CN109666651A - A kind of CAR-T cell and its preparation method and application of secreting type targeting Lewis-Y - Google Patents
A kind of CAR-T cell and its preparation method and application of secreting type targeting Lewis-Y Download PDFInfo
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- CN109666651A CN109666651A CN201910075310.2A CN201910075310A CN109666651A CN 109666651 A CN109666651 A CN 109666651A CN 201910075310 A CN201910075310 A CN 201910075310A CN 109666651 A CN109666651 A CN 109666651A
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Classifications
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5434—IL-12
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
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- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
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Abstract
The invention discloses the CAR-T cells of secreting type targeting Lewis-Y a kind of, the CAR-T cell including targeting Lewis-Y and secretion IL12 cell factor；It targets Lewis-Y and secretes the CAR-T cell of PD-1 antibody.The invention also discloses the CAR-T cell preparation methods of secreting type targeting Lewis-Y a kind of and the CAR-T cell to prepare the application in antitumor cellular therapeutic agent.The PD-1 antibody of CAR-T cell secretion of the present invention, it is specific to be combined with the PD-1 on T cell surface, the inhibition reaction active to T cell of PD-1/PD-L1 signal is blocked, is conducive to CAR-T and effective inhibit is generated to growth of tumour cell for a long time；The IL12 cell factor of CAR-T cell secretion of the present invention, may act on the immunosuppressant cell that tumor-inhibitory is immunized in microenvironment, reduce its immunosuppression capability, and then enhance the fragmentation effect of CAR-T cell.
Description
Technical field
The present invention relates to immunology, Celluar and Molecular Biology field more particularly to one kind can secrete IL12 or
PD-1 antibody and the CAR-T cell for targeting Lewis-Y, moreover, it relates to which a kind of prepare this carefully using slow virus carrier
The application of the preparation method of born of the same parents and the cell.
Technical background
" Cancer Statistics in China, the 2015 " display delivered for 2016, China in 2015 in advance in respect of
429.2 ten thousand new hair tumor cases and 281.4 ten thousand deaths.Wherein, the highest cancer of the death rate are as follows: lung cancer, bronchus
Cancer, gastric cancer, liver cancer, cancer of the esophagus and colorectal cancer have accounted for 3/4ths of all cancer mortalities.According to the newest announcement of WHO
Data, China's tumor mortality number account for about 23%, are only second to cardiovascular disease.With China human mortality aging aggravation, industrialize into
The bring living habit of the factors such as bring environmental pollution and urbanization changes in journey, the tumour illness rate of China resident and death
Rate significantly improves.The traditional treatment mode of tumour, including operation, radiotherapy, chemotherapy and targeted therapy, these types of method have respectively
Limitation.The tumor patient performed the operation primarily directed to early stage, it is bad for advanced stage and metastatic tumo(u)r patient outcome；Chemicotherapy
Clinical efficacy is relatively limited and toxic side effect is larger；Although targeted therapy effect is obvious, quick, it is easy to generate drug resistance
Property.Immunotherapy of tumors is the new treatment means after operation, radiotherapy, chemotherapy, especially immunologic test point monoclonal antibody treatment with
The curative effect of the emergence of CAR-T cellular immunotherapy, immunization therapy is gradually affirmed.2013, " Science " magazine was by tumour
Immunization therapy is chosen as first of annual ten big sciences breakthrough.In January, 2016, the U.S. is based on immunization therapy and targeted therapy develops,
It proposes cancer " Lunar Probe Project ".
1985, Rosenberg SA outwardly disclosed the clinic of LAK joint IL-2 treatment malignant mela noma for the first time
Result of study.Later, developed kinds of schemes in succession on this basis, as the Tumor-infiltrating lymphocytes of the first generation are treated
Method (Lymphokine Activated Killer, LAK), the cytokine induced kill cell therapy of the second generation
(Cytokine Induced Killer cell, CIK), the tumor infiltrating lymphocyte therapy (Tumor of the third generation
Infiltrating Lymphocytes, TIL), cytotoxic T cell therapy (the Cytotoxic T of forth generation
Lymphocytes, CTL) and the 5th generation Chimeric antigen receptor T cell therapy (Chimeric Antigen Receptor-
Modified T cells, CAR-T).
Chimeric antigen receptor T cell (CAR-T cell): CAR-T cell therapy is to utilize technique for gene engineering by subject T
Lymphocytes in vitro transfects Chimeric antigen receptor CAR, forms the effector T cell for having specific recognition capability to specific antigen, warp
It is fed back in subject's body after crossing amplification in vitro, the CAR-T cell of feedback passes through efficiently special non-MHC restrictive one identification
Specific tumor cell surface antigen, and tumour cell is completed to kill.CAR usually encodes one and includes single-chain antibody scFv,
To combine tumor cell surface specific antigen；It is connected with intracellular signal transduction structural domain simultaneously, usually CD28 or/and 4-
1BB costimulatory molecules and CD3 ζ intracellular signal domain, the activation of mediate T cell.2017, FDA have approved targeting CD19 based on
The two generation CAR-T launch of 4-1BB.
Lewis Y antigen is also known as CD174, CD174 antigen category blood group antigens (blood group antigen, BGA)
One of ABH and Lewis antigen family member, be H2 dimerization fucose (α 1-2 galactolipin β 1-4 acetylglucosamine), CD174 and
ABH antigen is closely related, they are regulated and controled by different genes, and common precursor generates.2001, Lewis Y was formally ordered
Entitled CD174.As a kind of blood group antigens, CD174 plays a role in cell recognition, differentiation, growth regulating, and
Material alterations occur in good process of malignant transformation.Wakabayashi etc. is to 46 liver cancer patients the study found that 20 tumour cells
In there is CD174 to express (43.5%), the CD174 expression rate of AFP>200ng/ml patient is higher than AFP<200ng/ml patient.Right
The Immunohistochemical detection discovery of patient with breast cancer's operation Operated Specimens, the expression of the patient with breast cancer CD174 in advanced stage are higher than
Early stage patient, the researchs such as Inufusa find that 5 years survival rates of CD174 positive patient are significantly lower than negative patient.In patients with lung cancer
In, it is again seen that there is height to express by CD174.Tanaka etc. has studied 236 Patients with Non-small-cell Lung, CD174 expression sun
179 of property, accounting 75.8%.Mehdi etc. has found 234 in 260 Patients with Non-small-cell Lung researchs in I, II phase
A degree of CD174 positive expression can be detected in the patients surgery sample of position.
Scott is equal to the CD174 antibody hu3S193 for having delivered humanization for 2000, gives the mouse of heteroplastic transplantation MCF-7
The discovery of hu3S193 antibody, hu3S193 are able to suppress the growth of mouse interior tumor.At one with CD174 monoclonal antibody (hu3S193)
In the II phase clinical research for treating the oophoroma of recurrent and refractory, carcinoma of fallopian tube and Primary peritoneal carcinoma, 31 subjects receive
The monoclonal antibody treatment of hu3S193, it was demonstrated that the safety of hu3S193 monoclonal antibody targeting CD174 antigen, that observes is main secondary anti-
It should be fatigue, nausea and allergic reaction.
Westwood etc. is the study found that anti-CD174CAR-T cell is trained altogether with CD174 antigen positive expression tumour cell
When supporting, T cell can kill cracking tumour cell, and the water-soluble CD174 antigen in serum has no effect on the work of CAR-T cell
Property.In mouse experiment in vivo, the discovery such as Westwood, Peinert can be killed based on the CAR-T cell that hu3S193 is constructed
The intracorporal tumour of mouse is to inhibit the growth of tumour.Ritchie etc. completes one and is controlled with the CAR-T cell of anti-CD174
The Phase I clinical trial of patients with acute myeloid leukemia is treated, 4 subjects receive 1.3 × 109T cell, do not find 3 grades
Or 4 grades of toxicity, it was demonstrated that the CAR-T of anti-CD174 is safe for clinical treatment.
Currently, the CAR structure for treating CD174 positive tumor is two generation CAR structures, though the CAR-T cell of the structure
There is stronger antigen recognizing binding ability, but due to the tumor microenvironment of solid tumor complexity, CAR-T cell cannot soak well
Enter in tumor tissues, and CAR-T cell due in conjunction with the PD-L1 molecule of tumor cell surface its function will receive inhibition, because
This, the CAR-T in two generations is limited to the identification killing ability of tumour cell.The technical program using forth generation CAR structure,
CAR-T cell is capable of the antibody of secrete cytokines such as IL12 or PD-1 in identification CD174 simultaneously, and the cell factor of secretion is such as
IL12 can reinforce the killing ability of CAR-T cell itself, while it is thin to tumour simultaneously to recruit other immunocytes such as DC cell
Born of the same parents' killing；The PD-1 antibody of secretion can block the combination of tumor cell surface PD-L1 molecule and CAR-T cell, to release
PD-L1 to the inhibiting effect of CAR-T cell, make by therefore killing that the technical program can be improved CAR-T cells against tumor cells
With.
Summary of the invention
One of the technical problem to be solved in the present invention is to provide the CAR-T cell of secreting type targeting Lewis-Y a kind of.It should
Secrete cytokines or antibody while CAR-T cell expresses Lewis-Y-CAR can preferably overcome the immune of tumor microenvironment
Inhibit, enhances the killing ability of CAR-T cells against tumor cells.
The second technical problem to be solved by the present invention is to provide the CAR-T cell of above-mentioned secreting type targeting Lewis-Y a kind of
Preparation method, this method can prepare existing cell killing toxicity, but can high level expression cell factor or antibody it is thin
Born of the same parents.
The CAR-T cell that the third technical problem to be solved by the present invention is to provide a kind of secreting type targeting Lewis-Y exists
Prepare the application in antitumor cellular therapeutic agent.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
In the first aspect of the present invention, the CAR-T cell of secreting type targeting Lewis-Y, including following cell are provided:
The T cell of secretory antibody while expressing Lewis-Y-CAR；
The T cell of secrete cytokines while expressing Lewis-Y-CAR.
In the present invention, term Lewis-Y-CAR refers to Lewis-Y Chimeric antigen receptor, includes targeting Lewis-Y
ScFv sequence.
ScFv: full name single-chain antibody fragment, i.e. single chain antibody fragments.
In the present invention, term Lewis-Y-scFv refers to anti-Lewis-Y single chain antibody fragments, after carrying out codon optimization
Nucleotide sequence is as shown in SEQ ID NO.1；The nucleotide sequence is used for Lentiviral, retroviral vector
On upper, adenovirus expression carrier, glandular associated virus expression vector or other kinds of expression vector, preferably slow virus is expressed
On carrier.
As currently preferred technical solution, the T cell of secretory antibody while expressing Lewis-Y-CAR,
Its antibody is preferably PD-1 antibody.
As currently preferred technical solution, the T of secrete cytokines is thin while the described expression Lewis-Y-CAR
Born of the same parents, cell factor are preferably IL12 cell factor.
As a second aspect of the invention, the preparation method of the CAR-T cell of secreting type targeting Lewis-Y, packet are provided
Include following steps:
(1) PBMC is separated from the peripheral blood that donor provides；
(2) it activated from PBMC, sub-elect CD3 positive T cell；
(3) building carries Lewis-Y-CAR and antibody or the slow virus plasmid backbone of cytokine gene obtains recombinant slow virus
Plasmid；
(4) by the resulting recombinant slow virus plasmid for carrying Lewis-Y-CAR and antibody or cytokine gene of step (3) and auxiliary
Plasmid-infected host cells are helped, preparation can infect the recombined lentivirus vector of T cell；
(5) the resulting recombined lentivirus vector transduction of step (4) is entered step into the T cell through activating that (2) sub-elect, generated
Secretory antibody or the CAR-T cell of cell factor while expressing Lewis-Y-CAR；
(6) by cell injuring model obtained by step (5)；
(7) by cell massive amplification obtained by step (6)；
(8) secretory antibody or the CAR-T cell of cell factor while collecting expression Lewis-Y-CAR.
It include a tumor associated antigen combined area (Single chain in the Basic Design of Chimeric antigen receptor (CAR)
Antibody Fragment, scFv), an extracellular hinge area, a transmembrane region and an intracellular signal transduction area.
The outer tumor associated antigen combined area of the after birth of Lewis-Y-CAR described in step (3) of the present invention is for combining
The Lewis-Y single-chain antibody (scFv) of Lewis-Y albumen is sequentially connected in series c-myc Epitope tag, CD8 Hinge Chimerical receptor hinge
Chain and CD8 Transmembrane Chimerical receptor transmembrane region.
The intracellular signal transduction area of Lewis-Y-CAR described in step (3) of the present invention includes coding costimulating factor area
Domain, the costimulating factor region be selected from 4-1BB, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, LIGHT, B7-H3,
Specifically bind ligand, ICAM-1, HVEM (LIGHTR), CD160, the relevant antigen -1(LFA- of lymphocyte function of CD83
1), IL2R α, CD103, CD11b, CD11c, TRANCE/RANKL, SLAMF4 (CD244,2B4), CD69, SLAM (SLAMF1,
CD150, IPO-3) one of or a variety of any combination.
As currently preferred technical solution, the costimulating factor region is CD28-4-1BB；The intracellular signal
Conducting region is CD28-4-1BB-CD3 ζ.
As currently preferred technical solution, in step (3), the sequence obtained after the optimization codon is the area scFv
Domain sequence, the nucleotide sequence is as shown in SEQ ID NO.1.
As currently preferred technical solution, in step (3), the recombinant slow virus plasmid is that the duplication after recombination lacks
Exogenous sequences can be integrated into host gene by swaged slow virus plasmid, disposable, can not be replicated and be proliferated, securely and reliably；
The recombinant slow virus plasmid is two generations or the slow virus transgenosis plasmid of three generations.
As currently preferred technical solution, in step (3), the recombinant slow virus plasmid is preferably that the third generation is slow
Viral transgene plasmid, the present invention used in third generation slow virus skeleton plasmid carrier be pCDH-CMV-MCS-EF1-copGFP(it is vast
Clever biology, P0980), as shown in Figure 1, the third generation slow virus plasmid includes ampicillin resistance gene AmpR sequence, original
It is core replicon pUC Ori sequence, Viral Replicon SV40 Ori sequence, RSV promoter, 5 Terminal LTR of slow virus, slow
3 terminal Self-Inactivating LTR of virus, Gag cis element, RRE cis element, env cis element, cPPT
The enhanced marmot hepatitis B posttranscriptional regulatory element of cis element, eWPRE.
As currently preferred technical solution, in step (3), the recombinant slow virus plasmid is targeting Lewis-Y
The slow virus transgenosis plasmid of antigen, recombinant slow virus plasmid successively include CMV promoter sequence, Lewis-Y single-chain antibody
(scFv), Flag, CD8 Transmembrane Chimerical receptor transmembrane region, intracellular signal transduction area, NFAT promoter sequence,
IL12 cytokine sequence, NFAT promoter nucleotide sequence is as shown in SEQ ID NO.7, IL12 cell factor nucleotide sequence
As shown in SEQ ID NO.8；Recombined lentivirus vector described in step (5) will target the Chimeric antigen receptor table of Lewis-Y
Up on T cell (or NK cell) surface, while IL12 cell factor can be secreted.
As currently preferred technical solution, in step (3), the recombinant slow virus plasmid is targeting Lewis-Y
The slow virus transgenosis plasmid of antigen, recombinant slow virus plasmid successively include CMV promoter sequence, Lewis-Y single-chain antibody
(scFv), Flag, CD8 Transmembrane Chimerical receptor transmembrane region, intracellular signal transduction area, EF1 α promoter sequence, PD-
1 antibody sequence, EF1 α promoter nucleotide sequence is as shown in SEQ ID NO.9, IL2 SS nucleotide sequence such as SEQ ID
Shown in NO.10, Anti-PD-1 scFv, that is, PD-1 antibody nucleotide sequences are as shown in SEQ ID NO.11；Described in step (4)
Recombined lentivirus vector the Chimeric antigen receptor for targeting Lewis-Y is expressed on T cell (or NK cell) surface, while can
Express PD-1 antibody.
As currently preferred technical solution, in step (3), PD-1 signal peptide itself is used in the slow virus plasmid
Strong secreting signal peptide IL2 SS substitution, is remarkably improved the outer secretion capacity of PD-1.
As currently preferred technical solution, host cell described in step (5) is 293FT or 293T cell.
In the third aspect of the present invention, provide the CAR-T cell of above-mentioned secreting type targeting Lewis-Y prepare it is antitumor
Application in cellular therapeutic agent.The cell can be used for preparing antitumor cellular therapeutic agent, be especially targeted
The cellular therapeutic agent of the tumor stem cell of the Lewis-Y positive；A kind of mix preparation, including above-mentioned expression Lewis- can be made
Secretion IL12 cell factor while secreting the CAR-T cell and expression Lewis-Y-CAR of PD-1 antibody while Y-CAR
The drug that CAR-T cell mixes in varing proportions.
Compared with prior art, the invention has the following beneficial effects:
The present invention be can secrete cytokines IL12 or PD-1 blocking antibody CAR-T cell.Wherein, PD-1 signal peptide itself is used
Strong secreting signal peptide IL2 SS substitution, is remarkably improved the outer secretion capacity of PD-1, IL12 is conducive to the work of T cell and NK cell
Change, enhances the anti-tumor capacity of T cell.The PD-1 antibody of CAR-T cell secretion can be with the PD-1 molecule knot on T cell surface
It closes, to block the combination of tumor cell surface PD-L1 receptor and T cell surface PD-1, plays T cell more fully anti-swollen
Tumor ability.
One or more combinations of factors of costimulating factor used in the present invention can be such that T cell continuous activation is proliferated, carefully
Intracellular cytokine continuous release, the effect of enhancing killing tumor cell and immunological memory.
Detailed description of the invention
Fig. 1 is pCDH-CMV-MCS-EF1-copGFP skeleton carrier plasmid schematic diagram in the embodiment of the present invention 1,2；
Fig. 2 is the slow virus skeleton plasmid of carrying Lewis-Y-CAR and cell factor IL12 gene described in the embodiment of the present invention 1
SV305 structure chart；
Fig. 3 is helper plasmid pRSV-Rev described in the embodiment of the present invention 1,2；
Fig. 4 is helper plasmid pCMV-VSV-G described in the embodiment of the present invention 1,2；
Fig. 5 is helper plasmid pMDLg-pPRE described in the embodiment of the present invention 1,2；
Fig. 6 is point that IL12 in the CAR-T cell of IL12 is secreted while expression LewisY-CAR described in the embodiment of the present invention 1
Secrete ELISA testing result figure；
Fig. 7 is the slow virus skeleton plasmid that Lewis-Y-CAR and PD-1 antibody gene is carried described in the embodiment of the present invention 2
SV306 structure chart；
PD-1 antibody in the CAR-T cell of PD-1 antibody while Fig. 8 is expression LewisY-CAR described in the embodiment of the present invention 2
Secretion ELISA testing result figure；
Fig. 9 is the CAR-T of secretion IL12 while expressing LewisY-CAR in the embodiment of the present invention 4 under the conditions of different effect target ratios
The CAR-T cell of secretion PD-1 antibody is to target cell killing-efficiency line chart while cell and expression CD133-CAR.
Specific embodiment
The present invention is described further with attached drawing with reference to embodiments, and the embodiment for illustrating invention should not solve
It is interpreted as this clearly demarcated range of limitation.Present disclosure can be improved from material, method and reaction condition simultaneously,
All these improvement should all be fallen within spirit and scope of the invention.
The experimental method of detailed conditions is not specified in the following example, usually according to condition proposed by manufacturer.Unless
It is otherwise noted, otherwise percentage is calculated by weight.Experimental material used in following embodiment and reagent are equal unless otherwise instructed
It can be obtained from commercially available channel.
Although can be used in an embodiment of the present invention to of the present invention similar or of equal value any method and material,
Preferred material and method are enumerated by place herein.
Embodiment 1: the CAR-T cell of IL12 is secreted while preparation expression Lewis-Y-CAR.
It is of the present invention expression Lewis-Y-CAR while secretion IL12 CAR-T cell the preparation method is as follows:
One, PBMC is separated from the peripheral blood that donor provides.
1, with anticoagulant tube or blood taking bag acquisition human peripheral (while acquisition while rock so that peripheral blood and anti-coagulants sufficiently mix
Close) 80-100mL, PBMC is separated in sterile cell preparation workshop.Peripheral blood is transferred in 50ml centrifuge tube, room temperature 800g,
Centrifugation 15 minutes, draw upper layer yellow autologous plasma in new 50ml centrifuge tube, 56 DEG C inactivate 30 minutes, be placed in -20 DEG C it is standby
With；Isometric PBS is added to the cellular layer of lower layer, mixing of turning upside down；
2, it draws 15ml lymphocyte separation medium (Ficoll-PaquePlus, GE) and is centrifuged bottom of the tube in new 50ml, and along pipe
Wall is slowly added to 30ml dilute blood to lymph separating liquid upper layer, centrifuge tube is placed in centrifuge, 450g is slow to rise slow drop centrifugation 25
Minute, no break；
3, after being centrifuged, the careful tunica albuginea confluent monolayer cells drawn above lymphocyte separation medium are transferred to a new 50ml centrifugation
Guan Zhong, is added 50ml PBS cleaning, and 300g is slow to rise slow drop centrifugation 10min；
4, supernatant is abandoned, the cell precipitation of centrifugation bottom of the tube is retained；PBS 50ml, 160g are added again, it is slow to rise slow drop centrifugation
15min；
5, supernatant is abandoned；It is eventually adding PBS, 300g, it is slow to rise slow drop centrifugation 10min, supernatant is abandoned to get PBMC cell is arrived.
Two, it activated from PBMC, sub-elect CD3 positive T cell.
Kit (EasySep is sorted according to CD3 positive T cellTMHuman T Cell Isolation Kit article No.: #
17951, manufacturer: STEMCELL) CD3 positive T cell in sorting (5) PBMC, specific as follows:
1, adjustment cell density is 5 × 107Cell is added to the cell cryopreservation tube of 5mL by cells/mL；
2, separating liquid Isolation Cocktail 50ul/mL is added, stands 5 minutes after mixing；
3, with vortex instrument by RapidSheresTMReagent is vortexed 30 seconds, is uniformly mixed particle, adds according to the ratio of 40ul/mL
It is added in cell and mixes；
4, it adds PBS to 2.5mL and blows and beats 2 ~ 3 times up and down, be placed on magnetic frame 3 minutes；
5, liquid is transferred to new Tissue Culture Dish to get the CD3 positive T cell of sorting is arrived by inclination magnetic frame.
Three, optimize Lewis-Y-scFv codon.
Prepare Lewis-Y Chimeric antigen receptor (CAR), i.e. Lewis-Y-CAR, scFv segment is delivered into company and carries out password
Son optimization, be allowed to be easier to express in human body cell, after codon optimization sequence be SEQ ID NO.1 shown in nucleotide
Sequence.
Four, building carries Lewis-Y-CAR and the slow virus plasmid of cell factor IL12 gene obtains recombinant slow virus matter
Grain.
Building recombinant slow virus skeleton plasmid (SV305 is shown in Fig. 2): the CAR structure of synthesis secretion IL12 is simultaneously cloned into
On pCDH-CMV-MCS-EF1-copGFP skeleton plasmid (vast spirit biology, P0980 are shown in Fig. 1), SV305 is obtained after being sequenced correctly
Skeleton plasmid is in series with CMV promoter-Lewis-YscFv-Flag-CD8TM-CD28-4-1BB-CD3 ζ-NFAT according to this
Promoter-IL12, wherein the nucleotide sequence after Lewis-YscFv optimization is as shown in SEQ ID NO.1, Flag nucleotides sequence
Column are as shown in SEQ ID NO.2, and CD8TM nucleotide sequence is as shown in SEQ ID NO.3, CD28 nucleotide sequence such as SEQ ID
Shown in NO.4,4-1BB nucleotide sequence as shown in SEQ ID NO.5, CD3 ζ nucleotide sequence as shown in SEQ ID NO.6,
NFAT promoter nucleotide sequence is as shown in SEQ ID NO.7, and IL12 nucleotide sequence is as shown in SEQ ID NO.8.
Five, by the resulting recombinant slow virus plasmid sense for carrying Lewis-Y-CAR and cell factor IL12 gene of step 4
293T cell is contaminated, preparation can infect the slow virus of T cell.
1. the cell after secondary culture 2-3 generation after 293T cell recovery to be used for the working cardial cell of slow virus packaging, pass
Generation number sum was no more than for 10 generations.
2. liposome transfection (for 10cm culture dish).
1) day before transfection is inoculated with 293T cell into a diameter 10cm culture dish, and inoculating cell quantity should be worked as with transfection
It cell growth reaches 90% ~ 95% fusion and is preferred；
2) the transfection same day removes culture solution, and 5ml virus is added and is packed for culture solution；
3) prepare DNA-Lipofectamine2000 compound:
A. prepare a sterile 5mL centrifuge tube and 1.5mL serum-free Opti-MEM culture solution, helper plasmid (pRSV-Rev is added
(addgene, #12253), pCMV-VSV-G (addgene, #8454), pMDLg-pRRE(addgene, #12251), see respectively
Fig. 3-5) and slow virus expression plasmid (SV305), sufficiently piping and druming mixing；
B. prepare in other one 5 sterile mL centrifuge tubes, 1.5 mL serum-free Opti-MEM culture solutions and 68 μ L are added
Lipofectamine2000, gently piping and druming mixes, and is incubated at room temperature 10 minutes；
C. after 30 minutes, the serum-free Opti-MEM culture solution containing Plasmid DNA is added to containing Lipofectamine2000
Serum-free Opti-MEM culture solution, gently piping and druming mix, be incubated at room temperature 30 minutes;
D. DNA- Lipofectamine2000 compound is drop by drop added in 293T cell, is lightly rocked back and forth
Culture dish is shaken to mix compound.Place 37 DEG C, 5% CO2 It is cultivated in saturated humidity incubator.
3. the collection of recombined lentivirus vector is concentrated.
1) it 48 hours after transfecting, collects and contains virulent culture solution.Culture solution, which is drawn onto one, sterile has 50 mL of lid
In centrifuge tube, then adds 8 mL viruses and be packed for culture solution extremely and can also produce in the cell of poison；
2) 4 DEG C, 3000 rpm centrifugation 15 minutes, low-speed centrifugal viral supernatants remove cell fragment, recycle supernatant；
3) it 72 hours after transfecting, collects and contains virulent culture solution.4 DEG C, 3000rpm be centrifuged 15 minutes, low-speed centrifugal virus on
Clearly, cell fragment is removed, supernatant is recycled.The vial supernatant that 48 hours are collected and 72 hours vial supernatants collected, are used
0.45 μm of small filter filtering, collects filtrate with 50 mL centrifuge tubes；
4) viral supernatants of collection are concentrated with ultracentrifuge, 4 DEG C, 72000g/min is centrifuged 120 minutes；It is fresh with 500 μ l
Viral pellet is resuspended in culture solution, freezes after packing, and part is taken to measure virus titer.
Six, two T cells sub-elected are entered step using the resulting recombined lentivirus vector transduction of step 5, generates expression
The CAR-T cell of secrete cytokines IL12 while Lewis-Y-CAR.
The activation of 1.T cell
(1) by the T cell of sorting with density 0.5 ~ 1 × 106Cells/ml is resuspended in 581 culture medium of KBM (article No. 88-581-
CM, Corning) in, add 1% autologous plasma；
(2) Dynabeads is washed:
A) Dynabeads is taken out from refrigerator, is vibrated 30 seconds with vortex instrument, the magnetic bead for being deposited in bottom is resuspended uniform；
B) according to the T cell number in (1), Dynabeads required for being calculated according to Dynabeads:T=1:1 ~ 1:3 ratio
, Dynabeads is transferred in a new 15ml centrifuge tube using sterile pipette, and the DPBS of same volume is added, is made
It is blown and beaten and is mixed with pipettor.Centrifuge tube is placed on magnetic frame, stands 5 minutes, liquid is inhaled with pipettor and is abandoned；
C) DPBS of 2ml is added in 15ml centrifuge tube, repeats the cleaning step in b)；
D) beads is resuspended in 581 culture mediums that 100ul is added, and is added dropwise in cell culture fluid, and piping and druming mixes and is placed on 37
DEG C, 5%CO2It is cultivated 3 days in cell incubator.
The T cell 2. recombined lentivirus vector is transduceed.
(1) after t cell activation 3 days, trypan blue staining counts the number of T cell.Required for being calculated according to MOI=3-10
Virus quantity: required virus quantity (ml)=(MOI* cell number)/virus titer (Tu/ml)；
(2) from -80 DEG C of ultra low temperature freezers, nozzle (is not deep by quick-thawing in 37 DEG C of water-baths after taking-up slow virus
Below liquid level)；
(3) T cell culture dish is taken out, polybrene to final concentration of 6ug/ml is added, required virus quantity is added, uses liquid relief
Device gently blows and beats mixing, and the cell mass of aggregation is made to be uniformly dispersed, under room temperature after culture dish is sealed using sealed membrane,
800g is centrifuged 1 hour；
(4) remove sealed membrane, culture dish is placed in 37 DEG C, 5%CO2Continue culture 24 hours in incubator；
(5) room temperature 250g is centrifuged 10 minutes, is removed containing virulent culture medium supernatant, and it is heavy that cell is resuspended with fresh culture medium
It forms sediment, cell is transferred in new culture dish, continues to cultivate.
Seven, by cell injuring model obtained by step 6.
The cell mass gently blown and beaten in cultivating system daily counts cell number to dispersity, and with trypan blue staining
Mesh makes cell density maintain 1 × 106A/ml.
Eight, by cell massive amplification obtained by step 7.
A) when cell number is greater than 100 × 106When, CAR-T cell is transferred in cell culture bags and is cultivated.Platform is used every other day
Expect that blue counting method to cell count, maintains cell density 1 × 106A/ml；
B) when CAR-T cell culture was to 10 days, can extraction section cell culture fluid do Sterility testing；
C) when total number of cells reach 500-1000 × 106When, that is, CAR-T cell is collected, and remove magnetic bead with magnetic pole.
D) take part cell suspension, positive with target cell OVCAR-3(LewisY) co-culture after, then stimulated two days with PMA,
ELISA detects the secretion situation (as shown in Figure 6) of IL12 in supernatant, and experimental group successful secretion IL12, says compared with the control group
The bright CAR-T cell that secrete cytokines IL12 while expressing Lewis-Y-CAR is successfully made, by cell expansion culture and freezes
It deposits.
Nine, the CAR-T cell of secrete cytokines IL12 while collecting expression Lewis-Y-CAR.
Embodiment 2: the CAR-T cell of PD-1 antibody is secreted while preparation expression Lewis-Y-CAR.
It is of the present invention expression Lewis-Y-CAR while secretion PD-1 antibody CAR-T cell the preparation method is as follows:
One, PBMC is separated from the peripheral blood that donor provides.
1, with anticoagulant tube or blood taking bag acquisition human peripheral (while acquisition while rock so that peripheral blood and anti-coagulants sufficiently mix
Close) 80-100mL, PBMC is separated in sterile cell preparation workshop.Peripheral blood is transferred in 50ml centrifuge tube, room temperature 800g,
Centrifugation 15 minutes, draw upper layer yellow autologous plasma in new 50ml centrifuge tube, 56 DEG C inactivate 30 minutes, be placed in -20 DEG C it is standby
With；Isometric PBS is added to the cellular layer of lower layer, mixing of turning upside down；
2, it draws 15ml lymphocyte separation medium (Ficoll-Paque Plus, GE) and is centrifuged bottom of the tube in new 50ml, and along pipe
Wall is slowly added to 30ml dilute blood to lymph separating liquid upper layer, centrifuge tube is placed in centrifuge, 450g is slow to rise slow drop centrifugation 25
Minute, no break；
3, after being centrifuged, the careful tunica albuginea confluent monolayer cells drawn above lymphocyte separation medium are transferred to a new 50ml centrifugation
Guan Zhong, is added 50ml PBS cleaning, and 300g is slow to rise slow drop centrifugation 10min；
4, supernatant is abandoned, the cell precipitation of centrifugation bottom of the tube is retained；PBS 50ml, 160g are added again, it is slow to rise slow drop centrifugation
15min；
5, supernatant is abandoned；It is eventually adding PBS, 300g, it is slow to rise slow drop centrifugation 10min, supernatant is abandoned to get PBMC cell is arrived.
Two, it activated from PBMC, sub-elect CD3 positive T cell.
Kit (EasySep is sorted according to CD3 positive T cellTMHuman T Cell Isolation Kit article No.: #
17951, manufacturer: STEMCELL) CD3 positive T cell in sorting (5) PBMC, specific as follows:
1, adjustment cell density is 5 × 107Cell is added to the cell cryopreservation tube of 5mL by cells/mL；
2, separating liquid Isolation Cocktail 50ul/mL is added, stands 5 minutes after mixing；
3, with vortex instrument by RapidSheresTMReagent is vortexed 30 seconds, is uniformly mixed particle, adds according to the ratio of 40ul/mL
It is added in cell and mixes；
4, it adds PBS to 2.5mL and blows and beats 2 ~ 3 times up and down, be placed on magnetic frame 3 minutes；
5, liquid is transferred to new Tissue Culture Dish to get the CD3 positive T cell of sorting is arrived by inclination magnetic frame.
Three, optimize Lewis-Y-scFv codon.
Prepare Lewis-Y Chimeric antigen receptor (CAR), i.e. Lewis-Y-CAR, scFv segment is delivered into company and carries out password
Son optimization, be allowed to be easier to express in human body cell, after codon optimization sequence be SEQ ID NO.1 shown in nucleotide
Sequence.
Four, the slow virus plasmid backbone that building carries Lewis-Y-CAR and PD-1 antibody gene obtains recombinant slow virus matter
Grain.
Building recombinant slow virus skeleton plasmid (SV306, as shown in Figure 7): the CAR structure of synthesis antibody containing PD-1 is simultaneously cloned
On to pCDH-CMV-MCS-EF1-copGFP skeleton plasmid (vast spirit biology, P0980 are shown in Fig. 1), obtained after being sequenced correctly
SV306 skeleton plasmid is in series with CMV promoter-Lewis-YscFv-Flag-CD8TM-CD28-4-1BB-CD3 ζ-EF1- according to this
IL2SS-Anti-PD-1 scFv, wherein the nucleotide sequence after Lewis-YscFv optimization is as shown in SEQ ID NO.1, Flag
Nucleotide sequence is as shown in SEQ ID NO.2, and CD8TM nucleotide sequence is as shown in SEQ ID NO.3, and CD28 nucleotide sequence is such as
Shown in SEQ ID NO.4,4-1BB nucleotide sequence is as shown in SEQ ID NO.5, CD3 ζ nucleotide sequence such as SEQ ID NO.6
Shown, IL2 SS nucleotide sequence is as shown in SEQ ID NO.10, Anti-PD-1 scFv nucleotide sequence such as SEQ ID
Shown in NO.11.
Five, the resulting recombinant slow virus plasmid for carrying Lewis-Y-CAR and PD-1 antibody gene of step 4 is infected
293T cell, preparation can infect the recombined lentivirus vector of T cell.
1. the cell after secondary culture 2-3 generation after 293T cell recovery to be used for the working cardial cell of slow virus packaging, pass
Generation number sum was no more than for 10 generations.
2. liposome transfection (for 10cm culture dish).
1) day before transfection is inoculated with 293T cell into a diameter 10cm culture dish, and inoculating cell quantity should be worked as with transfection
It cell growth reaches 90% ~ 95% fusion and is preferred；
2) the transfection same day removes culture solution, and 5ml virus is added and is packed for culture solution；
3) prepare DNA-Lipofectamine2000 compound:
A. prepare a sterile 5mL centrifuge tube and 1.5mL serum-free Opti-MEM culture solution, helper plasmid (pRSV-Rev is added
(addgene, #12253), pCMV-VSV-G (addgene, #8454), pMDLg-pRRE(addgene, #12251), see respectively
Fig. 3-5) and slow virus expression plasmid (SV306 is shown in Fig. 7), sufficiently piping and druming mixing；
B. prepare in other one 5 sterile mL centrifuge tubes, 1.5 mL serum-free Opti-MEM culture solutions and 68 μ L are added
Lipofectamine2000, gently piping and druming mixes, and is incubated at room temperature 10 minutes；
C. after 30 minutes, the serum-free Opti-MEM culture solution containing Plasmid DNA is added to containing Lipofectamine2000
Serum-free Opti-MEM culture solution, gently piping and druming mix, be incubated at room temperature 30 minutes；
D. DNA- Lipofectamine2000 compound is drop by drop added in 293T cell, is lightly rocked back and forth
Culture dish is shaken to mix compound.Place 37 DEG C, 5% CO2 It is cultivated in saturated humidity incubator.
3. the collection of recombined lentivirus vector is concentrated.
1) it 48 hours after transfecting, collects and contains virulent culture solution.Culture solution, which is drawn onto one, sterile has 50 mL of lid
In centrifuge tube, then adds 8 mL viruses and be packed for culture solution extremely and can also produce in the cell of poison；
2) 4 DEG C, 3000 rpm centrifugation 15 minutes, low-speed centrifugal viral supernatants remove cell fragment, recycle supernatant；
3) it 72 hours after transfecting, collects and contains virulent culture solution.4 DEG C, 3000 rpm centrifugation 15 minutes, low-speed centrifugal virus
Supernatant removes cell fragment, recycles supernatant.The vial supernatant that 48 hours are collected and 72 hours vial supernatants collected,
It is filtered with 0.45 μm of small filter, collects filtrate with 50 mL centrifuge tubes；
4) viral supernatants of collection are concentrated with ultracentrifuge, 4 DEG C, 72000g/min is centrifuged 120 minutes；It is new with 500ul
Viral pellet is resuspended in fresh culture solution, freezes after packing, and part is taken to measure virus titer.
Six, two T cells sub-elected are entered step using the resulting recombined lentivirus vector transduction of step 5, generates expression
The CAR-T cell of PD-1 antibody is secreted while Lewis-Y-CAR.
The activation of 1.T cell.
(1) by the T cell of sorting with density 0.5 ~ 1 × 106Cells/ml is resuspended in 581 culture medium of KBM (article No. 88-
581-CM, Corning) in, add 1% autologous plasma；
(2) Dynabeads is washed:
A) Dynabeads is taken out from refrigerator, is vibrated 30 seconds with vortex instrument, the magnetic bead for being deposited in bottom is resuspended uniform；
B) according to the T cell number in (1), Dynabeads required for being calculated according to Dynabeads:T=1:1 ~ 1:3 ratio
, Dynabeads is transferred in a new 15ml centrifuge tube using sterile pipette, and the DPBS of same volume is added, is made
It is blown and beaten and is mixed with pipettor.Centrifuge tube is placed on magnetic frame, stands 5 minutes, liquid is inhaled with pipettor and is abandoned；
C) DPBS of 2ml is added in 15ml centrifuge tube, repeats the cleaning step in b)；
D) beads is resuspended in 581 culture mediums that 100ul is added, and is added dropwise in cell culture fluid, and piping and druming mixes and is placed on 37
DEG C, 5%CO2It is cultivated 3 days in cell incubator.
The T cell 2. recombinant viral vector is transduceed.
(1) after t cell activation 3 days, trypan blue staining counts the number of T cell.Required for being calculated according to MOI=3-10
Virus quantity: required virus quantity (ml)=(MOI* cell number)/virus titer (Tu/ml)；
(2) from -80 DEG C of ultra low temperature freezers, nozzle (is not deep by quick-thawing in 37 DEG C of water-baths after taking-up slow virus
Below liquid level)；
(3) T cell culture dish is taken out, polybrene to final concentration of 6ug/ml is added, required virus quantity is added, uses liquid relief
Device gently blows and beats mixing, and the cell mass of aggregation is made to be uniformly dispersed, under room temperature after culture dish is sealed using sealed membrane,
800g is centrifuged 1 hour；
(4) remove sealed membrane, culture dish is placed in 37 DEG C, 5%CO2Continue culture 24 hours in incubator；
(5) room temperature 250g is centrifuged 10 minutes, is removed containing virulent culture medium supernatant, and it is heavy that cell is resuspended with fresh culture medium
It forms sediment, cell is transferred in new culture dish, continues to cultivate.
Seven, by cell injuring model obtained by step 6.
The cell mass gently blown and beaten in cultivating system daily counts cell number to dispersity, and with trypan blue staining
Mesh makes cell density maintain 1 × 106A/ml.
Eight, by cell massive amplification obtained by step 7.
A) when cell number is greater than 100 × 106When, CAR-T cell is transferred in cell culture bags and is cultivated；Platform is used every other day
Expect that blue counting method to cell count, maintains cell density 1 × 106A/ml；
B) when CAR-T cell culture was to 10 days, can extraction section cell culture fluid do Sterility testing；
C) when total number of cells reach 500-1000 × 106When, that is, CAR-T cell is collected, and remove magnetic bead with magnetic pole；
D) part cell suspension is taken, after BFA24h is added, ELISA detects the secretion situation (as shown in Figure 8) of PD-1 in supernatant,
Experimental group secretes PD-1 compared with the control group, secretion PD-1 antibody while illustrating that expression Lewis-Y-CAR is successfully made
CAR-T cell by cell expansion culture and freezes.
Embodiment 3: the targeting Lewis-Y-CAR-T cell of IL12 or PD-1 antibody can be secreted for immunization therapy.
1. the whether high expression Lewis-Y of tumor patient tumor tissue cell is detected by immunohistochemical method,
Lewis-Y has high expression in kinds of tumors, such as lung cancer, gastric cancer, the tumor patient of all high expression Lewis-Y antigen
Using the targeting Lewis-Y-CAR-T cell therapy that can secrete IL12 or PD-1 antibody.
2. acquisition peripheral blood in patients simultaneously separates T cell.
3. prepared by CAR-T cell, CAR-T cell is prepared in the workshop GMP.Isolated T cell is swashed with CD3/CD28 beads
After work, according to MOI=3-10, with lentiviruses transduction T cell, and the CAR-T cell that spreads cultivation in vitro.
4.CAR-T cell is cultivated in vitro meet treatment cell number after, extraction section T cell is Sterility testing, T
Cell phenotype detection, just can be used for treating after meeting clinical treatment CAR-T cell release standard.
5. feeding back CAR-T cell by way of venoclysis to patient.
The CAR-T cell and expression Lewis-Y- of secretion IL12 while expression Lewis-Y-CAR prepared by embodiment 4
The CAR-T cells in vitro that PD-1 antibody is secreted while CAR kills verifying.
The CAR-T cell of IL12, table are secreted while cultivating Raji cell and effector cell expression Lewis-Y-CAR respectively
The CAR-T cell of the CAR-T cell of secretion PD-1 antibody and expression Lewis-Y-CAR while up to Lewis-Y-CAR.
Collect target cell Raji4 × 105Cells and effector cell's (CAR-T cell) each 3 × 106Cells, 300g, from
Heart 10min, it is slow to rise slow drop, abandon supernatant；Target cell and effector cell are resuspended respectively with 1ml PBS solution, 300g is centrifuged 10min,
It is slow to rise slow drop, abandon supernatant；It is repeated once；Effector cell is resuspended with 700 μ l culture mediums (AIM-V culture medium+10%FBS), uses 2ml
Target cell is resuspended in culture medium (1640 culture medium+10%FBS).
Effect target is set than the experimental port for 1:1,5:1,10:1,20:1, and control group is set, every group of 3 multiple holes, 37 DEG C
5% CO22h is cultivated in incubator；500g is centrifuged 5min, slow to rise slow drop plate centrifugation；Take the 20 μ l supernatants in each hole to new 96
In orifice plate, and 50 μ l substrate solutions (being protected from light operation) are added in every hole, and room temperature, which is protected from light, is incubated for 15min；Every hole is added 50 μ l and terminates
Liquid, microplate reader detect 490nm absorbance.Under the conditions of difference effect target ratio, the CAR- of IL12 is secreted while expressing Lewis-Y-CAR
T cell, the CAR-T cell of secretion PD-1 antibody killing-efficiency in Raji target cell is obvious while expressing Lewis-Y-CAR
It is better than the CAR-T cell of expression Lewis-Y-CAR, when imitating target ratio is 20:1, expression Lewis-Y-CAR's produced by the present invention
The CAR-T cell of IL12 is secreted simultaneously, the CAR-T cell tumour killing of secretion PD-1 antibody while expressing Lewis-Y-CAR
Ability is respectively that 58% and 64%(is shown in Fig. 9).
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Sequence table
<110>Suzhou Mao Hang Biotechnology Co., Ltd
<120>a kind of CAR-T cell and its preparation method and application of secreting type targeting Lewis-Y
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 795
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atggagttcg ggctgagatg ggtgtttctg gtggccatcc tgaaggacgt gcagtgcgag 60
gtgcagctgg tggagagcgg ggggggagtg gtgcagcctg gaaggagcct gagactgagc 120
tgtagcacaa gcggctttac cttttctgat tattacatgt actgggtgag gcaggccccc 180
ggaaagggcc tggagtgggt ggcttatatg tccaacgtgg gcgccattac agactacccc 240
gacacagtga agggcagatt caccattagc cgggacaaca gcaaaaacac actgttcctg 300
cagatggaca gcctgagacc cgaggacacc ggcgtgtact tctgcgccag aggcaccaga 360
gacggcagct ggttcgccta ctggggccag ggaacacccg tgaccgtgag cagcggcggc 420
ggaggaagcg gaggaggagg aagcggcgga ggaggcagcg acatccagat gacccagagc 480
cctagcagtc tgagtgccag cgtgggcgac agagtgacca tcacctgcag aagcagtcag 540
agaatcgtgc acagcaacgg aaatacatac ctggagtggt accagcagac acccggcaaa 600
gcccccaaac tgctgatcta caaggtgagc aatagattca gcggcgtgcc cagcagattc 660
agcggaagcg gaagcggcac cgacttcacc ttcactatca gcagcctgca gcccgaagat 720
atcgcaacct actactgctt ccaggggagc cacgtgccat tcaccttcgg ccagggaacc 780
aagctgcaga tcacc 795
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gattacaagg acgacgatga caag 24
<210> 3
<211> 249
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttcgtccccg tgttcctgcc tgccaagcca acaactaccc ctgctccacg accacctact 60
ccagcaccta ccatcgcaag tcagcccctg tcactgcgac ctgaggcttg ccggccagca 120
gctggaggag cagtgcacac ccgaggcctg gacttcgcat gcgatatcta catttgggca 180
ccactggctg gaacctgtgg ggtcctgctg ctgagcctgg tcatcaccct gtattgtaac 240
cacagaaat 249
<210> 4
<211> 123
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aggagcaaac gctcccgact gctgcattcc gactacatga acatgacacc tcggagacca 60
ggccccacta gaaagcatta ccagccatat gccccaccca gggatttcgc agcctatcgg 120
agc 123
<210> 5
<211> 141
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cggttcagcg tcgtgaaaag ggggcgcaag aaactgctgt acatcttcaa gcagcctttt 60
atgcgcccag tgcagacaac tcaggaggaa gacggatgct cttgtcggtt cccagaggag 120
gaggaaggag gctgcgagct g 141
<210> 6
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
agagtgaagt tcagccggag cgccgatgca ccagcatatc agcagggaca gaatcagctg 60
tacaacgagc tgaatctggg caggcgcgag gaatatgacg tgctggataa gcgacgagga 120
cgggaccccg aaatgggagg aaaacccaga aggaagaacc ctcaggaggg gctgtataat 180
gaactgcaga aagacaagat ggctgaggca tacagcgaaa ttggaatgaa aggagagcgc 240
cgacggggga agggacacga tgggctgtac cagggactgt caaccgccac taaagatacc 300
tacgacgcac tgcacatgca ggctctgccc ccaaga 336
<210> 7
<211> 174
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ggaggaaaaa ctgtttcata cagaaggcgg gaggaaaaac tgtttcatac agaaggcggg 60
aggaaaaact gtttcataca gaaggcggga ggaaaaactg tttcatacag aaggcgggag 120
gaaaaactgt ttcatacaga aggcgggagg aaaaactgtt tcatacagaa ggcg 174
<210> 8
<211> 1746
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
attttgacac ccccataata tttttccaga attaacagta taaattgcat ctcttgttca 60
agagttccct atcactctct ttaatcacta ctcacagtaa cctcaactcc tgccacaacc 120
ggtatgtgtc accagcagtt ggtcatctct tggttttccc tggtttttct ggcatctccc 180
ctcgtggcca tatgggaact gaagaaagat gtttatgtcg tagaattgga ttggtatccg 240
gatgcccctg gagaaatggt ggtcctcacc tgtgacaccc ctgaagaaga tggtatcacc 300
tggaccttgg accagagcag tgaggtctta ggctctggca aaaccctgac catccaagtc 360
aaagagtttg gagatgctgg ccagtacacc tgtcacaaag gaggcgaggt tctaagccat 420
tcgctcctgc tgcttcacaa aaaggaagat ggaatttggt ccactgatat tttaaaggac 480
cagaaagaac ccaaaaataa gacctttcta agatgcgagg ccaagaatta ttctggacgt 540
ttcacctgct ggtggctgac gacaatcagt actgatttga cattcagtgt caaaagcagc 600
agaggctctt ctgaccccca aggggtgacg tgcggagctg ctacactctc tgcagagaga 660
gtcagagggg acaacaagga gtatgagtac tcagtggagt gccaggagga cagtgcctgc 720
ccagctgctg aggagagtct gcccattgag gtcatggtgg atgccgttca caagctcaag 780
tatgaaaact acaccagcag cttcttcatc agggacatca tcaaacctga cccacccaag 840
aacttgcagc tgaagccatt aaagaattct cggcaggtgg aggtcagctg ggagtaccct 900
gacacctgga gtactccaca ttcctacttc tccctgacat tctgcgttca ggtccagggc 960
aagagcaaga gagaaaagaa agatagagtc ttcacggaca agacctcagc cacggtcatc 1020
tgccgcaaaa atgccagcat tagcgtgcgg gcccaggacc gctactatag ctcatcttgg 1080
agcgaatggg catctgtgcc ctgcaggggc ggaggcggaa gcggaggcgg aggaagcggc 1140
ggtggcggca gcagaaacct ccccgtggcc actccagacc caggaatgtt cccatgcctt 1200
caccactccc aaaacctgct gagggccgtc agcaacatgc tccagaaggc cagacaaact 1260
ctagaatttt acccttgcac ttctgaagag attgatcatg aagatatcac aaaagataaa 1320
accagcacag tggaggcctg tttaccattg gaattaacca agaatgagag ttgcctaaat 1380
tccagagaga cctctttcat aactaatggg agttgcctgg cctccagaaa gacctctttt 1440
atgatggccc tgtgccttag tagtatttat gaagacttga agatgtacca ggtggagttc 1500
aagaccatga atgcaaagct tctgatggat cctaagaggc agatctttct agatcaaaac 1560
atgctggcag ttattgatga gctgatgcag gccctgaatt tcaacagtga gactgtgcca 1620
caaaaatcct cccttgaaga accggatttt tataaaacta aaatcaagct ctgcatactt 1680
cttcatgctt tcagaattcg ggcagtgact attgatagag tgatgagcta tctgaatgct 1740
tcctaa 1746
<210> 9
<211> 546
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60
cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120
aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180
gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240
acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300
ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360
tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420
cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480
actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540
gcctac 546
<210> 10
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaactcg 60
<210> 11
<211> 2196
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
atggagttct ggttgtcatg ggtctttctg gtagctattc ttaagggagt acagtgtcag 60
gtgcagctgg tgcagagcgg cgtggaagtg aaaaaaccgg gcgcgagcgt gaaagtgagc 120
tgcaaagcga gcggctatac ctttaccaac tattatatgt attgggtgcg ccaggcgccg 180
ggccagggcc tggaatggat gggcggcatt aacccgagca acggcggcac caactttaac 240
gaaaaattta aaaaccgcgt gaccctgacc accgatagca gcaccaccac cgcgtatatg 300
gaactgaaaa gcctgcagtt tgatgatacc gcggtgtatt attgcgcgcg ccgcgattat 360
cgctttgata tgggctttga ttattggggc cagggcacca ccgtgaccgt gagcagcgcg 420
agcaccaaag gcccgagcgt gtttccgctg gcgccgtgca gccgcagcac cagcgaaagc 480
accgcggcgc tgggctgcct ggtgaaagat tattttccgg aaccggtgac cgtgagctgg 540
aacagcggcg cgctgaccag cggcgtgcat acctttccgg cggtgctgca gagcagcggc 600
ctgtatagcc tgagcagcgt ggtgaccgtg ccgagcagca gcctgggcac caaaacctat 660
acctgcaacg tggatcataa accgagcaac accaaagtgg ataaacgcgt ggaaagcaaa 720
tatggcccgc cgtgcccgcc gtgcccggcg ccggaatttc tgggcggccc gagcgtgttt 780
ctgtttccgc cgaaaccgaa agataccctg atgattagcc gcaccccgga agtgacctgc 840
gtggtggtgg atgtgagcca ggaagatccg gaagtgcagt ttaactggta tgtggatggc 900
gtggaagtgc ataacgcgaa aaccaaaccg cgcgaagaac agtttaacag cacctatcgc 960
gtggtgagcg tgctgaccgt gctgcatcag gattggctga acggcaaaga atataaatgc 1020
aaagtgagca acaaaggcct gccgagcagc attgaaaaaa ccattagcaa agcgaaaggc 1080
cagccgcgcg aaccgcaggt gtataccctg ccgccgagcc aggaagaaat gaccaaaaac 1140
caggtgagcc tgacctgcct ggtgaaaggc ttttatccga gcgatattgc ggtggaatgg 1200
gaaagcaacg gccagccgga aaacaactat aaaaccaccc cgccggtgct ggatagcgat 1260
ggcagctttt ttctgtatag ccgcctgacc gtggataaaa gccgctggca ggaaggcaac 1320
gtgtttagct gcagcgtgat gcatgaagcg ctgcataacc attataccca gaaaagcctg 1380
agcctgagcc tgggcaaaga attcgaagga tccgcggccg ctgagggcag aggaagtctt 1440
ctaacatgcg gtgacgtgga ggagaatccc ggcccttccg ggatggagtt ctggttgtca 1500
tgggtctttc tggtagctat tcttaaggga gtacagtgtg aaattgtgct gacccagagc 1560
ccggcgaccc tgagcctgag cccgggcgaa cgcgcgaccc tgagctgccg cgcgagcaaa 1620
ggcgtgagca ccagcggcta tagctatctg cattggtatc agcagaaacc gggccaggcg 1680
ccgcgcctgc tgatttatct ggcgagctat ctggaaagcg gcgtgccggc gcgctttagc 1740
ggcagcggca gcggcaccga ttttaccctg accattagca gcctggaacc ggaagatttt 1800
gcggtgtatt attgccagca tagccgcgat ctgccgctga cctttggcgg cggcaccaaa 1860
gtggaaatta aacgcaccgt ggcggcgccg agcgtgttta tttttccgcc gagcgatgaa 1920
cagctgaaaa gcggcaccgc gagcgtggtg tgcctgctga acaactttta tccgcgcgaa 1980
gcgaaagtgc agtggaaagt ggataacgcg ctgcagagcg gcaacagcca ggaaagcgtg 2040
accgaacagg atagcaaaga tagcacctat agcctgagca gcaccctgac cctgagcaaa 2100
gcggattatg aaaaacataa agtgtatgcg tgcgaagtga cccatcaggg cctgagcagc 2160
ccggtgacca aaagctttaa ccgcggcgaa tgctag 2196
Claims (13)
1. a kind of CAR-T cell of secreting type targeting Lewis-Y, which is characterized in that including dividing while expression Lewis-Y-CAR
The CAR-T cell of cell factor while secreting the CAR-T cell and expression Lewis-Y-CAR of antibody.
2. CAR-T cell as described in claim 1, which is characterized in that the antibody is PD-1 antibody, and cell factor is
IL12 cell factor.
3. CAR-T cell as claimed in claim 1 or 2, which is characterized in that the Lewis-Y-CAR includes the region scFv,
Lewis-Y antigen, the scFv regional sequence are by the nucleotide sequence of optimization, sequence such as SEQ for identification in the region scFv
Shown in ID NO.1.
4. the preparation method of the CAR-T cell of secreting type targeting Lewis-Y as claimed in claim 1 or 2, which is characterized in that
Include the following steps:
(1) PBMC is separated from the peripheral blood that donor provides；
(2) it activated from PBMC, sub-elect CD3 positive T cell；
(3) building carries Lewis-Y-CAR and antibody or the slow virus plasmid backbone of cytokine gene obtains recombinant slow virus
Plasmid；
(4) by the resulting recombinant slow virus plasmid for carrying Lewis-Y-CAR and antibody or cytokine gene of step (3) and auxiliary
Plasmid-infected host cells are helped, preparation can infect the recombined lentivirus vector of T cell；
(5) the resulting recombined lentivirus vector transduction of step (4) is entered step into the T cell through activating that (2) sub-elect, generated
Secretory antibody or the CAR-T cell of cell factor while expressing Lewis-Y-CAR；
(6) by cell injuring model obtained by step (5)；
(7) by cell massive amplification obtained by step (6)；
(8) secretory antibody or the CAR-T cell of cell factor while collecting expression Lewis-Y-CAR.
5. method as claimed in claim 4, which is characterized in that the region scFv that the Lewis-Y-CAR is included is excellent
Change the sequence obtained after codon, as shown in SEQ ID NO.1.
6. method as claimed in claim 4, which is characterized in that in step (3), the recombinant slow virus plasmid be two generations or
The slow virus transgenosis plasmid of person three generations.
7. method as claimed in claim 4, which is characterized in that in step (3), the recombinant slow virus plasmid is the third generation
Slow virus transgenosis plasmid；The recombinant slow virus plasmid is to target Lewis-Y antigen and secrete the slow of IL12 cell factor
Viral transgene plasmid, recombinant slow virus plasmid successively include CMV promoter sequence, Lewis-Y single-chain antibody (scFv),
Flag, CD8 Transmembrane Chimerical receptor transmembrane region, intracellular signal transduction area, NFAT promoter sequence, IL12 cell because
Subsequence；Recombined lentivirus vector described in step (5) is thin in T by the Chimeric antigen receptor expression for targeting Lewis-Y antigen
Cellular surface, while IL12 cell factor can be secreted.
8. method as claimed in claim 4, which is characterized in that in step (3), the recombinant slow virus plasmid is the third generation
Slow virus transgenosis plasmid；The recombinant slow virus plasmid is the slow virus for targeting Lewis-Y antigen and secreting PD-1 antibody
Transgenosis plasmid, recombinant slow virus plasmid successively include CMV promoter sequence, Lewis-Y single-chain antibody (scFv), Flag, CD8
Transmembrane Chimerical receptor transmembrane region, intracellular signal transduction area, EF1 α promoter sequence, IL2SS sequence, PD-1 antibody
Sequence；Recombined lentivirus vector described in step (5) expresses the Chimeric antigen receptor for targeting Lewis-Y antigen in T cell
Surface, while PD-1 antibody can be secreted.
9. method as claimed in claim 7 or 8, which is characterized in that the intracellular signal transduction area include coding costimulation because
Subregion, the costimulating factor region are selected from 4-1BB, CD28, CD27, OX40, CD30, CD40, PD-1, ICOS, LIGHT, B7-
H3, ligand, ICAM-1, HVEM (LIGHTR), CD160, the relevant antigen -1 of lymphocyte function for specifically binding CD83
(LFA-1)、IL2Rα、CD103、CD11b、CD11c、TRANCE/RANKL、SLAMF4(CD244,2B4)、CD69、SLAM
One of (SLAMF1, CD150, IPO-3) or a variety of any combination.
10. method as claimed in claim 7 or 8, which is characterized in that the intracellular signal transduction area is CD28-4-1BB-CD3
ζ。
11. method as claimed in claim 4, which is characterized in that in step (4), there are three the helper plasmids, respectively
PRSV-Rev, pCMV-VSV-G, pMDLg-pRRE, the host cell are 293FT or 293T cell.
12. the purposes of the CAR-T cell of secretion PD-1 antibody or IL12 as claimed in claim 1 or 2, which is characterized in that be used for
Antitumor cellular therapeutic agent is prepared, the cellular therapeutic agent of the tumor stem cell of the Lewis-Y positive is especially targeted.
13. a kind of mix preparation, secretion is anti-while including the described expression Lewis-Y-CAR as claimed in claim 1 or 2
The CAR-T cell of secrete cytokines while the CAR-T cell and expression Lewis-Y-CAR of body.
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