CN105925536B - T lymphocyte modified by Trop2 chimeric antigen receptor and application thereof - Google Patents
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Abstract
The invention discloses a T lymphocyte modified by a Trop2 specific chimeric antigen receptor, wherein the surface of the T lymphocyte expresses a Trop2 specific chimeric antigen receptor, and the Trop2 specific chimeric antigen receptor is formed by connecting a human anti-Trop 2 single-chain antibody, CH2CH3 of an FC (immunoglobulin G) region of human IgG1, an intracellular region of CD28, an intracellular region of CD137 and an intracellular region of CD3 zeta in series. The invention also discloses an amino acid sequence and a nucleic acid sequence of the Trop2 specific chimeric antigen receptor. The invention also discloses application of the T lymphocyte in preparing an anti-tumor medicament.
Description
Technical Field
The invention relates to the technical field of cellular immunity, in particular to a T lymphocyte modified by a Trop2 specific chimeric antigen receptor and application thereof.
Background
T lymphocyte-mediated specific immune responses against tumors play an important role in the response of the body to eliminate tumor cells. However, the killing of tumor cells by T cells is often limited due to factors such as host immune tolerance and the local immunosuppressive microenvironment of the tumor. Researches find that a T cell technology modified by a Chimeric Antigen Receptor (CAR) can effectively improve the proliferation capability and survival time of T lymphocytes in vivo and the targeted killing effect on tumors, and becomes one of the recent popular cellular immunotherapy technologies. The CAR comprises an extracellular region, i.e., a specific receptor for a tumor antigen, a transmembrane region, and an intracellular costimulatory signal region. With the evolution of intracellular costimulatory signal regions, CARs were divided into five generations: the first generation CARs contained only one intracellular signaling molecule, CD3 ζ or fcepsilon RI γ. Second generation CARs added a co-stimulatory factor; third generation CARs contain two co-stimulatory factors; and fourth generation CARs introduce cytokines, suicide genes, etc.; the fifth generation CARs are now universal CARs that knock out T lymphocyte TCRs and CD 52.
At present, adoptive immunotherapy for tumors mainly comprises NK cells, DC-CIK, genetically modified T cells and the like, wherein the clinical application of the adoptive immunotherapy for tumors is limited by the treatment methods of DC-CIK, NK and the like due to the difficult in-vitro preparation, the unsatisfactory tumor killing effect and the like. The genetic modification technology for modifying T lymphocytes mainly comprises a TCR transgenic technology and a CAR modified T Cell (CART) technology. The TCR transgenic technology is to integrate a tumor-specific TCR gene into a T cell via a vector, thereby allowing the receptor T cell to express the tumor-specific TCR and obtain antigen specificity. However, TCR-modified T cells are MHC-restricted in their function and only recognize proteinaceous antigens, and have no effect on carbohydrate and glycolipid antigens. In addition, transgenic TCRs may be potentially at risk for mismatches with the TCR genes endogenously produced by T cells. CAR-modified T cells express CAR molecules that specifically kill tumor cells by CAR molecules recognizing associated tumor surface antigens. Such engineered T cells are not subject to major histocompatibility complex limitations (MHC) in addition to better targeting, greater proliferation, killing and longer survival, and are able to target proteins, sugars and glycolipids.
Human trophoblast cell surface antigen 2 (Trop-2) is a single transmembrane surface glycoprotein, is not expressed or is low expressed in normal human tissues, but is found to be over-expressed in various epithelial cancers, such as cervical cancer, breast cancer, ovarian cancer, prostate cancer and the like, is a tumor-associated antigen closely related to the invasive behaviors of various tumors, has the effect of promoting the invasion and metastasis of tumor cells, and can be used as a target for targeted therapy of a plurality of malignant tumors.
Disclosure of Invention
Based on the technical problems in the prior art, the invention aims to provide a Trop 2-specific chimeric antigen receptor modified T lymphocyte.
The invention also aims to provide an amino acid sequence and a nucleic acid sequence of the Trop2 specific chimeric antigen receptor.
The invention also aims to provide application of the Trop2 specific chimeric antigen receptor modified T lymphocyte.
In order to achieve the aim, the invention provides a Trop 2-specific chimeric antigen receptor modified T lymphocyte, which expresses a Trop 2-specific chimeric antigen receptor on the surface.
Preferably, the Trop 2-specific chimeric antigen receptor is composed of a human anti-Trop 2 single-chain antibody, a CH2CH3 region of the FC region of a human IgG1 molecule, an intracellular region of CD28, an intracellular region of CD137, and an intracellular region of CD3 zeta in tandem.
Preferably, the amino acid sequence of the Trop2 specific chimeric antigen receptor is shown as SEQ ID NO. 1.
Preferably, the nucleic acid sequence of the Trop2 specific chimeric antigen receptor is shown as SEQ ID NO. 2.
Preferably, the amino acid sequence of the human anti-Trop 2 single-chain antibody is shown in SEQ ID NO. 3.
Preferably, the nucleic acid sequence of the human anti-Trop 2 single-chain antibody is shown as SEQ ID NO. 4.
Preferably, a connecting peptide is arranged between the heavy chain molecule and the light chain molecule of the human anti-Trop 2 single-chain antibody, and the amino acid sequence of the connecting peptide is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser.
Preferably, the structure formed by connecting CH2CH3 of the FC region, the intracellular region of CD28, the intracellular region of CD137 and the intracellular region of CD3 zeta of the human IgG1 molecule in series is a signaling domain, and the amino acid sequence of the signaling domain is shown as SEQ ID NO. 5.
Preferably, the nucleic acid sequence of the signaling domain is as set forth in SEQ ID NO. 6.
The application of the T lymphocyte modified by the Trop2 specific chimeric antigen receptor in preparing antitumor drugs is provided.
According to the invention, a Trop2 antibody Fab is screened from a fully humanized antibody library in a laboratory, a signal peptide is added at the 5' end through codon optimization, a chimeric antigen receptor Trop2-ScFv-CH2CH3-CD28-CD3 zeta is synthesized through a whole gene, and a synthesized ScFv sequence is cloned into a retrovirus vector to construct an anti-human Trop2-CAR expression plasmid. And cloning the synthesized CD137 fragment into Trop2-ScFv-CH2CH3-CD28-CD3 zeta by Gateway recombinant cloning technology to obtain the chimeric antigen receptor Trop2-ScFv-CH2CH3-CD28-CD137-CD3 zeta.
The chimeric antigen receptor and a packaging plasmid RD114env of the retrovirus are used for packaging the virus in GP293T cells, and the retrovirus is used for infecting T lymphocytes so that the T cells express the chimeric antigen receptor. The obtained CART cell is co-cultured with tumor cells in vitro, the expression condition of the CART cell surface antigen is detected by flow cytometry, and the specific killing activity of the CART cell to the tumor cells is detected by a CCK8 method, so as to confirm the specific killing effect of the T lymphocyte modified by the chimeric antigen receptor to the tumor. Therefore, the chimeric antigen receptor Trop2-ScFv-CH2CH3-CD28-CD137-CD3 zeta can be applied to relevant tumor treatment.
The chimeric antigen receptor Trop2-ScFv-CH2CH3-CD28-CD137-CD3 zeta provided by the invention adopts a retrovirus technology, human T lymphocytes can be infected in vitro, the obtained CART cells can specifically recognize tumor cells expressing Trop2 through the single-chain antibody part of CAR, and simultaneously, the CART cells are activated to release various cytokines, so that the specific killing effect on the tumor cells is realized.
Drawings
FIG. 1 is an electrophoretogram of the sequence band of Trop2-scFv variable region obtained in example 1 of the present invention, in which 1 is a 500kD nucleic acid molecular weight standard, 2 is a part of Trop2-ScFv V kappa sequence, and 3 is a part of Trop2-ScFv VH sequence.
FIG. 2 is an electrophoretogram of a desired fragment of a Trop 2-specific chimeric antigen receptor obtained in example 1 of the present invention, wherein 1 is DL10000kD nucleic acid molecular weight standard, 2 is an uncut CAR vector, 3 is a retroviral vector released by the CAR vector cleaved with XbaI and Bam HI, and 4 is Trop2-ScFv-CH2CH3-CD28-CD137-CD3 ζ released by the CAR vector cleaved with XbaI and Bam HI. .
FIG. 3 shows the expression of the Trop 2-specific chimeric antigen receptor obtained by the invention after transfection of 293T cells by Western Blotting detection in example 2 of the invention, wherein 1 is 293T cells transfected with a vector of CD19-CAR, 2 is 293T cells transfected with a vector of Trop2-CAR, and 3 is 293T cells of a control.
FIG. 4 shows Western Blotting detection of Trop2 expression in tumor cells of different logarithmic growth phases, wherein 1 is HO8910 cell protein, 2 is SKOV3 cell protein, and 3 is A375 cell protein.
FIG. 5 shows the detection of the killing of tumor cells by the T cells modified by the Trop 2-specific chimeric antigen receptor obtained by the invention.
FIG. 6 shows the expression of IFN-. gamma.of the T cells modified by the Trop 2-specific chimeric antigen receptor of the invention after stimulation by the Trop2 antigen.
Detailed Description
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1: construction of Trop2 specific chimeric antigen receptor lentiviral vector
1. The amino acid sequences of the VH chain and the V kappa chain of the human Fab sequence of the anti-Trop 2 extracellular region screened by the laboratory phage display technology optimize the codon sequence by using OptimumGene gene (TM) gene design software, so that the amino acid sequence is more suitable for a human expression system under the condition of not changing the amino acid sequence. The ScFv part structure of the obtained chimeric antigen receptor is constructed by adding connecting peptide between VH and V kappa: vkappa- (Gly4Ser)3-VH (corresponding nucleic acid sequence is shown in SEQ ID NO.4), the fragment is cloned in pUC57 vector after synthesis, Xba I and Bam HI enzyme cutting sites are introduced at the junction, and the vector is named as: pUC57-Trop2-ScFv
The Trop2-scFv-pUC57 was digested with restriction enzymes NcoI and BamH I as follows: mu.g of trop2-scFv-pUC57, 1 mu.L of NcoI, 1 mu.L of BamH I and 2 mu.L of 10 Xenzyme digestion buffer solution, adding water to 20 mu.L, standing overnight in a water bath at 37 ℃, electrophoresing the enzyme digestion product with 1% agarose gel, cutting the target band under ultraviolet, and recovering the target fragment by using a DNA gel recovery kit (AXYGEN company); as a result, as shown in FIG. 1, the pUC57-Trop2-ScFv vector was cleaved with NcoI and BamH I to release the desired band.
The pSFG-CH2CH3-CD28-CD3 zeta vector was digested with NcoI and BamH I in the same manner, and the desired fragment was recovered by agarose gel electrophoresis, and the recovered Trop2-ScFv was ligated to the digested vector with T4DNA ligase in the reaction system of 2. mu.L of Trop2-ScFv, 2. mu.L of the vector digested product, 1. mu.L of 10 Xligation buffer, 1. mu.L of ligase, supplemented with water to 10. mu.L, overnight in a 16 ℃ water bath, and the ligation product was added to DH5 α competence [ see molecular cloning, third edition E.coli competence preparation (CaCl)2Method)]The cells were cultured overnight in a37 ℃ bacterial incubator. Picking single colony, enlarging culturing, extracting positive clone plasmid, enzyme cutting and sequencing to name the right carrier as Trop2-scFv-CH2CH3-CD28-CD3 zeta. .
3 cloning the sequence of CD137 between the CD28 and CD3 zeta molecules of Trop2-scFv-CH2CH3-CD28-CD3 zeta by gateway recombination technology, (technical method reference application No. 201310053109 chimeric antigen receptor and its use, published as 12.06.12.2013, published as 103145849A). After the plasmid construction was completed, Xba I and Bam HII were used for enzyme digestion identification, and the results are shown in FIG. 2. As can be seen from fig. 2: positive clones released the band of interest. And then carrying out sequencing verification, wherein the sequencing result is correct.
Example 2: trop2 specific chimeric antigen receptor expression identification
The extracted plasmid was transfected into human embryonic kidney cell 293T with PI transfection reagent after 48h, washed once with PBS, lysed with cell protein extraction Reagent (RIPA), the protein of the transfected 293T cell was separated by 10% SDS-PAGE, transferred to PVDF membrane at constant current (300mA, 1h), incubated with anti-CD 3 ζ (1: 1000) antibody, and incubated overnight at 4 ℃ with reference to the instructions in endotoxin-free plasmid macroextraction kit (Tiangen). After 3 washes with PBST, the cells were incubated with secondary HRP goat anti-mouse antibody (1: 5000) for 1h at room temperature. After color development by adding ECL, imaging was performed by using a ChemiDoc XRS System from Bio-Rad, and the results are shown in FIG. 3.
As can be seen from fig. 3: the recombinant plasmid constructed by the invention can detect the expression of a target band, the size of the target band is consistent with that of the male ginseng CD19, and the untransfected 293T cell has no band.
Example 3: preparation of Trop 2-specific chimeric antigen receptor-modified T lymphocytes
1. Packaging of anti-Trop 2 chimeric antigen receptor lentivirus
Extraction of retroviral packaging plasmid pRD114 and Trop2-scFv-CH2CH3-CD28-CD137-CD3 zeta retroviral plasmids in LB medium were cultured in bulk using the instructions in the endotoxin-free plasmid Tibet kit (Tiangen), and plasmids were extracted in bulk using the instructions in the endotoxin-free plasmid Tibet kit (Tiangen). The plasmid was co-transfected into GP-293T cells, and cell supernatants were collected 48h after transfection and centrifuged at 4000rpm for 10 min. Collecting supernatant, filtering with 0.45 μm filter membrane, and freezing at-80 deg.C.
Preparation of T lymphocytes
Fresh anticoagulated blood from 20mL healthy volunteers was collected and Peripheral Blood Mononuclear Cells (PBMC) were isolated using lymph separation medium (GE). The isolated cells were stimulated with CD3 and CD28 plates for 48h, and cultured in T lymphocyte medium GT-T551 (TAKARA) plus 1: 5000IL2 was subjected to induction culture to obtain T lymphocytes.
Preparation of CAR-T cells
Non-tissue culture plates 24-well plates were coated with 50. mu.g/mL of RetroNectin (TAKARA), 500. mu.L per well, overnight at 4 ℃. mu.L of virus supernatant was added to each well and incubated at 37 ℃ for 30 min. The viral supernatant was removed, 500. mu.L of viral supernatant was added, incubation was carried out at 37 ℃ for 30min, and 1.5mL of viral supernatant and 0.5mL of diluted T-lymphocytes were added to each well to remove viral supernatant. Thus obtaining Trop2-scFv-CH2CH3-CD28-CD137-CD3 zeta T cells, i.e. Trop2 specific CAR-T cells.
Example 4: killing effect of Trop2 specific CAR-T cells on Trop2 related tumors
Western Blotting detection of Trop2 expression in tumor cells
Ovarian cancer cells HO8910, ovarian cancer cells SKOV3 and melanoma cells A375 in logarithmic growth phase are selected, cell protein extraction Reagent (RIPA) is used for cell lysis, cell protein is extracted, WesternBlotting detection is carried out, and the detection result is shown in figure 4.
Killing detection of tumors by CAR-T cells
Adjusting the tumor cell culture medium to 5X 106mL, 50 μ L per well, as per E: t (effector cell to target cell ratio) is 16: 1. 8: 1. 4: 1. 2: 1, tumor cells are added in 2.5X 1061.25X 1066.25X 10 pieces53.125X 105A plurality of; collecting T cells and CAR-T cells after the cells are completely attached to the wall, adjusting the cell concentration to 1 × 10650. mu.L/well for 12 h. And (3) discarding the supernatant, adding 100 mu L of 10-fold diluted CCK8, incubating for 4-6 hours, and detecting the light absorption value of OD450 by using an enzyme-labeling instrument.
The tumor cells are ovarian cancer cells HO8910, ovarian cancer cells SKOV3 and melanoma cells A375.
The detection results are shown in fig. 5: the killing rate of CAR-T cells on the ovarian cancer cells HO8910 with high expression of Trop2 is higher than that of the ovarian cancer cells SKOV3 with low expression of Trop2, and the killing rate of the CAR-T cells on the ovarian cancer cells SKOV3 with low expression of Trop2 is higher than that of melanoma cells A375 with no detectable expression of Trop 2. In addition, the killing effect of the CAR-T cells on tumor cells is higher than that of T cells and higher than that of a pure culture medium.
ELISPOT detection of expression of IFN-gamma in CAR-T cells stimulated by Trop2 antigen
IFN-. gamma.antibody was diluted with sterile coating and added to an ELISPOT (Millipore, Cat. No. MAIPS4510) plate at 100. mu.L/well and left overnight at 4 ℃. The next day the plates were washed 2 times with sterile coating solution. Add 200. mu.L of complete medium to each well and block for 1h at room temperature. The serum-containing cell culture solution RPMI-1640 was washed off and washed three times with PBS. The Trop2 extracellular domain polypeptide was prepared, diluted in serum-containing cell culture medium RPMI-1640, and adjusted to a final concentration of 1. mu.g/ml. Each well was 100. mu.L. Adjusting the concentration of CAR-T cells to be detectedIs 1 × 106Per mL, 100. mu.L of 5% CO at 37 ℃ per well2After 24h, the cells and medium were discarded and washed 3 times with ELISPOT wash. Biotin-labeled detection antibody was added to each well at 100. mu.L/well and left overnight at 4 ℃. The plate was washed 4 times with ELISPOT wash. HRP-labeled avidin was added to each well at 100. mu.L/well and left at room temperature for 45 min. After washing 3 times with ELISPOT wash, the cells were washed 2 times with PBS. Adding 100 mu L of ACE chromogenic substrate into each hole, and developing for 20-60 min at room temperature. After the appearance of a distinct colony point, the reaction was terminated by 3 washes with sterile water. The plate was air dried and the number of spots formed was counted by an ELISPOT plate reader as shown in FIG. 6. As can be seen from fig. 6: the results show that the CAR-T cells can secrete IFN-gamma under the stimulation of the specific Trop2 antigen.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A Trop 2-specific chimeric antigen receptor-modified T lymphocyte, wherein the surface of the T lymphocyte expresses the Trop 2-specific chimeric antigen receptor;
wherein, the Trop2 specific chimeric antigen receptor is formed by connecting a human anti-Trop 2 single-chain antibody, a CH2CH3 of an FC region of a human IgG1 molecule, an intracellular region of CD28, an intracellular region of CD137 and an intracellular region of CD3 zeta in series;
wherein the amino acid sequence of the Trop2 specific chimeric antigen receptor is shown in SEQ ID NO. 1;
wherein the amino acid sequence of the human anti-Trop 2 single-chain antibody is shown in SEQ ID NO. 3;
wherein a connecting peptide is arranged between a heavy chain molecule and a light chain molecule of the human anti-Trop 2 single-chain antibody, and the amino acid sequence of the connecting peptide is Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser;
wherein, the structure formed by connecting CH2CH3 of the FC region of the human IgG1 molecule, the intracellular region of CD28, the intracellular region of CD137 and the intracellular region of CD3 zeta in series is a signal transduction domain, and the amino acid sequence of the signal transduction domain is shown as SEQ ID NO. 5.
2. The T lymphocyte of claim 1, wherein the nucleic acid sequence of the Trop 2-specific chimeric antigen receptor is shown as SEQ ID No. 2.
3. The T lymphocyte of claim 1 or 2, wherein the nucleic acid sequence of the human anti-Trop 2 single-chain antibody is shown as SEQ ID No. 4.
4. The T lymphocyte of claim 1, wherein the signaling domain has the nucleic acid sequence of seq id No. 6.
5. The use of the T lymphocyte of claim 1 in the preparation of a medicament for treating ovarian cancer and melanoma.
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| CN106399550A (en) * | 2016-11-04 | 2017-02-15 | 深圳市第二人民医院 | TaqMan real-time fluorescent quantitative PCR kit for detecting peripheral blood CAR (Chimeric Antigen Receptor)-T cells |
| US20180100016A1 (en) * | 2016-12-22 | 2018-04-12 | Xiaotong Song | Use of car-modified human natural killer cells to treat cancer |
| US11649294B2 (en) | 2017-11-14 | 2023-05-16 | GC Cell Corporation | Anti-HER2 antibody or antigen-binding fragment thereof, and chimeric antigen receptor comprising same |
| TW202016148A (en) * | 2018-07-09 | 2020-05-01 | 大陸商艾比瑪特生物醫藥（上海）有限公司 | Antibodies specific to trophoblast antigen 2 (trop2) |
| CN110824158A (en) * | 2018-08-07 | 2020-02-21 | 上海恒润达生生物科技有限公司 | Method for detecting secretion of CART cells aiming at target antigen cytokine |
| CN109748975B (en) * | 2019-03-05 | 2020-02-14 | 南京市第一医院 | Bispecific chimeric antigen receptor and application thereof |
| CN110317822B (en) * | 2019-07-19 | 2020-06-05 | 英威福赛生物技术有限公司 | TROP2 chimeric antigen receptor, T cell thereof, and preparation method and application thereof |
| WO2022216811A2 (en) * | 2021-04-08 | 2022-10-13 | Artiva Biotherapeutics, Inc. | Chimeric antigen receptor comprising an anti-cd19 antibody or antigen-binding fragment thereof and natural killer cells comprising the same |
| CN115057934A (en) * | 2021-12-02 | 2022-09-16 | 浙江博锐生物制药有限公司 | anti-Trop-2 antibodies and uses thereof |
| CN118420787B (en) * | 2024-07-05 | 2024-09-20 | 南京医科大学 | Immunopotentiating trop2 CAR_ smbIL21 chimeric antigen receptor and application thereof |
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