CN106511379A - Preparation method of CAR-T cell preparation for treating breast cancer - Google Patents
Preparation method of CAR-T cell preparation for treating breast cancer Download PDFInfo
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Abstract
The invention discloses a preparation method for a CAR-T cell preparation for treating breast cancer. The method comprises the following steps: (S1) drawing blood from a patient, separating T cells, selecting 1-3mm<3> of T cells, putting into a centrifugal tube, adding 1mg/ml collagenase and soaking, and then digesting for 2-4 hours at 36-38 DEG C; (S2) adding D-Hank's balanced salt solution into the centrifugal tube acquired in the step (S1), washing the cells 2-6 times, applying 200-400g centrifugal force, continuing for 6-8min and acquiring a mixed solution; (S3) putting the mixed solution into a culture vessel, putting the culture vessel into a culture box and performing multiplication culture, wherein the temperature in the culture box is 36-38 DEG C and the culture box contains 5-7% of CO2; (S4) transfecting the T cells acquired in the step (S3) with slow viruses and performing multiplication culture, thereby acquiring CAR-T cells; and (S5) uniformly mixing the CAR-T cells prepared in the step (S4) with human albumin at a ratio of 1:(1-2). According to the invention, the ingredients are clear, the preparation is simple and convenient, the relapse of breast cancer patient is effectively reduced, the therapeutic effect is promoted and a more convenient method is provided for the clinic popularization and application of the CAR-T cells.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of preparation side of the CAR-T cell preparations for treating breast cancer
Method.
Background technology
In immunotherapy of tumors method, adoptive immunity effector cell treatment because have the advantages that it is numerous be subject to people
Attention.Chimeric antigen receptor modification T cell (CAR-T) is even more as the frontier of adoptive immunity cell therapy becomes research heat
Point.Cancer patient because caused by Radiotherapy chemotherapy function of immune system it is low, be the major reason for causing disease relapse, and puzzlement
The a great problem of medical circle.CAR-T cell therapies are then a kind of brand-new biological immune treatment methods, by extracting in patient's body
T lymphocytes, transform through the cultures of external 10 to 14 days, enter into normal T-cell gene order by vector integration, are formed
Chimeric antigen receptor T cell (CAR-T).The T cell being re-coded can just obtain specific recognition and attack killing tumour is thin
The ability of born of the same parents, the accurate killing tumor cell of energy, but normal cell will not be injured.Due to being to induce, activate autogenous cell, therefore
The therapy does not have the toxicity of usual Radiotherapy chemotherapy, the drug resistance occurred in yet not having traditional treatment.
CAR-T cells are formed by connecting by the single-stranded variable region and T cell signal transduction area of monoclonal antibody, antibody and phase
The tumour antigen answered can make T cell activation with major histocompatibility complex after combining in a non-limiting manner, and then play
GVT.The structure of CAR is divided into：Extracellular antigen binding domain, hinge area, transmembrane region and intracellular signal area.Target antigen
Select all be crucial decision for the specificity of CAR, validity and the genetic modification T cell security of itself because
Element.Extracellular antigen binding domain：Generally it is formed by connecting by the light chain and heavy chain of the monoclonal antibody for tumor associated antigen.Hinge
Area：For the epitope away from cell membrane, the CAR-T cells of hingeless sequence can be recognized and conjugated antigen；And it is near for film
The epitope at end, a flexible identification of the hinge area to antigen is necessary.Therefore need for different tumor associated antigens
Hinge section length is adjusted, binding ability is targetted with more preferable performance.Transmembrane region：Transmembrane region from CD3, CD28, CD8 is commonly used to
CAR is built, and is played a significant role in the dimerization and T cell activation of CAR.Intracellular signal area：By costimulatory molecules such as
CD28, CD134 and CD137 are constituted, and wherein CD28 can raise PI3K, Grb2 equimolecular to adjust the activity of key transcription factor；
CD134 can promote T cell in-vitro multiplication and increase the secretion of proleulzin；CD137 is tnf family cytokines acceptor, can be swashed
The JNK in downstream living, p38, MAPK and NF- κ B signal approach.Collaboration costimulatory signal of the most researchers CD28 or CD137
Molecule is fused to the upstream of CD3 domains.3 generation CAR-T cells can be divided into according to the composition difference in intracellular signal transduction region.The
Intracellular part only ζ containing the CD3 domains of 1 generation CAR-T cell, although the activation of energy inducing T cell and initial cytotoxicity are anti-
Should, but the time-to-live is short and there was only generation that is very low or not having proleulzin；2nd generation CAR-T cell is by costimulatory molecules
Intracellular part such as CD28, CD137 is fused to the upstream of CD3 domains, and effect is remarkably reinforced than 1st generation thus most widely used
It is general；3rd generation CAR-T cell then merges 2 kinds of costimulatory moleculeses simultaneously to the upstream of CD3 domains, its antitumor activity and 2nd generation
CAR-T cells are compared and there is no final conclusion.CAR-T cells serve important function in the immunization therapy of neoplastic hematologic disorder, with CD19-
CAR-T cell therapy lymphocytic tumours are projected the most, and some come into II clinical trial phase.CAR-T cells are swollen in entity
Research major part in knurl is still in animal experiment stage, but has the clinical testing of some small samples and Case report to achieve not
Wrong curative effect.
Breast cancer is to endanger one of major malignant tumor of WomanHealth, with the acceleration of China human mortality urbanization, mammary gland
Carcinogenesis rate and case fatality rate have the trend for rising year by year.The conventional treatments of breast cancer mainly have：Operative treatment, Chemo-Therapy
Treatment, radiotherapy, endocrine therapy.Complex treatment in recent years improves the therapeutic effect of breast cancer, but still has quite a few
Patients on Recurrence or to conventional therapy resistance, we have proposed a kind of preparation side of the CAR-T cell preparations for treating breast cancer for this
Method.
The content of the invention
Based on the technical problem that background technology is present, the present invention proposes a kind of CAR-T cell preparations for treating breast cancer
Preparation method.
A kind of preparation method of CAR-T cell preparations for treating breast cancer proposed by the present invention, comprises the following steps：
S1, extracts the blood of patient itself, isolates T cell, chooses 1-3mm3T cell, be put in centrifuge tube, add
The clostridiopetidase A infiltration of 1mg/ml, digests 2-4 hours at 36-38 DEG C；
S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 2-6 time in S1, centrifugal force 200-400g holds
Continuous 6-8min, obtains mixed liquor；
S3, mixed liquor is placed in culture dish, and culture dish is put in incubator, and the temperature in incubator is 36-38
DEG C, the CO containing 5-7%2, amplification cultivation；
S4, slow virus is carried out transfecting and amplification cultivation to culture gained T cell in S3, CAR-T cells are obtained；
S5, by the CAR-T cells prepared in S4 and human serum albumin with 1：The ratio of 1-2 is mixed, and adds physiological saline,
Make the CAR-T cell preparations that can treat breast cancer.
Preferably, the S1, extracts the blood of patient itself, chooses 2mm3Blood be put in centrifuge tube, add 1mg/
The clostridiopetidase A infiltration of ml, digests 3 hours at 37 DEG C, gentle per 15 minutes to shake once.
Preferably, in the S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 4 times, centrifugal force in S1
300g, continues 7min, obtains mixed liquor.
Preferably, in the S3, the incubator is 94-96% saturated humidities.
Preferably, the target cell antigen of the CAR-T cells is CA153-HER2.
Preferably, the slow virus is built by the way of carrier package.
Preferably, in the S2, centrifugation rate is 1500r/min.
Preferably, in the S3, the number of days of amplification cultivation is 10-15 days.
The present invention first extracts the blood of patient itself, isolates T cell and is put in centrifuge tube, adds clostridiopetidase A infiltration to disappear
Change, add D-Hank ' s balanced salt solutions and rinse cell, expanded in being then centrifuged for and being placed on the culture dish in incubator
Culture, then slow virus is carried out transfecting to T cell and amplification cultivation, obtain CAR-T cells；The CAR-T cells that will finally prepare
With human serum albumin with 1：The ratio of 1-2 is mixed, and adds physiological saline, makes the CAR-T cell systems that can treat breast cancer
Agent.Present component is clearly, simple for production, effectively reduces the recurrence of patient with breast cancer and improves therapeutic effect, is CAR-T cells
Clinical application provide more easily method.
Specific embodiment
The present invention is further explained with reference to specific embodiment.
Embodiment one
A kind of preparation method of CAR-T cell preparations for treating breast cancer proposed by the present invention, comprises the following steps：
S1, extracts the blood of patient itself, isolates T cell, chooses 1mm3T cell, be put in centrifuge tube, add
The clostridiopetidase A infiltration of 1mg/ml, digests 2 hours at 36 DEG C；
S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 2 times in S1, centrifugal force 200g continues 6min,
Obtain mixed liquor；
S3, mixed liquor is placed in culture dish, and culture dish is put in incubator, and the temperature in incubator is 36 DEG C,
Containing 5% CO2, amplification cultivation；
S4, slow virus is carried out transfecting and amplification cultivation to culture gained T cell in S3, CAR-T cells are obtained；
S5, by the CAR-T cells prepared in S4 and human serum albumin with 1：1 ratio is mixed, and adds physiological saline, is made
Into the CAR-T cell preparations that can treat breast cancer.
Embodiment two
A kind of preparation method of CAR-T cell preparations for treating breast cancer proposed by the present invention, comprises the following steps：
S1, extracts the blood of patient itself, isolates T cell, chooses 1mm3T cell, be put in centrifuge tube, add
The clostridiopetidase A infiltration of 1mg/ml, digests 2.5 hours at 36.5 DEG C；
S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 3 times in S1, centrifugal force 250g continues
6.5min, obtains mixed liquor；
S3, mixed liquor is placed in culture dish, and culture dish is put in incubator, and the temperature in incubator is 36.5
DEG C, containing 5.5% CO2, amplification cultivation；
S4, slow virus is carried out transfecting and amplification cultivation to culture gained T cell in S3, CAR-T cells are obtained；
S5, by the CAR-T cells prepared in S4 and human serum albumin with 1：1.2 ratio is mixed, and adds physiological saline,
Make the CAR-T cell preparations that can treat breast cancer.
Embodiment three
A kind of preparation method of CAR-T cell preparations for treating breast cancer proposed by the present invention, comprises the following steps：
S1, extracts the blood of patient itself, isolates T cell, chooses 2mm3T cell, be put in centrifuge tube, add
The clostridiopetidase A infiltration of 1mg/ml, digests 3 hours at 37 DEG C；
S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 4 times in S1, centrifugal force 300g continues 7min,
Obtain mixed liquor；
S3, mixed liquor is placed in culture dish, and culture dish is put in incubator, and the temperature in incubator is 37 DEG C,
Containing 6% CO2, amplification cultivation；
S4, slow virus is carried out transfecting and amplification cultivation to culture gained T cell in S3, CAR-T cells are obtained；
S5, by the CAR-T cells prepared in S4 and human serum albumin with 1：1.5 ratio is mixed, and adds physiological saline,
Make the CAR-T cell preparations that can treat breast cancer.
Example IV
A kind of preparation method of CAR-T cell preparations for treating breast cancer proposed by the present invention, comprises the following steps：
S1, extracts the blood of patient itself, isolates T cell, chooses 2.5mm3T cell, be put in centrifuge tube, add
The clostridiopetidase A infiltration of 1mg/ml, digests 3.5 hours at 37.5 DEG C；
S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 5 times in S1, centrifugal force 350g continues
7.5min, obtains mixed liquor；
S3, mixed liquor is placed in culture dish, and culture dish is put in incubator, and the temperature in incubator is 37.5
DEG C, containing 6.5% CO2, amplification cultivation；
S4, slow virus is carried out transfecting and amplification cultivation to culture gained T cell in S3, CAR-T cells are obtained；
S5, by the CAR-T cells prepared in S4 and human serum albumin with 1：1.8 ratio is mixed, and adds physiological saline,
Make the CAR-T cell preparations that can treat breast cancer.
Embodiment five
A kind of preparation method of CAR-T cell preparations for treating breast cancer proposed by the present invention, comprises the following steps：
S1, extracts the blood of patient itself, isolates T cell, chooses 3mm3T cell, be put in centrifuge tube, add
The clostridiopetidase A infiltration of 1mg/ml, digests 4 hours at 38 DEG C；
S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 6 times in S1, centrifugal force 400g continues 8min,
Obtain mixed liquor；
S3, mixed liquor is placed in culture dish, and culture dish is put in incubator, and the temperature in incubator is 38 DEG C,
Containing 7% CO2, amplification cultivation；
S4, slow virus is carried out transfecting and amplification cultivation to culture gained T cell in S3, CAR-T cells are obtained；
S5, by the CAR-T cells prepared in S4 and human serum albumin with 1：2 ratio is mixed, and adds physiological saline, is made
Into the CAR-T cell preparations that can treat breast cancer.
Present component is clearly, simple for production, effectively reduces the recurrence of patient with breast cancer and improves therapeutic effect, is CAR-
The clinical application of T cell provides more easily method.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any those familiar with the art the invention discloses technical scope in, technology according to the present invention scheme and its
Inventive concept equivalent or change in addition, should all be included within the scope of the present invention.
Claims (8)
1. a kind of preparation method of the CAR-T cell preparations for treating breast cancer, it is characterised in that comprise the following steps：
S1, extracts the blood of patient itself, isolates T cell, chooses 1-3mm3T cell, be put in centrifuge tube, add 1mg/
The clostridiopetidase A infiltration of ml, digests 2-4 hours at 36-38 DEG C；
S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 2-6 time in S1, centrifugal force 200-400g continues 6-
8min, obtains mixed liquor；
S3, mixed liquor is placed in culture dish, and culture dish is put in incubator, and the temperature in incubator is 36-38 DEG C, is contained
There is the CO of 5-7%2, amplification cultivation；
S4, slow virus is carried out transfecting and amplification cultivation to culture gained T cell in S3, CAR-T cells are obtained；
S5, by the CAR-T cells prepared in S4 and human serum albumin with 1：The ratio of 1-2 is mixed, and adds physiological saline, is made
The CAR-T cell preparations of breast cancer can be treated.
2. the preparation method of a kind of CAR-T cell preparations for treating breast cancer according to claim 1, it is characterised in that
The S1, extracts the blood of patient itself, chooses 2mm3Blood be put in centrifuge tube, add 1mg/ml clostridiopetidase A infiltration,
Digest 3 hours at 37 DEG C, it is gentle per 15 minutes to shake once.
3. the preparation method of a kind of CAR-T cell preparations for treating breast cancer according to claim 1, it is characterised in that
In the S2, centrifuge tube addition D-Hank ' s balanced salt solution flushings cell 4 times in S1, centrifugal force 300g continues 7min, obtains
Obtain mixed liquor.
4. the preparation method of a kind of CAR-T cell preparations for treating breast cancer according to claim 1, it is characterised in that
In the S3, the incubator is 94-96% saturated humidities.
5. the preparation method of a kind of CAR-T cell preparations for treating breast cancer according to claim 1, it is characterised in that
The target cell antigen of the CAR-T cells is CA153-HER2.
6. the preparation method of a kind of CAR-T cell preparations for treating breast cancer according to claim 1, it is characterised in that
The slow virus is built by the way of carrier package.
7. the preparation method of a kind of CAR-T cell preparations for treating breast cancer according to claim 1, it is characterised in that
In the S2, centrifugation rate is 1500r/min.
8. the preparation method of a kind of CAR-T cell preparations for treating breast cancer according to claim 1, it is characterised in that
In the S3, the number of days of amplification cultivation is 10-15 days.
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| CN201611221435.4A CN106511379A (en) | 2016-12-26 | 2016-12-26 | Preparation method of CAR-T cell preparation for treating breast cancer |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523550A (en) * | 2017-08-08 | 2017-12-29 | 安徽惠恩生物科技股份有限公司 | A kind of Car T cell preparation methods for ED-SCLC |
| CN107686831A (en) * | 2017-08-23 | 2018-02-13 | 安徽惠恩生物科技股份有限公司 | A kind of Car T cell preparation methods for cancer of pancreas |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105640990A (en) * | 2016-01-06 | 2016-06-08 | 奥思达干细胞有限公司 | CAR-T cell preparation for treating breast cancer and preparation method thereof |
| CN105640991A (en) * | 2016-01-06 | 2016-06-08 | 奥思达干细胞有限公司 | CAR-T cell preparation for treating prostatic cancer and preparation method thereof |
-
2016
- 2016-12-26 CN CN201611221435.4A patent/CN106511379A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105640990A (en) * | 2016-01-06 | 2016-06-08 | 奥思达干细胞有限公司 | CAR-T cell preparation for treating breast cancer and preparation method thereof |
| CN105640991A (en) * | 2016-01-06 | 2016-06-08 | 奥思达干细胞有限公司 | CAR-T cell preparation for treating prostatic cancer and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107523550A (en) * | 2017-08-08 | 2017-12-29 | 安徽惠恩生物科技股份有限公司 | A kind of Car T cell preparation methods for ED-SCLC |
| CN107686831A (en) * | 2017-08-23 | 2018-02-13 | 安徽惠恩生物科技股份有限公司 | A kind of Car T cell preparation methods for cancer of pancreas |
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