistical significance (P ＜ 0.01), shows that high expressed CD47 and TAZ promotes breast cancer cell Propagation growth.

24 orifice plate Transwell cells are used to carry out.Cell aperture 8 μm, after upper room is coated with 100 μ l Matrigel glue Ultraviolet irradiates 2h.CD47-MCF7, TAZ-MCF7 and MCF-7 cell is with 1 × 10⁶The density of/ml is inoculated in 6 orifice plates, after 24h, Each group absorption 200l cell respectively is inoculated in upper strata cell and is placed in 24 orifice plates, and lower room adds containing 10% hyclone RMPI1640 culture fluid cultivates 24h.Matrigel glue and the cell in face, room wiped by cotton swab, and methanol fixes 10min, and crystal violet contaminates Cell number in 5 visuals field of random counter under high power lens after color, experiment is repeated 3 times.Invasion and attack suppression ratio (%)=(invade by 1-experimental group Attack the quantity of the quantity/matched group invasion and attack cell of cell) × 100%.

Table 1 high expressed CD47 and the TAZ effect to breast cancer cell MCF-7 invasion and attack transfer ability

It can be seen that the breast cancer cell MCF-7 of high expressed CD47 and TAZ has attacks the most by force transfer ability from table 1, It is significantly higher than MCF-7 cell.

Effect example four CD47 and TAZ process LAN breast cancer cell has tumor stem cell characteristic

1 × 10 prepared in effect example three⁶CD47-MCF7, TAZ-MCF7 and MCF-7 cell, carries out streaming antibody staining Detection is analyzed, and experimental group is respectively 20 μ L mouse-anti people CD24-FITC and 20 μ L mouse-anti people CD44-PE；Isotype control group is 20 μ L MIgG1-FITC and 20 μ L mIgG1-PE；Place and wash 3 times, then at FACS with PBS after 4 DEG C of refrigerators dye 30 minutes The upper machine testing of Calibur instrument (U.S. company BD) and analysis.

Fig. 6 is CD47-MCF7, TAZ-MCF7 and MCF-7 cell FCM analysis of preparation, San Zhexi in effect example three The CD44+CD24-percentage ratio of born of the same parents is respectively 42.00%, 70.54% and 13.62%；CD47-MCF7 and TAZ-MCF7 breast carcinoma Stem Cell Phenotypic CD44+CD24-percentage ratio, apparently higher than MCF-7 cell, shows that the MCF-7 of high expressed CD47 and TAZ has breast Adenocarcinoma stem Cell Phenotypic.

45 8 weeks SCID nude mice abdominal part flank subcutaneous injections 10²、10³、10⁴、10⁵With 10⁶Individual CD47-MCF7, TAZ-MCF7 With MCF-7 cell, volume is 100 μ l, 3 nude mices of each injection dosage, raises 30 days continuously and observes into tumor situation, being specifically shown in Table 2.

Table 2 becomes tumor situation in inoculating breast cancer cell nude mouse

Note :-it is into tumor ,+, ++ with +++ different becomes tumor degree, and tumor size is the biggest.

From table 2 it can be seen that CD47-MCF7 and TAZ-MCF7 be vaccinated with 100 the 7th day existing tumor, and MCF-7 10⁶The order of magnitude tumor and 10⁴The order of magnitude became tumor at the 14th day；Show that only a few CD47-MCF7 and TAZ-MCF7 in vivo may be used Become tumor, there is tumor stem cell characteristic.

The preparation of the T lymphocyte of embodiment one bispecific chimeric antigen receptor genetic modification

By people's IFN γ-signal peptide-scFv (CD47)-CD8a hinge region-CD28-CD137-CD3 ζ intracellular region, and scFv (TAZ)-IgD hinge region-TET2 is consisted of the genetic fragment of Furin cleavage site and catenation sequence.

By people's IFN γ-signal peptide-scFv (CD47)-CD8a hinge region-CD28-CD137-CD3 ζ intracellular region, and scFv (TAZ)-IgD hinge region-TET2 is linked together by Furin cleavage site and catenation sequence (Linker), forms restructuring base Because of sequence, gene order is obtained by chemosynthesis, forms the cDNA of complete bispecific chimeric antigen receptor gene, i.e. Di-CAR；Di-CAR slow virus carrier builds with packing method as the method described in effect example three, it is thus achieved that Di-CAR is the most sick Poison takes supernatant fluorescence spectrometry titre, and virus, according to 50 μ l 2E+8TU/ml subpackages, is stored in-80 DEG C of ultra cold storage freezers, i.e. makes Standby one-tenth Di-CAR slow virus.

Gather patients with breast cancer 50ml (signing Informed Consent Form with it), add equivalent 1 × PBS dilution, along centrifugal Tube wall is superimposed on lymphocyte separation medium gently, forms sharp interface, and under room temperature, 2000rpm is centrifuged 20min, draws interface The PBMC of layer, washes twice with 1 × PBS (pH value is 7.4).After tongue dish orchid counting, resuspended also with VIVO-15 serum-free medium It is diluted to 3 × 10⁶Cell/ml, and supplementary 50ng/ml IL-1 α, 500IU/ml IL-2 and 5% autoserum, take 10ml and add To mouse-anti people CD3 (OKT3) coated 75cm²In culture bottle, in 37 DEG C, containing 5%CO₂Incubator cultivates the 3rd day；3rd day from Heart results T lymphocyte, and expect blue counting by tongue.

The T lymphocyte serum-free medium of the 3rd day results is resuspended for 5-10 × 10⁶Cell/ml, takes 6ml and joins The coated 75cm of OKT3²In culture bottle, and adding 100 μ l 1E+7TU/ml restructuring Di-CAR slow viruss, system is 6ml, mixing, 37 DEG C, 5%CO₂Incubator in hatch 8-12 hour after, change serum-free medium 10ml, described complete culture solution is preferred Containing 50ng/ml IL-1 α, 500IU/ml IL-2 and 5% autoserum；When T lymphocyte growth density reaches 10 × 10⁶Carefully During born of the same parents/ml, proceed to new 75cm²Culture bottle grows, and supplements complete serum-free medium to 50ml cultivating system；In 37 DEG C, containing 5%CO₂Cultivating by the 14th day in incubator, period changes supplementary containing according to T lymphocyte density and culture medium color 500IU/ml IL-2 and the fresh complete serum-free medium of 5% autoserum, transfer to grow in 2L culture bag；

Fig. 7 is the expression after Di-CAR slow-virus transfection T lymphocyte after cultivating.By quantitative fluorescent PCR skill The T lymphocyte of the 14th day is cultivated in art detection, has transfected its expression Di-CAR gene of T lymphocyte of Di-CAR viral vector It it is 125.6 times higher than the T lymphocyte of untransfected.

The T lymphocyte protein trace of embodiment two bispecific chimeric antigen receptor genetic modification and fluidic cell inspection Survey

Example one preparation advanced breast cancer patient and normal healthy people originate the 14th day cultivate obtain T lymph thin Born of the same parents each 1 × 10⁶, by carrying out western blotting western blot analysis after ultrasonication, described method take effect fruit example one.

Fig. 8 is that western blotting technique detection Di-CAR fusion protein is originated Di-CAR slow-virus transfection patient with breast cancer T lymphocyte, and Di-CAR fusion protein expression in escherichia coli level (positive control), i.e. western blotting ties Fruit figure, all occurs two bands in two example patients, and wherein one is CD47-CAR, and about 479 amino acid residues, molecular weight is about 53kD；Another is TAZ-CAR, and about 417 amino acid residues, molecular weight is about 46kD.1st band is Di-CAR slow-virus transfection Breast carcinoma T lymphocyte, the 2nd band is Di-CAR fusion protein expression in escherichia coli, and band molecular weight is about 99kD, i.e. CD47-CAR and TAZ-CAR molecular weight sum.

The patient with breast cancer of Example one preparation originates the 14th day and cultivates 1 × 10 obtained⁶T lymphocyte, flows The detection of formula antibody staining is analyzed.First group of T lymphocyte be respectively 20 μ L mouse-anti people CD3-FITC, 20 μ L mouse-anti people CD4-PE and 20 μ L mouse-anti people CD8-PerCP；Second component is not 20 μ L mouse-anti people FoxP3-FITC, 20 μ L mouse-anti people CD4-PE and 20 μ L Mus Anti-human CD25-PerCP；Isotype control group is 20 μ L mIgG1-FITC, 20 μ L mIgG1-PE and 20 μ L mIgG1-PerCP；Put Put and wash 3 times, then at the upper machine testing of FACS Calibur instrument (U.S. company BD) with PBS after 4 DEG C of refrigerators dye 30 minutes And analysis.

Fig. 9 is patient with breast cancer's Di-CAR slow-virus transfection T lymphocyte FCM analysis of embodiment one preparation, should T lymphocyte CD 3 and CD8 positive rate is 91.09%, and CD4+CD25+FoxP3+ positive rate is 2.71%.

Embodiment three

The T lymphocytes in vitro of bispecific chimeric antigen receptor genetic modification kills tumor test

Respectively with the breast carcinoma stem cell of the CD44+CD24-of preparation in breast cancer cell line MCF-7 and effect example two (Breast stem cells, BSC) is target cell, makees with the Di-CAR slow-virus transfection T lymphocyte of preparation in embodiment two For effector lymphocyte, it is separately added in 96 well culture plates than 40: 1,20: 1,10: 1,5: 1 and 2.5: 1 by effect target, is subsequently placed in 37 DEG C, cultivate 4h under the conditions of 5%CO2, be all provided with 3 multiple holes.Employing LDH cellulotoxic experiment method mensuration T lymphocyte activation: Aspirate supernatant 50 μ L, are placed in 96 orifice plates, add LDH base fluid 50 μ L, room temperature lucifuge 30min, add 50 μ L stop buffers and terminate reaction, 490nm Absorbance (OD) value is surveyed at wavelength.The killing rate of PC-3 and MCF-7 cell is calculated respectively: killing rate=(experiment by following equation Group OD value-effector lymphocyte's Spontaneous release group OD value-target cell Spontaneous release group OD value)/(target cell maximum release group OD value-target Cell Spontaneous release group OD value) × 100%.

Figure 10 is that patient with breast cancer originates Di-CAR slow-virus transfection T lymphocyte (Di-CAR-T) to MCF-7's and BSC Killing activity, along with effect target ratio improves, cellular cytoxicity activity also improves constantly, and Di-CAR slow-virus transfection patient with breast cancer T drenches Bar cell killing MCF-7 and BSC kills toxicity and is significantly higher than the T lymphocyte of untransfected, and p value is 0.016, and Di-CAR-T Kill MCF-7 with BSC to compare zero difference.

Embodiment five

The effect in tumor experiment is killed in the T lymphocyte body of bispecific chimeric antigen receptor genetic modification

Taking the nude mice model using CD47-MCF7, TAZ-MCF7 and MCF7 to set up in effect example four, above-mentioned three kinds of cells connect Planting the Breast Carcinoma in nude mice model set up to be grouped, A group is the Di-CAR slow-virus transfection patient with breast cancer T of embodiment one preparation Lymphocyte treatment group；B group is patient with breast cancer's T lymphocyte treatment group of the untransfected of embodiment one preparation；C group is physiology Saline placebo group.A group is cultivated the 14th day and the 16th day tail is quiet at Di-CAR slow-virus transfection patient with breast cancer's T lymphocyte Arteries and veins injection 2 × 10⁴Cell/gram, B group C group is cultivated the 14th day and the 16th day tail is quiet at untransfected patient with breast cancer's T lymphocyte Arteries and veins injection 2 × 10⁴Cell/gram, C group and A and B treatment group same time, the normal saline of tail vein injection same volume, each 1mL.Control Draw neck to put to death respectively at 7d, 14d, 21d and 28d after treatment, take breast carcinoma SCID mouse model tumor tissues, calculate mouse model tumor big Little.

The SCID mouse model T lymphocyte of Figure 11 CD47-MCF7, TAZ-MCF7 and MCF7 has treated tumor size afterwards Change curve, normal saline group C is compared with Di-CART cell therapy group A, T lymphocyte treatment group B, newborn in treatment group A and B Adenocarcinoma tumor size reduces, but treatment group A breast cancer tumour size compared with treatment group B reduces substantially, and p value is respectively 0.004, The mouse model that the most anti-CD47-MCF7 and TAZ-MCF7 sets up becomes apparent from, and treatment group B kills this two kinds of mouse model effects ten Divide inconspicuous, show that Di-CAR slow-virus transfection patient with breast cancer's T lymphocyte is equal to breast cancer cell and breast carcinoma stem cell Powerful cytotoxicity can have been caused.

Claims

1. the preparation side of the T lymphocyte of the bispecific chimeric antigen receptor genetic modification of a targeting breast carcinoma stem cell Method, it is characterised in that targeting breast carcinoma stem cell antigen CD4 7 and TAZ extracellular domain, immunity produces specific binding monoclonal Strain, obtains the single-chain antibody with both specific bond by gene recombinaton, is built into the double distinctive embedments containing anti-human CD47 and TAZ Antigen receptor gene (double chimeric antigen receptor, Di-CAR), recombinates on viral vector and transfects Human T lymphocyte, can specific bond express people CD47 and TAZ breast carcinoma stem cell；

The cDNA of described bispecific chimeric antigen receptor (Di-CAR) gene builds in viral vector, and it is thin to transfect T lymph Born of the same parents, then T lymphocyte is cultivated to the 14th day, it is thus achieved that Di-CAR-T cell；Detected by fluorescent quantitative PCR technique, transfect Its expression Di-CAR gene of the T lymphocyte of Di-CAR viral vector is more than 50 times higher than the T lymphocyte of untransfected；

The T lymphocyte obtained, this bispecific chimeric antigen receptor gene of high expressed, can specific bond express CD47 and The breast carcinoma stem cell of TAZ, activates the first signal and costimulatory signal simultaneously and causes antitumor cell toxic activity, internal body Outer test antitumor has and kills the most by force toxicity；Its high expressed its specific markers CD3+/CD8+, positive rate higher than 90% with On, and Autoimmune disease CD4+/CD25+/Foxp3+ positive rate is less than less than 5%.

Method the most according to claim 1, it is characterised in that by people's IFN γ-signal peptide-scFv (CD47)-CD8a hinge District's-CD28-CD 137-CD3 ζ intracellular region, and scFv (TAZ)-IgD hinge region-TET2 is by Furin cleavage site and connection Sequence links together, and forms recombination sequence, and gene order is obtained by chemosynthesis, forms complete bispecific embedding Close the cDNA, i.e. Di-CAR of antigen receptor gene；

Described CD47 and TAZ gene and protein high expressed in breast cancer tissue, by fluorescence quantitative PCR detection, and just Often mammary gland tissue is compared, and both are respectively 89.00 and 134.55 times in expression in breast；Western blot analysis detects, Both are respectively 23.49 and 35.67 times of normal galactophore tissue in expression in breast.

Method the most according to claim 1 and 2, it is characterised in that described CD47 and TAZ gene and protein are at mammary gland High expressed in cancer stem cell, the CD44+CD24-breast carcinoma stem cell fluorescence quantitative PCR detection that In vitro culture obtains, with breast carcinoma Cell is compared, and both express in breast carcinoma stem cell and are respectively 23.45 and 54.33 times；Western blot analysis detects, Liang Zhe Breast carcinoma stem cell is expressed be respectively breast cancer cell 11.56 and 25.61 times.

4. according to the method described in any one of claim 1-3, it is characterised in that by building two slow viruss of CD47 and TAZ Carrier, transfects breast cancer cell, its high expressed CD47 and TAZ gene and albumen respectively after packaging；Body outer cell proliferation is cultivated and is divided Analysis, the breast cancer cell of these two kinds of CD47 and TAZ virus transfections is respectively the breast cancer cell of untransfected at the 7th day proliferation times 9.44 and 12.56 times of propagation；In Transwell Cell migration assay, these two kinds of cells there occurs significantly migration, and does not turns The breast cancer cell of dye migrates inconspicuous.

5. according to the method described in any one of claim 1-4, it is characterised in that the breast of two slow-virus transfections of CD47 and TAZ Adenocarcinoma cell passes through flow cytomery, finds that both express CD44+CD24-phenotype thin apparently higher than the breast carcinoma of untransfected The CD44+CD24-positive rate of born of the same parents, i.e. these two kinds of breast cancer cells is respectively 67.56% and 78.90%, and the mammary gland of untransfected Cancerous cell is only 1.45%；

Become in tumor experiment in nude mouse, when the breast cancer cell of only 100 CD47 and TAZ virus transfections is inoculated, at the 7th day with regard to shape Become tumor, and the breast cancer cell of untransfected inoculates square one-tenth tumor during 106 order of magnitude, shows high expressed CD47's and TAZ Breast cancer cell has phenotype and the characteristic of tumor stem cell.

6. according to the method described in any one of claim 1-5, it is characterised in that described bispecific chimeric antigen receptor contains There is CD47 and TAZ Chimeric antigen receptor gene, including the single-chain antibody of CD47 and linked the genetic transcription of specific bond IL-6 and press down The genetic fragment of preparation DNA modification enzyme, can suppress the expression of IL-6 gene；The single-chain antibody of specific bond TAZ has also connected two Individual costimulatory molecules intracellular territory and the genetic fragment in CD3 ζ intracellular signal district；Said two costimulatory molecules is CD28 and CD137； Having linked furin cleavage site between bispecific chimeric antigen receptor gene, it can produce after intracellular protein is translated The Chimeric antigen receptor of raw two independent functions.

Method the most according to any one of claim 1 to 6, it is characterised in that

Collection derives from patient with breast cancer autologous peripheral blood 50-100ml, obtains single core through lymphocyte separation medium is isolated and purified After cell, resuspended with serum-free medium and be diluted to 2-5 × 10⁶Cell/ml, and supplementary 1-500ng/ml IL-1 α, 10- 2000IU/ml IL-2 and 5% autoserum, take 10ml and join mouse-anti people CD3 (OKT3) coated 75cm²In culture bottle, in 37 DEG C, containing 5%CO₂Incubator cultivates the 3rd day；3rd day harvested by centrifugation T lymphocyte, and expect blue counting by tongue；

The T lymphocyte serum-free medium of the 3rd day results is resuspended for 5-10 × 10⁶Cell/ml, takes 6ml and joins OKT3 bag The 75cm of quilt²In culture bottle, and adding 100 μ l 1E+7 TU/ml restructuring Di-CAR slow viruss, system is 6ml, mixing, 37 DEG C, 5%CO₂Incubator in hatch 8-12 hour after, change serum-free medium 10ml, described complete culture solution is preferably containing 1- 500ng/ml IL-1 α, 10-2000IU/ml IL-2 and 5% autoserum；When T lymphocyte growth density reaches 10 × 10⁶ During cell/ml, proceed to new 75cm²Culture bottle grows, and supplements complete serum-free medium to 50ml cultivating system；In 37 DEG C, containing 5%CO₂Cultivating by the 14th day in incubator, period changes supplementary containing according to T lymphocyte density and culture medium color 10-2000IU/ml IL-2 and the fresh complete serum-free medium of 5% autoserum, or expand bottle to 175cm²In culture bottle or turn Move on to 1-3L culture bag grows；

The T lymphocyte cultivated the 14th day is detected by fluorescent quantitative PCR technique, and the T having transfected Di-CAR viral vector drenches Its expression Di-CAR gene of bar cell is more than 50 times higher than the T lymphocyte of untransfected；Western blotting technique detects its Di- CAR expressing fusion protein level, can express the fusion egg of scFv (CD47)-CAR 53kDa and scFv (TAZ)-CAR 42kDa In vain.

8. the preparation side of the T lymphocyte of the bispecific chimeric antigen receptor genetic modification of a targeting breast carcinoma stem cell Method, it is characterised in that

By people's IFN γ-signal peptide-scFv (CD47)-CD8a hinge region-CD28-CD137-CD3 ζ intracellular region, and scFv (TAZ)-IgD hinge region-TET2 is linked together by Furin cleavage site and catenation sequence (Linker), forms restructuring base Because of sequence, gene order is obtained by chemosynthesis, forms the cDNA of complete bispecific chimeric antigen receptor gene, i.e. Di-CAR；Di-CAR slow virus carrier builds with packing method as the method described in effect example three, it is thus achieved that Di-CAR is the most sick Poison takes supernatant fluorescence spectrometry titre, and virus, according to 50 μ l 2E+8 TU/ml subpackages, is stored in-80 DEG C of ultra cold storage freezers, i.e. makes Standby one-tenth Di-CAR slow virus；

Gather patients with breast cancer 50ml, add equivalent 1 × PBS dilution, be superimposed on lymphocyte gently along centrifuge tube tube wall Separating on liquid, form sharp interface, under room temperature, 2000rpm is centrifuged 20min, draws the PBMC of boundary layer, washs two with 1 × PBS Secondary；After tongue dish orchid counting, resuspended with VIVO-15 serum-free medium and be diluted to 3 × 10⁶Cell/ml, and supplementary 50ng/ml IL-1 α, 500IU/ml IL-2 and 5% autoserum, take 10ml and join mouse-anti people CD3 (OKT3) coated 75cm²Culture bottle In, in 37 DEG C, containing 5%CO₂Incubator cultivates the 3rd day；3rd day harvested by centrifugation T lymphocyte, and expect blue counting by tongue；

The T lymphocyte serum-free medium of the 3rd day results is resuspended for 5-10 × 10⁶Cell/ml, takes 6ml and joins OKT3 bag The 75cm of quilt²In culture bottle, and adding 100 μ l 1E+7 TU/ml restructuring Di-CAR slow viruss, system is 6ml, mixing, 37 DEG C, 5%CO₂Incubator in hatch 8-12 hour after, change serum-free medium 10ml, described complete culture solution preferably contains 50ng/ml IL-1 α, 500IU/ml IL-2 and 5% autoserum；When T lymphocyte growth density reaches 10 × 10⁶Cell/ During ml, proceed to new 75cm²Culture bottle grows, and supplements complete serum-free medium to 50ml cultivating system；In 37 DEG C, contain 5%CO₂Cultivating by the 14th day in incubator, period changes according to T lymphocyte density and culture medium color supplements containing 500IU/ Ml IL-2 and the fresh complete serum-free medium of 5% autoserum, transfer to grow in 2L culture bag；

Cultivated to the T lymphocyte of the 14th day by fluorescent quantitative PCR technique detection, the T having transfected Di-CAR viral vector drenches Its expression Di-CAR gene of bar cell is 125.6 times higher than the T lymphocyte of untransfected；

Wherein, described Di-CAR slow virus carrier builds and packing method is as follows,

People CD47 and TAZ genetic fragment are designed the I/Hind III primer Han EcoR, is expanded from human peripheral respectively by PCR Increase and obtain, after pET-11c carrier EcoR I/Hind III double digestion, under T4DNA ligase effect, by CD47 and TAZ PCR Product respectively with the pET-11c after enzyme action in 4 DEG C, coupled reaction in 12 hours preparation clone is connected liquid, convert escherichia coli and experience Carry out positive colony PCR qualification after state cell DH5a and order-checking is identified；PCR primer detected through gel electrophoresis and order-checking qualification meet After CD47 and TAZ size and sequence, the correct bacterium solution that checks order is transferred in 10ml contains the LB fluid medium of corresponding antibiotic, 37 DEG C of overnight incubation, carry middle amount test kit with sky, Beijing root biology carry out plasmid extraction without endotoxin plasmid are little, and it is qualified to extract Recombiant plasmid places-80 DEG C of medium-term and long-term preservations of ultra cold storage freezer；Described CD47 and TAZ size is respectively 887bp and 1208bp；

After the target DNA fragment of CD47 and TAZ and GV358 carrier Pst I/Pst I double digestion, in T4DNA ligase effect Under, by both in 37 DEG C, coupled reaction in 12 hours preparation clone's connection liquid, carry out after conversion competent escherichia coli cell DH5a Positive colony PCR identifies and order-checking is identified；

Take cell state good, be in the 293T cell of exponential phase, after cell counting, according to the culture dish 6 of each 10cm ×10⁶Individual cell number is inoculated in culture dish, 37 DEG C, 5%CO₂Incubator in overnight incubation；Cultivation is removed before transfection in second day Liquid, changes 5ml Opti-MEM culture fluid；Take 9 μ g packaging mixed liquors and 3 μ g slow virus expression plasmids add 1.5ml Opti-MEM In, mixing gently, take 36 μ l lipofectamine2000 and add in 1.5ml Opti-MEM, mix gently, room temperature is placed 5min；Mixing plasmid solution and lipofectamine 2000 diluent, put room temperature 20min；Mixed liquor is slowly added dropwise to 293T In the culture fluid of cell, mixing, in 37 DEG C, 5%CO₂Cell culture incubator is cultivated；Discard after cultivating 6h containing transfection mixture Culture medium, the PBS liquid adding 10ml cleans once, softly rocks culture dish to abandon after the remaining transfection mixture of washing； It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO₂Continue in incubator to cultivate 72h；

According to cell state, collect the 293T cell supernatant of 48h after transfecting；In 4 DEG C, 4000g is centrifuged 10min, removes cell Fragment；With 0.45 μm filter filtering supernatant in 40ml ultracentrifugation pipe；Trim sample respectively, by with vial supernatant Ultracentrifugation pipe is put into one by one to Beckman supercentrifuge, and arranging parameter of noncentricity is 25000rpm, and centrifugation time is 2h, Centrifuging temperature controls at 4 DEG C；After centrifugal end, supernatant discarded, remove as far as possible and remain in the liquid on tube wall, add virus preservation Liquid, blows and beats resuspended the most repeatedly；After fully dissolving, high speed centrifugation 10000rpm, after centrifugal 5min, take supernatant fluorescence spectrometry Titre, virus, according to 50 μ l 2E+8 TU/ml subpackages, is stored in-80 DEG C of ultra cold storage freezers, has i.e. been prepared as CD47 and TAZ two Plant slow virus；

Described Di-CAR slow-virus transfection T lymphocyte FCM analysis, this T lymphocyte CD 3 and CD8 positive rate is 91.09%, CD4+CD25+FoxP3+ positive rate is 2.71%.

9. the reagent of the T lymphocyte of the bispecific chimeric antigen receptor genetic modification preparing targeting breast carcinoma stem cell Box, it is characterised in that described test kit includes:

(1) viral vector of the acquisition stable expression Di-CAR described in any one of claim 1-8；

(2) T lymphocyte factor, including IL-1 α and IL-2；

(3) OKT3 is coated 75cm²Culture bottle；

(4) lymphocytes culture medium, including serum-free medium；

(5) lymphocyte separation medium；

(6) normal saline；

(7) operation instructions；

Wherein, described operation instructions include the method according to any one of claim 1-8.

10. the bispecific chimeric antigen receptor gene of targeting breast carcinoma stem cell according to any one of claim 1-9 The purposes of the T lymphocyte modified.