CN105640990A - CAR-T cell preparation for treating breast cancer and preparation method thereof - Google Patents

Abstract

The invention discloses a CAR-T cell preparation for treating breast cancer and a preparation method thereof. The preparation disclosed by the invention is prepared from CAR-T cells, human serum albumin, IL-6 receptor antagonist Tocilizumab and normal saline in a certain proportion. The chimeric antigen receptor(CAR) prepared by the method disclosed by the invention is mainly composed of an antigen antibody binding region, a transmembrane region, a CD3zeta intracellular signal region and a co-stimulatory signal transmitting region. According to the CAR-T cell preparation for treating breast cancer disclosed by the invention, the CAR-T cells with chimeric breast cancer cell antigen receptors are used for treating breast cancer, the treatment effect is obvious, and the invention fundamentally provides a method for treating breast cancer.

Description

A kind of CAR-T cell preparation treating breast carcinoma and preparation method thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of CAR-T cell preparation treating breast carcinoma and preparation method thereof.

Background technology

In immunotherapy of tumors method, adoptive immunity effector lymphocyte treatment is subject to people's attention because having numerous advantages. Chimeric antigen receptor (ChimericAntigenRecepTor, CAR) modifies T cell (CAR-T) becomes study hotspot especially as the frontier of adoptive immunity cell therapy. The function of immune system that cancer patient causes because of Radiotherapy chemotherapy is low, is the major reason causing disease relapse, is also a great problem of puzzlement medical circle. CAR-T cell therapy is then a kind of brand-new biological immune Therapeutic Method, by extracting T lymphocyte in the patient, transform through the cultivations of external 10 to 14 days, enter into normal T-cell gene order by vector integration, form Chimeric antigen receptor T cell (CAR-T). The T cell being re-coded just can obtain specific recognition and attack the ability of killing tumor cell, and energy is killing tumor cell accurately, but will not injure normal cell. Owing to being induction, activating autogenous cell, therefore this therapy does not have the toxicity of usual Radiotherapy chemotherapy, also without the drug resistance occurred in traditional treatment.

CAR-T cell is the single-chain variable by monoclonal antibody and T cell signal conducting region is formed by connecting, and antibody can make T cell activation with corresponding tumor antigen in a non-limiting manner with major histocompatibility complex after combining, and then plays Graft Versus Tumor. The structure of CAR is divided into: born of the same parents' exoantigen land, hinge region, cross-film district and intracellular signal district. Selecting for the safety of the specificity of CAR, effectiveness and genetic modification T cell self of target antigen is all crucial determiner. Born of the same parents' exoantigen land: generally light chain and heavy chain by the monoclonal antibody for tumor associated antigen are formed by connecting. Hinge region: for the epitope away from cell membrane, the CAR-T cell of hingeless sequence may identify which and conjugated antigen; And for the epitope of film near-end, one flexibly hinge region be necessary to the identification of antigen. Need to adjust hinge region length hence for different tumor associated antigens, better to play targeting binding ability. Cross-film district: the cross-film district from CD3, CD28, CD8 is commonly used to build CAR, and plays a significant role in the dimerization and T cell activation of CAR. Intracellular signal district: be made up of costimulatory molecules such as CD28, CD134 and CD137, wherein CD28 can raise PI3K, Grb2 equimolecular to regulate the activity of key transcription factor;CD134 can promote T cell in-vitro multiplication and increase the secretion of interleukin-2; CD137 is tnf family cytokines receptor, can activate JNK, p38, MAPK and the NF-�� B signal approach in downstream. Most researchers is fused to the upstream of CD3 domain the collaborative costimulatory signal molecule of CD28 or CD137. Composition difference according to intracellular signal transduction region can be divided into 3 generation CAR-T cells. The interior part of the born of the same parents of 1st generation CAR-T cell is only containing CD3 �� domain, although activating and initial cell-cytotoxic reaction of energy inducing T cell, but the time-to-live is short and it is very low to only have or generation that is that do not have interleukin-2; 2nd generation CAR-T cell part will be fused to the upstream of CD3 domain in the born of the same parents of costimulatory molecules such as CD28, CD137, and effect is remarkably reinforced than 1st generation thus most widely used general; 3rd generation CAR-T cell then merges 2 kinds of costimulatory moleculeses upstream to CD3 domain simultaneously, and its anti-tumor activity there is no final conclusion compared with 2nd generation CAR-T cell. CAR-T cell serves important function in the immunization therapy of neoplastic hematologic disorder, and the most prominent with CD19-CAR-T cell therapy lymphocytic tumours, some comes into II clinical trial phase. CAR-T cell research major part in entity tumor is still in animal experiment stage, but has the clinical trial of some small samples and Case report to achieve good curative effect.

Breast carcinoma is one of major malignant tumor of harm WomanHealth, along with the acceleration of China's Population Urbanization, incidence of breast cancer and case fatality rate have the trend risen year by year. The conventional treatments of breast carcinoma mainly has: operative treatment, chemotherapy, radiotherapy, endocrine therapy. Comprehensive Treatment improves the therapeutic effect of breast carcinoma in recent years, but still has quite a few Patients on Recurrence or to conventional therapy drug resistance. Therefore find a kind of new Therapeutic Method to reduce the recurrence of patient with breast cancer and to improve therapeutic effect, there is the clinical meaning of particular importance.

Summary of the invention

In order to solve the problems referred to above, the invention provides a kind of CAR-T cell preparation treating breast carcinoma and preparation method thereof, the treatment of dry breast carcinoma is had the clinical meaning of particular importance.

For realizing above-mentioned technical purpose, reaching above-mentioned technique effect, the present invention is achieved through the following technical solutions:

A kind of CAR-T cell preparation treating breast carcinoma is formulated by a certain percentage by being fitted together to the CAR-T cell of target cell antigen receptor, human albumin, IL-6 receptor antagonist medicine Tocilizumab, normal saline.

Further, the specifically comprising of described cell injection: the concentration of CAR-T is (4��6) �� 10⁷Individual/ml, mass volume ratio is human albumin, the 8mg/mLIL-6 receptor antagonist medicine Tocilizumab of 2%, and surplus is normal saline.

Further, the target cell antigen of described CAR-T cell is the combination of following single antigen or several antigen: CA153 human epidermal growth factor receptor-2 (HER2), tissue factor (TF), EGF-R ELISA (EGFG), platelet derived growth factor receptor (PDGFR), it is preferable that the combination of HER2 and CA153.

Further, the CAR of described CAR-T cell is specifically comprising of HCA153-HER2scFv-CD8-OX40-CD3 ��, CAR: HCA153-HER2scFv, CD8 hinge region, antigen-antibody land, OX40 cross-film district and intracellular region and CD3 �� intracellular signal district are in series.

Further, described CAR-T cell is expressed by a kind of expression vector, and wherein, described expression vector is the one in slow virus, retrovirus, adenovirus, it is preferable that slow virus.

The preparation method of a kind of CAR-T cell preparation treating breast carcinoma, mainly comprises the steps that

(1) vector construction: build the carrier that can express HCA153-HER2scFv-CD8-OX40-CD3 ��;

(2) packaging of the virus of transfecting T cells: adopt the carrier package slow virus built in step (1), it is thus achieved that through the slow virus of packaging;

(3) T cell separates and amplification cultivation: extracts patient's self-blood, therefrom isolates T cell and carry out amplification cultivation;

(4) transfection of T cell and preparation: adopt in step (2) and carry out transfection amplification cultivation through the slow virus of packaging to cultivating gained T cell in step (3), it is thus achieved that CAR-T cell;

(5) CAR-T cell step (4) prepared and human albumin, IL-6 receptor antagonist medicine Tocilizumab mix in proportion, and surplus normal saline is supplied, and prepares into CAR-T cell preparation.

The present invention has the following advantages relative to prior art and effect:

(1) present invention will be fitted together to the CAR-T cell of breast cancer cell antigen receptor first, be used for treating breast carcinoma, and therapeutic effect is notable, fundamentally provides a kind of method treating breast carcinoma;

(2) present invention will be fitted together to the CAR-T cell of breast cancer cell antigen receptor and produce jointly into human albumin, IL-6 receptor antagonist medicine Tocilizumab etc. the immunocyte preparation of a kind of targeted therapy breast carcinoma first, and Tocilizumab can inflammatory reaction that effectively reduction of patient was with later at input CAR-T cell; Human albumin can maintain the activity of CAR-T cell. This formulation ingredients is clear and definite, and simple for production, the clinical application for CAR-T cell provides method more easily;

(3) target cell antigen selected by the present invention is the combination of following single antigen or several antigen: CA153, human epidermal growth factor receptor-2 (HER2), tissue factor (TF), EGF-R ELISA (EGFG), platelet derived growth factor receptor (PDGFR), it is preferable that the combination of CA153 and HER2. CA153 is the most important Specific marker of breast carcinoma. The CA153 of the patient with breast cancer of 30%-50% is significantly raised, and the change of its content is closely related with therapeutic effect, is that patient with breast cancer diagnoses and monitors postoperative recurrence, observes the optimal parameter of curative effect; HER2 is the important independent factor that Prognosis in Breast Cancer is bad, the breast cancer tissue of 24% ~ 30% exists HER2 receptor overexpression, its overexpression causes that tumor cell abnormality proliferation, aggressive and transfer danger increase, therefore adopt the combination of CA153 and HER2 can be greatly improved as target cell antigen make CAR-T cell-specific identification and kill the ability of breast cancer cell, reduce the recurrence of patient with breast cancer and improve therapeutic effect;

(4) that the present invention adopts is third generation CAR, and the restructuring T cell of third generation CAR can increase the secretion of gamma interferon and Anti-apoptotic proteins, is significantly increased in anti-tumor activity, time to live and release of cytokines;

Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, and can be practiced according to the content of description, below with the preferably case study on implementation of the present invention and coordinate accompanying drawing describe in detail as after. The specific embodiment of the present invention is shown in detail in by following example and accompanying drawing thereof.

Accompanying drawing explanation

Fig. 1 represents from the T cell cultured and amplified in vitro curve that peripheral blood in patients separation obtains.

The vitality test of Fig. 2 cultured and amplified in vitro gained T cell.

Fig. 3 shows the efficiency of infection (with the Protein Detection scFv of FITC-labelling expression rate in T cell) of the slow virus infection T cell by different MOI values.

Fig. 4 shows that CA153 and HER2 is expressed the specific killing rate of positive breast cancer cell T47D by CAR-T cell.

Detailed description of the invention

Embodiment 1CAR-T fibrocyte expression vector builds:

(1) anti-CA153 and the HER2 antibody of pcr amplification purpose fragment is with pCDNA3-scBW431/26-hFc plasmid for template design primer, at its 5', 3' end respectively plus Nhe I, Sal I restriction endonuclease sites, CEAUpNhe I forward primer 5'-AGGCTAGCATGGGATGGAGCTGTATCAT-3', CA153-HER2DnSal I downstream primer 5'-AGGTCGACGGTATCGATAAGCTTTG-3', 95 DEG C of denaturation 5min of reaction condition, 95 DEG C of degeneration 10s, 68 DEG C of annealing 15s, 72 DEG C extend 30s, 30 circulations, take out 2 �� L sample and identify after having expanded;

(2) double digestion LV-EF1 ��-Luciferase-PGK-FP635-WPRE carrier, purpose fragment CA153-HER2-CD3-2A-GFP carrier enzyme action system are 50 �� L:10 �� Greenbuffer5 �� L, carrier 3 �� L, Nhe I enzyme 1 �� L, Sal I enzyme 1 �� L, H₂O40 �� L; Purpose fragment enzyme action system is 50 �� L:10 �� Greenbuffer5 �� L, and purpose fragment reclaims product 2.5 �� L, Nhe I enzyme 1 �� L, EcoR V enzyme 1 �� L, H₂O39.5 �� L, 37 DEG C of water-bath 3h. Enzyme action takes out 2 �� L sample and identifies after completing;

(3) recombined lentivirus vector LV-EF1 ��-CA153-HER2-2A-GFPWPRE connection, conversion, extracting and enzyme action identify that linked system is 10 �� L:T4DNAligase10 �� buffer1 �� L, carrier segments 2 �� L, purpose fragment 6 �� L, T4DNAligase1 �� L, connects overnight. Take out in the 5 �� L 1.5mLEP pipe being equipped with competence antibacterial (frozen water admixture) TOP10 by connecting product, ice bath 30min, 42 DEG C of water-bath 90s, ice bath 3min, add 800 �� LLB fluid mediums, 30 DEG C, 280r/min shaken cultivation 1h; 5000r/min is centrifuged 2min, abandons supernatant, adds on the LB solid medium of ammonia benzyl resistance, smoothen with triangle Glass rod after staying 200 �� L piping and druming mixings, and overnight incubation in 30 DEG C of calorstats put into by flat board. The full bacterium colony of picking in the ammonia benzyl LB fluid medium of 1: 1000,30 DEG C, 280r/min, shaken cultivation 12h. Method for extracting, with reference to the plasmid extraction test kit extracting of American I nvitrogen company, takes out 2 �� L sample and identifies after completing.

Embodiment 2CAR-T virus is packed;

4 pUC pUCs are adopted to carry out virus packaging, 4 pUC pUCs expressing viral vector packaging is required respectively gag/pol, Rev, VSV-G, and artificial chimeric's antigen receptor that the single-chain antibody of engineering stability is constituted. 4 plasmids are carried out in proportion transient transfection. Gross mass is 10 �� g. Above-mentioned plasmid is added to the sterilized water of certain volume, is subsequently added 100 �� l2.5mmol/L's

CaCl₂, in mixed liquor, it is slowly added dropwise 2 �� HBS solution 500 �� l, above-mentioned solution is added to the culture dish being covered with 293T cell, mixes gently, be placed in 37 DEG C, 5%CO₂Incubator is cultivated 12h, adds the DMEM fluid medium 8mL containing 10%FBS, continue to cultivate 48 hours. Collect the T cell supernatant after 48h after transfecting, in 4 DEG C, 2000r/min, centrifugal 5 minutes, remove cell debris, with 0.45 ��m of frit supernatant subpackage, be saved in-70 DEG C.

The separation of embodiment 3T cell and amplification cultivation:

1), former blood pretreatment: extract peripheral blood in patients 100ml and add 100 �� L anticoagulant heparin agent, being then dispensed into the centrifuge tube of 4 50ml; With normal saline 1:1, blood it is diluted and mixes with pipet piping and druming;

2), Ficoll method separates: being carefully added in the 50ml centrifuge tube containing 15mlFicoll lymphocyte separation medium by the blood after dilution, often pipe is about 35ml, room temperature 2000rpm centrifugal 20 minutes;

3), the collection of T cell and washing: draw the cellular layer of two liquid level junctions in 50ml centrifuge tube, be supplemented to 30ml mixing, centrifugal 10 minutes of room temperature 2000rpm with normal saline, wash twice after cell precipitation;

4) amplification cultivation of T cell: T cell concentration is adjusted to 2 �� 10⁶Individual/ware is inoculated in 100mm culture dish, is placed in 37 DEG C, 5%CO₂In incubator cultivate, to be fused to 80-90% time, go down to posterity with 0.25% trypsinization, amplification culture T cell number is 10⁸-10⁹, collect T cell standby.

Result is as it is shown in figure 1, after amplification cultivation 12 days, T cell amplification ratio this concludes the description of through above-mentioned training method higher than T cells 100 again, it is possible to expands T cell at short notice; With determination of trypan blue staining T cell vigor, result is as in figure 2 it is shown, cell viability gradually steps up.

The transfection of embodiment 4CAR-T cell and preparation:

1) by the RetroNectin of 20 �� g, bag is standby in 6 orifice plates, is placed in 37 DEG C, 5%CO2 incubator interior cultivation 3h;

2) RetroNectin is drawn, 6 orifice plates after utilizing Hank ' the s closing bag containing 2.5%BSA standby, it is placed in 37 DEG C, 5%CO₂1h is cultivated in incubator;

3) draw confining liquid, utilize Hank ' s solution washing 6 orifice plate containing 2%Hepes. Add the DMEM fluid medium containing 10%FBS, and add the appropriate viral solution through packaging, 2000r/min, centrifugal 5 minutes;

4) abandon supernatant, add 2 �� 10⁶T cell, 1500r/min, centrifugal 5 minutes. It is placed in 37 DEG C, 5%CO₂Cultivate in incubator, repeat the above steps after 12h. Carry out cell harvesting after infecting 5 days, prepare CAR-T cell, infect the expression measuring scFv for latter 5 days, by the expression of flow cytomery scFv. Result is as it is shown on figure 3, the positive scFv of T cell surface expression after slow virus infection, and after again cultivating 15 days, the expression of its scFv is appointed 88.6%, does not lose expression simultaneously.

Breast cancer cell killing rate is detected by embodiment 5CAR-T cell

The present invention being prepared CAR-T cell action effect cell and carries out killing activity mensuration, experimental group target cell is that CA153 and HER2 expresses positive breast cancer cell T47D, and matched group target cell is that CA153 and HER2 expresses negative breast cancer cell T47D. It is that 5:1 adds in 96 well culture plates according to effector lymphocyte and target ration, at 37 DEG C of 5%CO₂Overnight incubation, adopting mtt assay to measure wavelength is 570nm place absorbance, and result is as shown in Figure 3.

Result shows, CA153 and HER2 is expressed the specific killing activity 51.3% of positive breast cancer cell T47D by the CAR-T cell prepared by experimental group of the present invention, is significantly higher than matched group 32.9%(P < 0.01). Illustrate that the specific CAR-T cell of CA153 and HER2 prepared by the present invention has the function killing CA153 and HER2 positive cell.

Claims (5)

1. the CAR-T cell preparation treating breast carcinoma, it is characterized in that: it is by being fitted together to the CAR-T cell of breast cancer cell antigen receptor HCA153-HER2scFv-CD8-OX40-CD3 ��, human albumin, IL-6 receptor antagonist medicine, the optimum ratio of preparation is: the concentration of CAR-T cell is (4��6) �� 107/ml, mass volume ratio is the human albumin of 2%, 8mg/mLIL-6 receptor antagonist medicine Tocilizumab, surplus is normal saline.

2. according to claim 1, the target cell antigen of CAR-T cell is the combination of following single antigen or several antigen: CA153 human epidermal growth factor receptor-2 (HER2), tissue factor (TF), EGF-R ELISA (EGFG), platelet derived growth factor receptor (PDGFR), it is preferable that the combination of HER2 and CA153.

3. according to claim 1, in CAR-T cell, CAR is specifically comprising of HCA153-HER2scFv-CD8-OX40-CD3 ��, CAR: HCA153-HER2scFv, CD8 hinge region, antigen-antibody land, OX40 cross-film district and intracellular region and CD3 �� intracellular signal district are in series.

4. according to claim 1, the target cell antigen of CAR-T cell is CA153-HER2.

5. according to claim 1, the preparation method of a kind of CAR-T cell preparation treating breast carcinoma, mainly comprise the steps that

(1) vector construction: build the carrier that can express HCA153-HER2scFv-CD8-OX40-CD3 ��;

(2) packaging of the virus of transfecting T cells: adopt the carrier package slow virus built in step (1), it is thus achieved that through the slow virus of packaging;

(3) T cell separates and amplification cultivation: extracts patient's self-blood, therefrom isolates T cell and carry out amplification cultivation;

(4) transfection of T cell and preparation: adopt in step (2) and carry out transfection amplification cultivation through the slow virus of packaging to cultivating gained T cell in step (3), it is thus achieved that CAR-T cell;

(5) CAR-T cell step (4) prepared and human albumin, IL-6 receptor antagonist medicine Tocilizumab mix in proportion, and surplus normal saline is supplied, and prepares into CAR-T cell preparation.