CN107557392B - Preparation method and application of anti-EGFR safe chimeric antigen receptor modified immune cells - Google Patents
Preparation method and application of anti-EGFR safe chimeric antigen receptor modified immune cells Download PDFInfo
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- CN107557392B CN107557392B CN201710916242.9A CN201710916242A CN107557392B CN 107557392 B CN107557392 B CN 107557392B CN 201710916242 A CN201710916242 A CN 201710916242A CN 107557392 B CN107557392 B CN 107557392B
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Abstract
The invention discloses a preparation method and application of an anti-EGFR safe chimeric antigen receptor modified immune cell, which comprises the steps of synthesizing scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta nucleotides, inserting a fusion gene segment into a lentivirus expression vector, packaging the lentivirus carrying scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta coding genes, infecting monocyte-induced immune cells to obtain the anti-EGFR safe chimeric antigen receptor modified immune cell, improving the safety of the chimeric antigen receptor modified immune cell, controlling toxic and side effects and avoiding adverse reactions.
Description
Technical Field
The invention relates to the field of biological genes, in particular to a preparation method and application of an anti-EGFR safe chimeric antigen receptor modified immune cell.
Background
EGFR (epidermal growth factor receptor) is an expression product of the protooncogene c-erbB1, and is one of the epidermal growth factor receptor (HER) family members. EGFR is expressed on the surface of normal epithelial cells as a cell surface protein, and is expressed at abnormally high levels on the surface of many types of cancer cells, such as kidney, lung, prostate, pancreatic, breast, and the like. EGFR is involved in the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis and apoptosis. It can be seen that EGFR is closely related to various cancers and is one of the ideal targets for tumor targeted therapy and diagnosis.
Chimeric antigen receptor-modified T cells (CAR-T cells for short) have significant efficacy in the treatment of acute leukemia and non-hodgkin lymphoma, are considered to be one of the most promising tumor treatment modalities, and show good application prospects in the treatment of other solid tumors, such as successful treatment of pancreatic cancer by using CAR-T constructed by using mesothelin as a target. However, in clinical application, CAR-T cells may cause false attack on normal tissue cells or cause toxic and side effects such as cytokine storm due to massive release of inflammatory cytokines.
Therefore, how to effectively control the expression level of the CAR-T cells to improve the therapeutic safety of the CAR-T cells is a big problem in current clinical application while ensuring that the CAR-T cells have good therapeutic effect.
Disclosure of Invention
In order to solve the potential safety hazard, the invention provides an anti-EGFR safe chimeric antigen receptor modified immune cell, so that the CAR modified CIK cell expresses a safe molecular switch in addition to the CAR expressing EGFR.
The scheme of the invention is as follows:
a preparation method of an anti-EGFR safe chimeric antigen receptor modified immune cell comprises the steps of synthesizing scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta nucleotides, inserting the synthesized scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta fusion gene segment into a lentivirus expression vector, packaging the lentivirus carrying the encoding gene of scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta, and infecting the monocyte induced immune cell carrying the encoding gene of scFv (EGFR) -CD8-CD137-CD3 zeta-T2-Leader-CD 20-CD8-CD3 zeta to obtain the anti-EGFR safe chimeric antigen receptor modified immune cell.
In a preferred embodiment, the nucleotides scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta are synthesized, wherein the CD20 and the chimeric antigen receptor gene against EGFR are integrated into the genome separately as two independent expression cassettes.
Preferably, the immune cell is one of a CIK cell, a T cell, an NK cell, a cytotoxic T lymphocyte, a regulatory T cell, and a memory T cell.
Preferably, the immune cells are CIK lymphocytes in CIK cells.
As a preferred technical scheme, the fusion gene fragment of scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta is a nucleotide sequence shown in a sequence table SEQ ID NO. 1.
The invention also discloses a medicament for treating cancer, which contains anti-EGFR safe chimeric antigen receptor modified immune cells.
The anti-EGFR safe chimeric antigen receptor modified immune cell is used for preparing a medicine for treating malignant tumor.
The preferred technical scheme is as follows: the malignant tumor is renal cancer, lung cancer, prostatic cancer, pancreatic cancer, and breast cancer.
By adopting the technical scheme, the preparation method of the immune cell modified by the anti-EGFR safe chimeric antigen receptor comprises the steps of synthesizing scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta nucleotide, inserting the synthesized scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta fusion gene segment into a lentivirus expression vector, packaging into lentivirus carrying the encoding gene of scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta, and inducing the immune cell infected by the EGFR by the lentivirus carrying the encoding gene of scFv (EGFR) -CD8-CD137-CD3 zeta-T2 zeta 2A-Leader-CD20-CD8-CD3 zeta), obtaining the immune cell modified by the anti-EGFR safe chimeric antigen receptor.
The invention has the advantages that:
the anti-EGFR safe chimeric antigen receptor modified immune cell is added with a safe 'switch' CD20 inducible suicide region, so that the toxic and side effects of CAR-CIK treatment can be effectively controlled in time, the safety is improved, the toxic and side effects are controlled, and the adverse reaction is avoided.
According to the anti-EGFR safety type chimeric antigen receptor, the active 'switch' element is formed by the interaction of an anti-CD 20 monoclonal antibody rituximab antibody and CD20, so that the aim of removing CAR modified CIK cells is fulfilled by mediating cytotoxicity (ADCC) and complement mediated cytotoxicity (CDC), namely, adverse reactions possibly caused by CAR modified CIK cells are prevented and effectively controlled by intravenous injection of rituximab.
Drawings
FIG. 1 is a diagram of the design of the fusion gene fragment of the chimeric antigen receptor scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta;
FIG. 2 is a schematic representation of the lentiviral pLent-scFv (EGFR) -T2A-CD20 expression plasmid of the present invention;
FIG. 3 is a CAR-expressing positive rate for CAR-CIK cells of the invention;
FIG. 4 is a graph of EGFR tumor growth following CAR-CIK inhibition and rituximab addition in accordance with the present invention.
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.
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used are not indicated by the manufacturer and are commercially available.
Example 1 insertion of the fusion Gene fragment scFv (EGFR) -CD8-CD137-CD3 ζ -T2A-Leader-CD20-CD8-CD3 ζ into the Lentiviral expression vector pLent-C-GFP
The CAR module of Anti-EGFR is schematically shown in FIG. 1 (nucleic acid sequence shown in appendix SEQ ID NO. 1).
CAR (sequence of modules) of Anti-EGFR (epidermal growth factor receptor)
(1) CD20 nucleic acid artificial sequence (SEQ ID NO.2)
(2) Linker nucleic acid artificial sequence (SEQ ID NO.3)
(3) T2A nucleic acid artificial sequence (SEQ ID NO.4)
(4) CAR Leader sequence (Leader) nucleic acid Artificial sequence (SEQ ID NO.5)
(5) Anti-EGFR single chain Fv antibody nucleic acid artificial sequence (SEQ ID NO.6)
(6) CD8Hinge region nucleic acid artificial sequence (SEQ ID NO.7)
(7) CD8 transmembrane region nucleic acid artificial sequence (SEQ ID NO.8)
(8) CD137 intracellular region nucleic acid artificial sequence (SEQ ID NO.9)
(9) CD3 zeta intracellular nucleic acid artificial sequence (SEQ ID NO.10)
The whole expression cassette was synthesized by committee biotechnology (Shanghai) Co., Ltd in the order of fusion gene fragment scFv (EGFR) -CD8-CD137-CD3 ζ -T2A-Leader-CD20-CD8-CD3 ζ, respectively, inserted into pLent-C-GFP vector (Invitrogen) NotI-AsiSI site (see FIG. 2), transformed into E.coli Top10, identified by bacterial liquid PCR, and plasmid was extracted using a plasmid extraction kit (purchased from Omega Co., Ltd.), and after the plasmid was correctly sequenced, the recombinant plasmid having the correct sequencing result was named as pLent-scFv (EGFR) -T2A-CD 20.
Example 2 Lentiviral packaging, Virus titer detection
Cell line 293T was seeded in 10cm dishes containing DMEM + 10% FBS at 37 ℃ in 5% CO2Culturing under the condition, and using the cultured lentivirus to transfect after the adherence rate is 70-80%. The recombinant plasmid pLent-scFv (EGFR) -T2A-CD20 and the unloaded plasmid pLent-C-GFP are respectively cotransfected with 293T cells by a calcium phosphate transfection method with a slow virus packaging plasmid, and the reference molecule is cloned by a specific method. After 24h after transfection, cells were significantly increasedBig and spherical, and easy to fall off due to reduced adherence ability. After 48h, the expression of green fluorescent protein in the cells was observed under an inverted fluorescence microscope. After 72h, the supernatant was collected into an EP tube, centrifuged at 2000g for 10min, transferred to a new EP tube, filtered through a 4.5 μm filter and stored at-80 ℃. According to Lenti-XTMGo StixTMThe kit (product of Beijing Huaxia ocean technology Co., Ltd.) determines the virus titer, and the result shows that the titer of the empty vector virus is 3.28 × 106pfu/mL, titer of recombinant lentivirus 3.08 × 106pfu/mL。
Example 3 amplification culture of Lentiviral-infected CIK cells and post-infection CIK cells
And (2) infecting the CIK cells by using the recombinant lentivirus, culturing the infected cells in a 37 ℃ and 5% CO2 culture box for 8 hours, collecting the cells, adding the virus solution again, centrifuging for 90 minutes, continuously culturing in a 37 ℃ and 5% CO2 culture box, and repeating multiple infection in the way to improve the infection efficiency of the CIK cells. 2ml of culture supernatant was aspirated off, 2ml of fresh 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 FLTC (isothiocyanate) channel of FC500 flow cytometer. The CIK cells infected by the recombinant lentivirus have a positive rate of 29.0% by taking uninfected CIK lymphocytes as a negative control (FIG. 3).
Example 4: CAR-CIK cell killing activity study with safety 'switch' system
1. Control of CAR-CIK cell Activity by a safety "switch" System
CAR-CIK cells were plated at a density of 1 × 105Inoculating 100ul of the strain/ml into a 96-well plate, and culturing in an incubator at 37 ℃ and 5% CO2 for 24 h; 10ng rituximab antibody (Shanghai Roche pharmaceutical Co., Ltd.) is added for culturing for 24h, 20 mu L CCK-8 is added into each well, and after incubation is continued for 2h, CAR-T cells without rituximab are used as negative control, and detection is carried out by using an enzyme labeling instrument. The death rate of CIK cells is [1- (OD value of rituximab group-blank control group OD value)/(OD value of non-rituximab group-blank control group OD value ]]× 100%, the death rate of CIK cells is 30.12%, the results show that the CAR-CIK cells designed by the invention have favorable activityThe control of the Tuximab and the safe 'switching' system can effectively control the activity of the CAR-CIK cells.
Analysis of killing Effect of CAR-CIK cells on EGFR-positive tumor cells
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%), and logarithmic phase lung cell line A549 cells were collected and diluted to 2 × 10 with Phosphate Buffered Saline (PBS)5one/mL. Under the aseptic condition, 0.2mL of lung cell line A549 cell suspension is inoculated to the left armpit of the mouse, observation is carried out for 3-5d, and the standard for successful modeling is used when a nodule with hard rice grain size appears in the armpit.
KM Lung 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 4 groups of 20 individuals each. The experimental groups were respectively:
a. in the control group, the tail part is injected with normal saline with the same volume;
b. treatment group, tail vein injection 2 × 106Each CAR-CIK cell is injected 7 days after the first injection for the second same dose;
c. treatment group, tail intravenous injection 2 × 106Each cell/CAR-CIK cell was injected with the same dose 7 days after the first injection, and 12h after the first treatment injection, rituximab antibody (10mg/kg) was injected intravenously every tail of the day for 14 days;
d. three groups of treatment, tail intravenous injection 2 × 106Each cell/CAR-CIK cell was injected with a second identical dose 7 days after the first injection, and 12h after the second treatment injection, rituximab (10mg/kg) was injected intravenously in the tail part of the day for a total of 14 days.
The subcutaneous tumor tissue mass of each experimental group of mice was measured by a vernier caliper every day and recorded, and the tumor growth curve was plotted using the mass mean, with the results shown in fig. 4. On day 3 after CAR-T cell injection, tumors began to diminish in 50% of mice, and at day 14, tumors were barely palpable in 80% of mice. The effect of CAR-T on tumors was almost completely inhibited after rituximab injection, suggesting that rituximab can eliminate CAR-T cells, preventing collateral damage to normal tissues and organs. The result shows that the CAR-CIK cells designed by the invention have obvious inhibition effect on the growth of mouse tumor, and the activity is controlled by a rituximab system, which shows that the safe 'switch' CD20 suicide inducible region is added, so that the toxic and side effects of CAR-CIK treatment can be effectively controlled in time.
Example 5: the clinical application method of the CAR-T cells comprises the following steps:
for the treatment of EGFR tumor patients, the treatment method is local tumor injection and intravenous infusion 2 × 106Individual CAR-T cells, weekly, were immunized continuously for two weeks. If adverse reactions occur, rituximab (0.2-10mg/kg) is injected into local tumors and is infused back into veins.
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> preparation method and application of anti-EGFR safe chimeric antigen receptor modified immune cell
<130>2017
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<400>9
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210>10
<211>336
<212>DNA
<213> ethnic species (Homo sapiens)
<400>10
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
Claims (6)
1. A preparation method of anti-EGFR safe chimeric antigen receptor modified immune cells is characterized in that: synthesizing scFv (EGFR) -CD8-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta nucleotide, inserting the synthesized scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta fusion gene segment into a lentivirus expression vector, packaging the lentivirus carrying scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta coding gene, infecting the lentivirus carrying scFv (EGFR) -CD8-CD137-CD3 zeta-T2 zeta-2A-Leader-CD 20-CD8-CD3 zeta coding gene with monocyte-induced immune cells to obtain anti-EGFR chimeric antigen receptor modified safe immune cells; the fusion gene segment of scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader-CD 20-CD8-CD3 zeta is a nucleotide shown in a sequence table SEQ ID NO. 1;
the sequence of the scFv (EGFR) is a nucleotide sequence shown in SEQ ID NO. 6;
the sequence of the CD20 is a nucleotide sequence shown in SEQ ID NO. 2.
2. The method of claim 1, wherein the anti-EGFR safe chimeric antigen receptor modified immune cell is prepared by the following steps: according to the fusion gene fragment scFv (EGFR) -CD8-CD137-CD3 zeta-T2A-Leader
The sequence of CD20-CD8-CD3 ζ was synthesized as a whole expression cassette and integrated into the genome.
3. The method of claim 1, wherein the anti-EGFR safe chimeric antigen receptor modified immune cell is prepared by the following steps: the immune cell is one of CIK cell, T cell and NK cell.
4. A medicament for treating cancer, comprising: an immune cell comprising a modification of the anti-EGFR safe chimeric antigen receptor of claim 1.
5. Use of the anti-EGFR safe chimeric antigen receptor modified immune cell of claim 1 for the preparation of a medicament for the treatment of a malignant tumor.
6. The use according to claim 5 for the preparation of a medicament for the treatment of malignant tumors, wherein: the malignant tumor is renal cancer, lung cancer, prostatic cancer, pancreatic cancer, and breast cancer.
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| CN105861531A (en) * | 2016-04-21 | 2016-08-17 | 汪治宇 | Chimeric antigen receptor T cell and preparation method thereof |
| CN106636003A (en) * | 2017-01-24 | 2017-05-10 | 北京普瑞金科技有限公司 | Completely humanized EGFRvIII chimeric antigen receptor T cell and preparation method thereof |
| WO2017084421A1 (en) * | 2015-11-20 | 2017-05-26 | 上海细胞治疗研究院 | Efficient killing initiated mechanism containing tumour precise t-cell and use thereof |
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