CN107557336B - Immune cell modified by anti-MUC16 safe chimeric antigen receptor and application thereof - Google Patents
Immune cell modified by anti-MUC16 safe chimeric antigen receptor and application thereof Download PDFInfo
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Abstract
The invention discloses an immune cell modified by an anti-MUC16 safe chimeric antigen receptor, wherein the immune cell is modified by an anti-MUC16 safe chimeric receptor to obtain the immune cell modified by an anti-MUC16 safe chimeric antigen receptor, and the anti-MUC16 safe chimeric antigen receptor comprises a CD8leader, a MUC16 binding region, a hinge region of CD8, a transmembrane-stimulating structure domain, a CD3 zeta stimulating signal conduction region, a chemical inducing element and application thereof, so that the toxicity is reduced, and the clinical effectiveness and the safety are improved.
Description
Technical Field
The invention relates to the field of biological genes, in particular to an immune cell modified by an anti-MUC16 safe chimeric antigen receptor and application thereof.
Background
Ovarian cancer is the fifth largest tumor causing death in women. Because of the concealment of the disease position and the lack of effective early detection indexes, the mortality rate of ovarian cancer is the first of all gynecological tumors, and the method poses serious threat to the life of women. Recent research data indicate that 90% of ovarian cancers are Epithelial Ovarian Cancers (EOC), and that five-year survival rates of ovarian cancer patients with high degrees of malignancy are even lower than 30%. MUC16, also known as CA125, is located on human chromosome 19p13.2, has size of 179000bp, is a high molecular weight glycoprotein expressed on the surface of various epithelial cells, and mainly plays a role in protecting and repairing epithelium. MUC16 is an important tumor index for early diagnosis of epithelial ovarian cancer, and is widely applied to clinic. Recent studies have found that MUC16 can promote distant metastasis of ovarian cancer, particularly in the peritoneal cavity of ovarian cancer, by binding to mesothelin; can help ovarian cancer cells realize immune escape by inhibiting the ovarian cancer cells from forming immune synapses with NK cells; the ovarian cancer can also be promoted by influencing epithelial-mesenchymal transition, proliferation, migration and metastasis of tumor cells through overexpression in ovarian cancer cells. Therefore, MUC16 can provide potential specific molecular targets for ovarian cancer treatment and also can provide a new strategy for ovarian cancer immunotherapy.
A Chimeric Antigen Receptor (CAR) is a recombinant antigen receptor and has functions of binding antigen and activating immune cells. The T cell transferred into CAR is mainly characterized in that the T cell carries a receptor for recognizing tumor antigen specificity and endows the T cell with personalized curative effect of targeted killing activity. Therefore, new strategies for cellular immunotherapy based on MUC16 as a target for chimeric antigen receptors would bring new hopes for ovarian cancer patients.
At present, CAR-T cells designed based on a series of different tumor surface antigens show significant anti-tumor effects in vitro and in vivo, and in particular CAR-T cells targeting CD19 have achieved good therapeutic efficacy in clinical patients with B-cell malignancies. However, these genetically modified T cells exhibit excessive activity, are difficult to control and may cause severe toxicity.
Disclosure of Invention
In order to make up for the defects, the invention provides an immune cell modified by an anti-MUC16 safe chimeric antigen receptor and application thereof.
The scheme of the invention is as follows:
an immune cell modified by an anti-MUC16 safe chimeric antigen receptor is modified by an anti-MUC16 safe chimeric receptor to obtain the anti-MUC16 safe chimeric antigen receptor modified immune cell, wherein the anti-MUC16 safe chimeric antigen receptor comprises a CD8leader, a MUC16 binding region, a hinge region of CD8, a transmembrane-stimulating domain, a CD3 zeta stimulating signal conducting region and a chemical inducing element.
As a preferred embodiment, the transmembrane-stimulating domain is selected from the group consisting of CD8, CD27, CD28, CD137/4-1BB, CD134/OX40, all or a partial fragment of an ICOS molecule.
As a preferred technical scheme, the chemical induction element is constructed based on an FK506binding protein 12-dimerization chemical induction subsystem (FK506binding protein 12-chemical indicators of dimerization, FKBP12-CID), comprises two copies of mutant F36V-FKBP of FKBP12 (Phe at position 36 is replaced by Val), and has a T2A sequence inserted in the middle.
As a preferred embodiment, the chemical inducing element is located between the transmembrane-stimulating domain of the recombinant chimeric antigen receptor and the CD3 zeta stimulatory signaling region.
Preferably, the immune cells modified by the anti-MUC16 safe chimeric antigen receptor are selected from autologous or transgenic T cells, NK cells, cytotoxic T lymphocytes or regulatory T cells, memory T cells, bispecific T cells and CIK cells.
Preferably, the fusion gene segment of the anti-MUC16 safe chimeric antigen receptor is a Leader-scFv (MUC16) -CD8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 zeta fusion gene segment.
As a preferred technical scheme, the Leader-scFv (MUC16) -CD8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 zeta fusion gene fragment is a nucleotide sequence shown in a sequence table SEQ ID NO. 1.
The invention also discloses a medicine for treating cancer, which contains immune cells modified by the anti-MUC16 safe chimeric antigen receptor.
Use of immune cells modified by anti-MUC16 safe chimeric antigen receptor for the preparation of a medicament for the treatment of malignant tumors.
In a preferred embodiment, the malignant tumor is ovarian cancer.
Due to the adoption of the technical scheme, the immune cell modified by the anti-MUC16 safe chimeric antigen receptor is modified by the anti-MUC16 safe chimeric receptor to obtain the immune cell modified by the anti-MUC16 safe chimeric antigen receptor, and the anti-MUC16 safe chimeric antigen receptor comprises a CD8leader, a MUC16 binding region, a hinge region of CD8, a transmembrane-stimulating domain, a CD3 zeta stimulating signal conducting region and a chemical inducing element.
The invention has the advantages that:
1. the invention provides an immune cell modified by an anti-MUC16 safe chimeric antigen receptor, which can utilize micromolecules to regulate and control the treatment function of the immune cell on the basis of retaining the antigen specificity. The structure of the split receptor is intact and functions only in the presence of the heterodimeric small molecule. This quantifiable drug modulation enables the physician to precisely control the intensity, timing and location of immune cell action, thereby reducing toxicity.
2. The invention is improved on the second generation and third generation CAR technology, and a chemical induction element is added, so that the toxic and side effects of treatment can be avoided while the treatment effect is ensured. The invention is applicable to any immune effector cells which can be modified by CAR technology, and greatly improves the clinical effectiveness and safety of the CAR technology.
Drawings
FIG. 1 is a schematic design diagram of the safety chimeric antigen receptor of the present invention
FIG. 2 is a design diagram of a fusion gene fragment of the chimeric antigen receptor Leader-scFv (MUC16) -CD8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 zeta.
FIG. 3 is a schematic diagram of lentivirus Leader-scFv (MUC16) -CD8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 zeta expression plasmid of the present invention.
FIG. 4 shows that the efficiency of the present CAR (MUC16) -CIK cells expressing CAR was 37.9%.
FIG. 5 shows that the killing efficiency of the CAR (MUC16) -CIK of the invention on MUC16 positive ovarian cancer cell line OVCAR3 in vitro increases with the increase of the concentration of AP1903, and the killing rate reaches more than 90% when the final concentration of AP1903 is 400 nM; the results show that the CAR (MUC16) -CIK cells designed by the invention have high killing effect on OVCAR3 cells, and the activity is regulated by AP 1903.
FIG. 6 shows that the ovarian cancer tumor inhibition rate of the CAR (MUC16) -CIK of the present invention increases with the increase of the concentration of AP1903, and no AP1903 was injected after the injection of CAR (MUC16) -CIK cells, the tumor mass became larger and the growth trend was completely consistent with that of the cells without the injection of CAR (MUC16) -CIK.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
Example 1 construction of lentivirus plasmids expressing chimeric antigen receptor proteins encoded by the nucleotides of the invention and viral packaging
1. The fusion gene sheet Leader-scFv (MUC16) -CD8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 zeta was inserted into the lentiviral expression vector pLent-C-GFP.
The CAR module of Leader-scFv (MUC16) -CD8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 ζ is schematically shown in FIG. 2 (see appendix SEQ ID NO.1 for the complete nucleic acid sequence).
CAR modular sequences of Leader-scFv (MUC16) -CD8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 zeta
(1) Leader nucleic acid artificial sequence (SEQ ID NO.2)
(2) Anti-MUC16single chain Fv antibody (scFv) nucleic acid artificial sequence (SEQ ID NO.3)
(3) CD8Hinge region nucleic acid artificial sequence (SEQ ID NO.4)
(4) CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO.5)
(5) CD137 intracellular region nucleic acid artificial sequence (SEQ ID NO.6)
(6) Linker nucleic acid artificial sequence (SEQ ID NO.7)
(7) F36V-FKBP nucleic acid artificial sequence (SEQ ID NO.8)
(8) Self-cleavage polypeptide T2A nucleic acid artificial sequence (SEQ ID NO.9)
(9) CD3 zeta intracellular nucleic acid artificial sequence (SEQ ID NO.10)
The whole expression frame is synthesized by the nucleic acid artificial sequence of Leader, the nucleic acid artificial sequence of Anti-MUC16, the nucleic acid artificial sequence of CD8Hinge region, the nucleic acid artificial sequence of CD8 transmembrane region, the nucleic acid artificial sequence of CD137, the nucleic acid artificial sequence of Linker1, the nucleic acid artificial sequence of F36V-FKBP, the nucleic acid artificial sequence of self-cleaving polypeptide T2A, the nucleic acid artificial sequence of F36V-FKBP, the nucleic acid artificial sequence of Linker2, the nucleic acid artificial sequence of CD3 zeta, the nucleic acid artificial sequence of the living organism engineering (Shanghai) Limited company, the pLent-C-GFP vector (Invitrogen) NotI-AsiSI site is inserted (see figure 3), the pLegli (5 α) is transformed, and after the sequencing is correct, the plasmid is extracted and purified by using the plasmid purification kit of the Qiagen company to obtain the high-quality plasmid of the recombinant expression vector.
2. Lentivirus packaging and titer detection
The lentivirus packaging cell line 293T was seeded in 10cm petri dishes containing DMEM + 10% FBS at 37 ℃ in 5% CO2And (5) culturing under the condition, and performing transfection after the anchorage rate is 70-80%. The recombinant plasmid and the no-load plasmid are co-transfected with the slow virus packaging plasmid through a calcium phosphate transfection method to 293T cells, and the reference molecule cloning is carried out through a specific method. After 24h after transfection, cells are obviously enlarged and spherical, cell nucleuses are enlarged and become round, and the wall-attaching capability is reduced and the cells are easy to fall off. After 48h, the expression of green fluorescent protein in the cells was observed under an inverted fluorescence microscope. After 72h, the supernatant was collected, filtered to remove bacteria, and stored in a low temperature freezer at-80 ℃ for further use. The virus titer is determined according to a Lenti-XTMGo StixTM kit (product of Beijing Huaxia ocean technologies Co., Ltd.), and the result shows that the titer of the recombinant lentivirus is 2.69 multiplied by 106pfu/mL。
Example 2 Lentiviral infection of CIK cells
Preparation of CIK cells
After inducing culture with a medium (purchased from CORNING, 88-551-CM) containing 1000IU/ml of recombinant interferon α 2a (purchased from sheng yang sansheng) for 24 hours, 1000IU/ml of recombinant interleukin 2 (purchased from sheng yang sansheng, pharmaceutics), 50ng/ml of OKT-3 and 5% of autologous plasma of the patient were added for further culture for 24 hours, and the solution was added every two days until day 14, and flow cytometry was performed to detect the positive expression rate of CD3+, CD56+ in CIK cells (CD3-FITC, CD16/CD56-PE antibody purchased from BECKMAN, a07735), CD3+ positive rate > 80%, CD3+ CD56+ double positive rate > 20%, and the CIK was considered to be successfully induced and the virus was left for infection.
2. Lentiviral-infected CIK (cytokine induced killer) cells and amplification culture of infected CIK cells
CIK cells were infected with recombinant and empty lentivirus as described above at MOI ═ 5, respectively. Infected cells at 37 ℃ with 5% CO2Culturing for 8 hr in incubator, collecting cells, adding virus solution, centrifuging at 1000g and 32 deg.C for 90 min, and centrifuging at 37 deg.C and 5% CO2And continuously culturing in the incubator, and repeatedly performing multiple infections in such a way, so that the infection efficiency of the CIK cells is improved. 2ml of culture supernatant was aspirated off, 2ml of fresh CORNING Medium was added, and the expansion culture was continued for 17 days until the cells were expanded to a sufficient amount. Chimeric antigen receptor expression was detected by FC500 flow cytometer (purchased from BECKMAN corporation) FL1 channel (fig. 4). The positive rate of the CIK cell infected by the recombinant lentivirus is 37.9 percent by taking the uninfected CIK lymphocyte as a negative control.
After the anti-MUC16 safe chimeric antigen receptor is transferred into a T cell and translated into protein, the T2A can lead the receptor to be split into two parts by self-cutting, wherein one part is an antigen binding region, and the other part is an intracellular signal transduction region.
Example 3: CAR (MUC16) -CIK cell killing activity study with chemically inducible elements
1. CAR (MUC16) -CIK cell in vitro killing activity study with chemical inducing element
The MUC16 positive ovarian cancer cell line OVCAR3 served as target cells, effector cells were CAR (MUC16) -CIK cells and empty lentivirus infected CIK.
Inoculation with 100. mu.l of 1X 105Target cells per well OVCAR3 to 96-well cell culture plates, effector cells, CAR-CIK cells, were added at a 1:2 effector-target ratio and AP1903 was added at final concentrations of 0nM, 50nM, 100nM, 200nM, 400nM, and effector and target cell controls were set. Placing in 5% CO2Adding 20 mu L of CCK-8 into each well after culturing in an incubator at 37 ℃ for 24h, continuously incubating for 2h, detecting by using an enzyme-linked immunosorbent assay, reading the OD value at the wavelength of 450nm, and reading the killing rate which is [1- (the OD value of the experimental group-the OD value of the effector cell control group)/the OD value of the target cell control group ═ the OD value of the experimental group]X 100%. CIK was infected with empty lentivirus as a control (see FIG. 5 for results). The killing efficiency of CAR (MUC16) -CIK cells on MUC16 positive ovarian cancer cell line OVCAR3 is increased along with the increase of the concentration of AP1903, and the killing rate reaches 90% at the final concentration of AP1903 of 400nM so as to achieve the effect of preventing and treating the ovarian cancer cell line OVCAR in vitro-mediated immune systemThe above step (1); the results show that the CAR (MUC16) -CIK cells designed by the invention have high killing effect on OVCAR3 cells, and the activity is regulated by AP 1903.
2. CAR (MUC16) -CIK cell in vivo killing activity study with chemically inducible elements
18-22g female KM mice (purchased from Guangzhou university of traditional Chinese medicine) were housed in an animal house (room temperature 23 + -2 deg.C, humidity 50% + -10%), logarithmic phase OVCAR3 cells were collected and diluted to 2X 10 with Phosphate Buffered Saline (PBS)5one/mL. Under aseptic conditions, the left axilla of the mice was inoculated with 0.2mL OVCAR3 cell suspension, observed for 3-5d, and the axilla was treated to develop hard rice-sized nodules as a criterion for successful modeling.
KM ovarian cancer transplantation tumor model mouse (the size of subcutaneous tumor tissue block measured by vernier caliper is 90-100 mm)3)
Treatment experiments with injections were started by randomized division into 5 groups of 20 individuals each. The experimental groups were respectively:
a. in the control group, the tail part of the patient is injected with normal saline with the same volume, and the patient is continuously observed for 14 d;
b. one treatment group, tail vein injection 2X 106Individual cells/CAR (MUC16) -CIK cells, injected 2 days after the first injection with a second isodose, and observed for 14 days;
c. two groups of treatment, tail intravenous injection 2X 106One cell/CAR (MUC16) -CIK cell, injected 2 days after the first injection with a second isodose, and 2h after each treatment injection, the tumor was injected locally with 0.1mg/kg AP1903 for 14 days;
d. three groups of treatment, tail intravenous injection 2X 106One cell/CAR (MUC16) -CIK cell, a second isodose injection 2 days after the first injection and 2h after each treatment injection, a local injection of 0.5mg/kg AP1903 into the tumor, with 14d observation continued.
e. Four groups of treatment, tail intravenous injection 2X 106One cell/CAR (MUC16) -CIK cell, a second isodose injection 2 days after the first injection and 2h after each treatment injection, the tumor was locally injected with 1mg/kg AP1903 for 14 d.
Body weight was measured 1 time every 3 days. The experiment was performed over a 14 day period. After the experiment was completed, the mice were dissected to isolate tumors and weighed (see figure 6 for results).
Injection of CAR (MUC16) -CIK cells followed by AP1903, the growth of mouse tumors was inhibited, and the extent of inhibition was directly proportional to the concentration of AP 1903. Without the injection of AP1903 after the injection of CAR (MUC16) -CIK cells, tumor masses became larger and grew in a trend identical to that of the non-injected CAR (MUC16) -CIK.
Conclusion the CAR (MUC16) -CIK cell designed by the invention has obvious inhibition effect on the growth of KM mouse tumor, and the action strength, time and position of the CAR (MUC16) -CIK cell can be accurately regulated and controlled by the AP1903 drug, so that the toxic and side effects of the CAR (MUC16) -CIK cell are avoided.
Under the action of a chemical inducer AP1903, the anti-MUC16 safe chimeric antigen receptor dimerizes F36V-FKBP, the split recombinant chimeric antigen receptor recombines, and after the complete receptor is combined with a specific antigen, ITAM phosphorylation activation downstream protein can be caused to activate and proliferate T cells, so that the opening effect is realized; without the action of a chemical inducer, the dividing recombinant chimeric antigen receptor cannot activate and proliferate T cells to realize the 'off' effect.
Example 4: CAR (MUC16) -CIK cell clinical application method
Can be used for treating patients with MUC16 positive tumor by local tumor injection and intravenous infusion of 2 × 106And (3) CAR (MUC16) -CIK cells. AP1903(0.01-100mg/kg) is injected into local tumor 2h after the treatment injection.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Shandonghui Biotechnology Ltd
<120> immune cell modified by anti-MUC16 safe chimeric antigen receptor and application thereof
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Claims (6)
1. An immune cell modified by an anti-MUC16 safe chimeric antigen receptor, which is characterized in that: the immune cell is modified by an anti-MUC16 safe chimeric receptor to obtain the anti-MUC16 safe chimeric antigen receptor modified immune cell, and the anti-MUC16 safe chimeric antigen receptor comprises a CD8leader, a MUC16 binding region, a hinge region of CD8, a transmembrane-stimulating domain, a CD3 zeta stimulating signal conduction region and a chemical induction element; the chemical inducing element is constructed based on the FK506binding protein 12-dimerization chemical inducing subsystem, and comprises two copies of mutant F36V-FKBP of FKBP12 and a T2A sequence inserted in the middle; the chemical-inducing element is located between the transmembrane-stimulating domain of the chimeric antigen receptor and the CD3 zeta stimulating signaling region.
2. The anti-MUC16 safe chimeric antigen receptor-modified immune cell of claim 1, wherein: the transmembrane-stimulating domain is selected from the group consisting of all or a partial fragment of one of CD8, CD27, CD28, CD137/4-1BB, CD134/OX40, ICOS molecules.
3. The anti-MUC16 safe chimeric antigen receptor-modified immune cell of claim 1, wherein: the immune cell is selected from one of autologous or transgenic T cells, NK cells, cytotoxic T lymphocytes, regulatory T cells, memory T cells, bispecific T cells and CIK cells.
4. The anti-MUC16 safe chimeric antigen receptor-modified immune cell of claim 1, wherein: the fusion gene segment of the anti-MUC16 safe chimeric antigen receptor is a fusion gene segment of CD8leader-scFv-CD8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 zeta, wherein the scFv is an anti-MUC16 single-chain fv antibody, CD8 is a hinge region, and CD137 is a transmembrane-stimulating domain.
5. The anti-MUC16 safe chimeric antigen receptor-modified immune cell of claim 4, wherein: the CD8 leader-CD 8-CD137-F36V-FKBP-T2A-F36V-FKBP-CD3 zeta fusion gene fragment is a nucleotide sequence shown in a sequence table SEQ ID NO. 1.
6. Use of the anti-MUC16 safe chimeric antigen receptor-modified immune cell of claim 1 for the preparation of a medicament for the treatment of ovarian cancer.
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| CN110526977A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of MUC1, Chimeric antigen receptor T cell and its preparation method and application |
| CN110526974A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | It is a kind of to target the single-chain antibody of MUC16, Chimeric antigen receptor T cell and its preparation method and application |
| CN110526991A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | Target Chimeric antigen receptor, the Chimeric antigen receptor T cell and its preparation method and application of FAP |
| CN110527667A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | A kind of Chimeric antigen receptor and Chimeric antigen receptor T cell and its preparation method and application targeting MUC16 |
| CN110526979A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of FAP |
| CN110526988A (en) * | 2018-05-25 | 2019-12-03 | 深圳宾德生物技术有限公司 | A kind of Chimeric antigen receptor and Chimeric antigen receptor T cell and its preparation method and application targeting MUC1 |
| CA3101641A1 (en) * | 2018-06-04 | 2019-12-12 | Intrexon Corporation | Muc16 specific chimeric antigen receptors and uses thereof |
| CN115819613B (en) * | 2022-05-10 | 2023-10-20 | 四川大学华西医院 | Preparation and application of chimeric antigen receptor immune cells constructed based on MSLN precursor protein |
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| WO2015179801A1 (en) * | 2014-05-23 | 2015-11-26 | University Of Florida Research Foundation, Inc. | Car based immunotherapy |
| AU2016312015A1 (en) * | 2015-08-21 | 2018-04-12 | Crage Medical Co., Limited | Fully human anti-mesothelin antibodies and immune effector cells targeting mesothelin |
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