CN112522295A - Recombinant CAR gene targeting human EpCAM, vector thereof, CAR-T cell, preparation method and application thereof - Google Patents

Abstract

The invention discloses a recombinant CAR gene targeting human EpCAM, a vector thereof, a CAR-T cell and a preparation method and application thereof, wherein the CAR-T cell expresses a fusion protein of a humanized anti-EpCAM antibody ScFv-CD8-4-1BB-CD3 zeta in the T cell. In the EpCAMhScFv-CD8-4-1BB-CD3 zeta, the EpCAMhScFv can specifically recognize and bind to human EpCAM protein. A batch of CAR-T cells capable of specifically recognizing and killing tumor cells with high EpCAM expression are prepared by modifying and transforming lentivirus infected and activated T cells, can be applied to tumor treatment with high EpCAM expression, and provides a new method for preventing and treating the tumor cells.

Description

Recombinant CAR gene targeting human EpCAM, vector thereof, CAR-T cell, preparation method and application thereof

Technical Field

The invention belongs to the technical field of tumor immunotherapy, and relates to a human EpCAM-targeted recombinant CAR gene and a vector thereof, CAR-T cells and a preparation method and application thereof.

Background

Cancer is a great threat to human health and public health safety due to its high incidence and mortality. The commonly used treatment methods include surgical resection, radiotherapy, chemotherapy, etc., however, these methods have limited efficacy and are not suitable for all patients, and therefore, treatments such as tumor immunotherapy, etc. have come into play. The research of CAR-T (clinical antigen receptor) cell therapy is very popular, and it is to genetically modify and modify T cells through an exogenous artificially designed Chimeric Antigen Receptor (CAR), so that T cells express a new protein aiming at Tumor-specific antigen (TSA) or Tumor-associated antigen (TAA), thereby improving the killing ability of T cells to tumors. The structure of the CAR mainly comprises three parts, namely an extracellular antigen binding region, a transmembrane region and an intracellular signal region, and the CAR has no MHC restriction, so that the immune escape caused by the reduction of the MHC expression of tumor cells is avoided. The extracellular region is a ScFv (Single chain variable fragment) fragment; the transmembrane region connects the extracellular antigen-binding region and the intracellular signaling region, anchors the CAR structure to the cell membrane of the T cell, and is involved in dimerization of the CAR structure; the intracellular domain is the T cell activation domain CD3 zeta chain and the intracellular costimulatory domain, providing the first and second signals for T cell activation. The first generation of CAR intracellular domain has only CD3 zeta chain, contains three immunoreceptor tyrosine-based activation motifs (ITAMs), and can only transmit the first signal of T cell activation, thus the antitumor effect is very limited. Second and third generation CARs are CAR structures with the addition of one and two costimulatory molecules, such as CD28, 4-1BB, OX40, CD27, ICOS, etc., respectively, to the first generation CAR, with CD28 and 4-1BB being more commonly used to enhance the proliferation signal of CAR-T cells. CAR-T cell killing using 4-1BB as a co-stimulatory molecule was more durable and cell depletion was lower. Fourth generation CARs in turn increased cytokine or costimulatory molecule ligands, such as IL-12, 4-1BBL, and the like. CAR-T cell immunotherapy is an important approach to tumor immunotherapy and currently has dramatic success in hematological tumors.

Epithelial cell adhesion molecule (EpCAM) is a type i transmembrane protein with a molecular weight of about 40kd, mainly plays an adhesion role, participates in biological processes such as proliferation and migration of cells, is one of the most concerned tumor-associated antigens, and promotes the occurrence and development of various tumors. EpCAM is often highly expressed in primary tumors and metastases, especially adenocarcinoma, certain squamous cell carcinomas, and retinoblastoma. Integrated analysis based on RNA-seq data in the TCGA and GTEx databases showed that EpCAM is overexpressed in a variety of cancers and its overexpression correlates with poor prognosis and treatment anergy in patients. In addition, EpCAM is also considered as one of tumor stem cell markers of various tumors, and is widely applied to characterization of tumor stem cells together with CD44, CD117, CD133 and the like. EpCAM + breast cancer cells have the ability to self-renew and differentiate, and are more aggressive than EpCAM-cells. Clonogenic and tumorigenic capacity of ovarian cancer stem cells highly expressing EpCAM is enhanced and unresponsive to doxorubicin and cisplatin treatment. In addition, EpCAM +/CD44+ tumor stem cells in tissues of colorectal cancer patients are obviously related to high invasiveness and tumor grading of tumors, and the EpCAM is shown to play an important role in promoting tumorigenesis and development, influencing survival of patients and resisting chemotherapeutic drugs. The results show that EpCAM is a tumor-associated antigen and can be used as a good target for treating tumors.

Currently, there are many therapeutic strategies targeting EpCAM. The first monoclonal antibody Edreclomab targeting EpCAM aiming at human tumor; panorex; mAb17-1A is a murine IgG2a antibody approved in 1995 in germany for use as an adjunct therapy after surgical resection of primary colorectal cancer. The efficacy of other anti-EpCAM monoclonal antibodies, such as Catumaxomab, Solitomab and Adecatumab, is not satisfactory, but their potential for application in tumor immunotherapy remains non-negligible. Adecatumab can kill 27-66% of tumor cells of high-grade ovarian cancer cell lines, and when the Adecatumab is used in combination with IL-2, the killing rate of the tumor cells is increased. Catumaxomab is a bispecific, trifunctional antibody that targets both EpCAM on the surface of tumor cells and the human T-cell antigen CD3, and whose antibody Fc fragment also mediates ADCC effects, thereby inhibiting tumor growth. However, studies have shown that in malignant ascites of patients who have been intraperitoneally injected with Catumaxomab, there is soluble EpCAM (sEpCAM) which adheres to Catumaxomab and greatly reduces the efficacy of the drug, resulting in increased dosages. Furthermore, the efficacy of the T cells recruited by Catumaxomab in the tumor microenvironment or Fc fragment-bound NK cells remains to be studied because the expression of immune checkpoint molecules allows the tumor to escape from the surveillance of the immune system.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a recombinant CAR gene targeting human EpCAM, a vector thereof, CAR-T cells, a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

the invention discloses a recombinant CAR gene targeting human EpCAM, which is a fusion gene consisting of EpCAMhScFv, CD8, 4-1BB costimulatory factor and CD3 zeta T cell activation domain, and the nucleotide sequence of the recombinant CAR gene is shown in SEQ ID NO. 1; wherein:

the nucleotide sequence of the EpCAMhScFv is shown in SEQ ID NO. 2;

the nucleotide sequence of the CD8 is shown as SEQ ID NO. 3;

the nucleotide sequence of the 4-1BB co-stimulatory factor is shown as SEQ ID NO. 4;

the nucleotide sequence of the CD3 zeta T cell activation domain is shown as SEQ ID NO. 5.

The invention also discloses a recombinant CAR gene vector which is a nucleic acid construct, plasmid, lentivirus or retrovirus incorporating a recombinant CAR gene targeting human EpCAM as described above.

Preferably, the recombinant CAR gene vector consists of EpCAMhScFv, CD8, 4-1BB costimulatory factor and CD3 zeta T cell activation domain, and the amino acid sequence is shown as SEQ ID NO. 6; wherein:

the amino acid sequence of the EpCAMhScFv is shown in SEQ ID NO. 7;

the amino acid sequence of the CD8 is shown as SEQ ID NO. 8;

the amino acid sequence of the 4-1BB co-stimulation factor is shown in SEQ ID NO. 9;

the amino acid sequence of the CD3 zeta T cell activation domain is shown in SEQ ID NO. 10.

Preferably, the recombinant CAR gene vector is an Anti-EpCAMhScFv-CD8-4-1BB-CD3 zeta chimeric antigen receptor and consists of an Anti-EpCAM single-chain antibody heavy chain VH, a Linker, an Anti-EpCAM single-chain antibody light chain VL, a CD8 Hinge chimeric receptor Hinge region and transmembrane region, a 4-1BB co-stimulatory factor and a CD3 zeta T cell activation region; wherein:

the amino acid sequence of the Anti-EpCAM single-chain antibody heavy chain VH is shown in SEQ ID NO. 11;

the amino acid sequence of the Linker is shown as SEQ ID NO. 12;

the amino acid sequence of the Anti-EpCAM single-chain antibody light chain VL is shown in SEQ ID NO. 13.

The invention also discloses a human EpCAM-targeted CAR-T cell prepared by transforming a T cell with the above-described recombinant CAR gene targeting human EpCAM or with the above-described recombinant CAR gene vector.

The invention also discloses a preparation method of the human EpCAM-targeted CAR-T cell, which comprises the following steps:

1) synthesizing Anti-EpCAMhScFv-CD8-4-1BB-CD3 zeta chimeric antigen receptor gene;

2) constructing a lentivirus expression vector which is provided with an Anti-EpCAMhScFv-CD8-4-1BB-CD3 zeta chimeric antigen receptor gene;

3) transfecting 293T cells by using the lentivirus expression vector constructed in the step 2) to obtain high-titer lentiviruses;

4) infecting the T cells by using the high-titer lentiviruses obtained in the step 3) to obtain CAR-T cells targeting human EpCAM.

The invention also discloses an application of the human EpCAM-targeted recombinant CAR gene, the recombinant CAR gene vector or the human EpCAM-targeted CAR-T cell in preparation of a medicine or a kit for treating diseases with high EpCAM molecule expression.

The invention also discloses an anticancer agent, which contains the CAR-T cell targeting the human EpCAM and pharmaceutically acceptable additives.

Compared with the prior art, the invention has the following beneficial effects:

the invention constructs an Anti-EpCAM CAR gene on the basis of an antibody aiming at a tumor-associated antigen EpCAM, prepares an Anti-EpCAM CAR-T cell and provides a new treatment approach for tumors with high EpCAM expression. The CAR-T cells of the invention are CAR-T cells targeting the extracellular domain of human EpCAM, which CAR-T cells are capable of expressing the Anti-epcamhsfv-CD 8-4-1BB-CD3 zeta fusion protein in T cells. The ScFv of EpCAM is used for constructing CAR-T cells, wherein the ScFv sequence carries out codon optimization on the scFv gene fragment of murine origin, which is a previous research result of the applicant team, so that the ScFv gene fragment is closer to the scFv gene fragment obtained by the codon usage mode of human cells. And the CAR-T cell is utilized, and an EpCAM molecule is taken as a target point to kill tumor cells with high EpCAM expression.

The Anti-EpCAM CAR-T is obtained by modifying and transforming T cells through lentivirus infection, so that the T cells specifically recognize and kill tumor cells with high EpCAM expression, such as Luminal-A type breast cancer cells with high EpCAM expression, and a new treatment way is provided for corresponding tumors.

Drawings

FIG. 1 is a schematic diagram of a chimeric antigen receptor vector according to the present invention;

FIG. 2 is magnetic bead sorting CD3⁺Detecting a T cell ratio chart by a front-back flow method of T;

FIG. 3 is a graph showing the effect of CCK8 on the proliferation of T cells after activation with CD3/CD28 antibody;

FIG. 4 is a graph of the results of flow cytometry for Anti-EpCAM CAR-T and control lentivirus infection efficiency; wherein A is EPCAM-CAR; b is EPCAM-CON;

FIG. 5 shows the case of the CAR-T cells secreting the cytokine IFN-gamma at different effective target ratios measured by ELISA; wherein A is MCF-7; b is SK-BR-3; c is MDA-MB-231; d is Hela.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the accompanying drawings:

the first embodiment is as follows: lentiviral expression vector preparation

The chimeric antigen receptor vector is constructed by adopting a single-chain antibody (ScFv) fragment for recognizing and binding EpCAM antigen, and fusing the fragment with a Hinge region (Hinge) and a transmembrane region (TM) of a CD8 alpha gene, CD137(4-1BB), an intracellular signal region (ICP) of a CD3 zeta gene, a self-splicing polypeptide 2A gene and a Green Fluorescent Protein (GFP) gene in vitro, so as to construct a chimeric receptor gene Anti-EpCAMhScFv-CD8-4-1BB-CD3 zeta, wherein the gene sequence is shown as SEQ ID NO:1, and the upstream and the downstream are synthesized with restriction enzyme digestion BamHI sites, and the structural schematic diagram of the constructed chimeric antigen receptor vector is shown as FIG. 1, and the sequences are synthesized by Shanghai Jikai gene chemistry Limited. The sequence is cut by BamHI enzyme and is connected to the Shanghai Jikai gene GV401 vector; transforming DH5 alpha competent cells to obtain positive clones; and performing shake culture and plasmid extraction to finally obtain the GV401-Anti-EpCAM-CAR lentiviral expression vector.

Example two: lentiviral preparation

The lentivirus package adopts a three-plasmid system, and comprises a GV401-Anti-EpCAM-CAR lentivirus expression vector and two lentivirus package plasmids. Three kinds of plasmid DNA in a lentivirus packaging system are extracted by referring to the specification of a plasmid extraction kit of Qiagen company and used as lentivirus packaging, and the concentration and the purity of the extracted plasmid DNA are measured by adopting an ultraviolet absorption method. The day before transfection 293T cells were trypsinized and cell density adjusted, passaged in 10cm petri dishes at 37 ℃ in 5% CO₂And the incubator can be used for transfection when the cell density reaches 70-80%, and the old culture solution is discarded and replaced by a serum-free DMEM culture medium. Mixing the prepared three plasmid DNA solutions with lipofectamine 2000 transfection reagents with corresponding volumes according to the instructions, incubating for 15min at room temperature, slowly and uniformly dripping the mixed solution into a culture dish, uniformly mixing, and standingAt 37 ℃ with 5% CO₂Culturing in an incubator, removing the culture medium containing the mixed solution after 6h, slowly adding a DMEM culture medium containing 10% fetal calf serum, and continuously culturing for 48-72h to collect cell supernatant containing the target virus particles. Then lentivirus concentration and purification are carried out, namely, the collected cell supernatant is placed into a 50mL centrifuge tube and centrifuged at the temperature of 4 ℃ to remove cell debris, and the rotation speed is 4000rpm for 10 min. Filtering the supernatant with 0.45 μm filter, placing the filtrate in ultracentrifuge tube, centrifuging at 4 deg.C and 25000rpm for 2h with Beckman ultracentrifuge, discarding the supernatant, adding cell culture medium to resuspend the precipitate, centrifuging at 10000rpm for 5min, collecting the supernatant (i.e. concentrated and purified virus), packaging, and placing in-80 deg.C refrigerator for use.

Example three: Anti-EpCAM CAR-T cell preparation

1. Isolation of Peripheral Blood Mononuclear Cells (PBMC)

Separating PBMC by adopting a polysucrose-diatrizoate density gradient centrifugation method, taking anticoagulated whole blood of a healthy person from the department of blood transfusion of the Chijing hospital, adding PBS with equal proportion and room temperature balance for dilution, taking a centrifuge tube, adding a human lymphocyte separation liquid, slowly adding the diluted blood onto the liquid level of the separation liquid along the tube wall of the centrifuge tube, and forming a clear interface between the two liquid levels so that the volume ratio of the anticoagulated blood, the PBS and the separation liquid is 1:1: 1. Using a horizontal centrifuge, centrifugation was carried out at 2000rpm at room temperature for 20min, and the acceleration/deceleration of the centrifuge was adjusted to 0. After centrifugation, different types of cells are layered due to different specific gravities of the cells, four layers can be observed in total, the uppermost layer is a plasma layer, the second cloud layer is a leucocyte layer and contains lymphocytes and mononuclear cells (PBMC), the third layer is human lymphocyte separation liquid, and the lowermost layer is red blood cells and granulocytes with larger specific gravities. Carefully sucking the cloud layer cells into a new centrifuge tube, adding more than five times of PBS for dilution and washing, centrifuging at 1500rpm for 10min, discarding supernatant, and repeatedly washing for 1-2 times. After obtaining PBMC, the PBMC were cultured in an appropriate amount of RPMI1640 medium containing 10% fetal bovine serum or L500 lymphocyte serum-free medium, and added with 200IU/mL IL-2. PBMC cells can be collected, antibodies are labeled, and the distribution of cell subsets is detected and analyzed by flow cytometry.

2. Magnetic bead sorting CD3⁺T cells (negative selection)

PBMC were collected, washed with PBS, resuspended cells using T cell magnetic bead isolation buffer, counted and adjusted to a cell concentration of 1X 10⁸and/mL. At 1 × 10⁷For example, 100. mu.L of cell suspension was transferred to a new sterile flow tube, 10. mu.L of Biotin-Antibody Cocktail was added, mixed well and allowed to stand on ice for 15min to allow the Antibody to bind to cell surface markers. mu.L of Streptavidin Nanobeads (which need to be vortexed for multiple times at the maximum rotation speed before use) are added into the sample, mixed evenly, and placed on ice for 15min to enable the magnetic beads to be combined with the antibody. Adding 2.5mL of separation buffer solution, mixing uniformly, placing the sterile flow tube in a magnetic frame for 5min, taking up the magnetic frame, pouring out the supernatant, collecting the supernatant in a new 15mL sterile centrifuge tube, adding 2.5mL of separation buffer solution in the centrifuge tube containing magnetic beads again, and collecting the target cells once again. The cell pellet was washed with PBS, centrifuged at 1000rpm for 5min, and repeated once. Adding RPMI1640 culture medium containing 10% fetal bovine serum or L500 lymphocyte serum-free culture medium, and adding IL-2 at 200 IU/mL. CD3⁺The activation of T cells adopts a CD3/CD28 antibody activation method, CD3 and CD28 antibody solutions are added to coat a 24-well plate, the temperature is kept overnight at 4 ℃, the antibody solution is discarded, the 24-well plate is washed by sterile PBS for 3 times, purified CD3+ T cells are added, and the distribution condition of T cell subsets is detected and analyzed by flow cytometry after the T cells are activated by the antibodies for 3 days.

The results are shown in FIG. 2: before magnetic bead sorting, the T cell subset distribution in PBMC, CD3, was detected by flow cytometry⁺The cells only account for 45.5% of all lymphocytes, of which CD3⁺/CD4⁺Cells account for only 22.2% of all lymphocytes. Sorting of CD3 using magnetic beads⁺After the cells were activated, CD3 was detected by flow cytometry⁺The cell proportion reaches 86.0%, the purity is high, and the cell can be used for the next research, wherein the cell proportion is CD3⁺/CD4⁺The number of cells was 50.7%.

3. T cell proliferation-toxicity assay

Detecting T cell proliferation by CCK-8 method, preparing cell suspension in 96-well plate, adding IL-2 and CD3/CD28 antibody, and comparing with control groupAdding corresponding IL-2-containing medium, and standing at 37 deg.C and 5% CO₂After incubation in the cell incubator for a suitable period of time, 10. mu.L of CCK-8 solution was added dropwise to each well, incubation was continued in the incubator for 2-3h, and then the absorbance value of the solution in the plate at 450nm was measured using a microplate reader.

The results are shown in FIG. 3: the proliferation rate of the T cells stimulated by the antibody is obviously improved (P < 0.05).

4. Lentiviral infection and CAR-T cell phenotype detection

CD3 to be activated⁺Spreading T cells on 24-well plate, taking out frozen virus solution from-80 deg.C, adding into cell culture solution, adding transfection aid solution P, centrifuging at 1200rpm for 1.5 hr, placing the culture plate at 37 deg.C and 5% CO₂Normally culturing in an incubator. One week later, cells were harvested and observed under a fluorescent microscope for CD3 post viral infection⁺Green fluorescence of T cells was analyzed by flow cytometry to identify gene expression.

The results are shown in FIG. 4: the Anti-EpCAM CAR-T and control lentivirus infection efficiencies were approximately 20.2% and 28.9%.

Example four: Anti-EpCAM CAR-T cell in vitro cytokine secretion assay

And (3) evaluating the release effect of cytokines when the Anti-EpCAM CAR-T cells and the tumor cells MCF-7, SK-BR-3, MDA-MB-231 and HeLa cells are cultured together by an ELISA detection method, incubating the CAR-T cells and the target cells respectively, setting the effective target ratio to be 1:1, 2:1, 4:1, 8:1 and 16:1, setting three multiple wells in each group, and collecting culture supernatant after 24 h. Coating an ELISA pore plate with an antibody of a cytokine to be detected, adding a cell culture supernatant to be detected after closing, adding an enzyme-labeled secondary antibody resisting the cytokine after washing, washing for multiple times, adding a substrate for color development, finally terminating the reaction, detecting an absorbance value at 450nm, and calculating the level of the cytokine.

The results are shown in figure 5, and the CAR-T cells have obviously higher IFN-gamma secretion level and higher cytokine secretion level along with higher effective target ratio compared with a control group under the stimulation of the Luminal-A type breast cancer cell line MCF-7 highly expressing EpCAM molecules. Triplex milk with low EpCAM molecular expressionThe secretion level of IFN-gamma is increased to some extent under the stimulation of the adenocarcinoma cell line MDA-MB-231, but the secretion level of the cytokine is limited. Furthermore, the CAR-T is directed against HER2⁺The SK-BR-3 cell line and the Hela cell line also generate a certain level of IFN-gamma, and the result indicates that the Anti-EpCAM CAR-T has obvious functional activity of promoting the secretion of cytokines.

The EpCAM-targeted CAR-T can not only target tumor cells through extracellular anti-EpCAM scFv, but also activate T cells' first and second signals through their intracellular segments, so as to ensure T cells to secrete cytokines and release killing media, thereby killing tumor cells.

Conventional murine antibodies may cause HAMA reactions in humans with serious consequences for the patient. According to the EpCAM-targeted CAR-T designed in the patent, the scFv fragment of anti-EpCAM is used as the extracellular segment, and the Fc segment of the antibody is not contained, so that the murine source of the antibody drug is greatly reduced, and the safety of the drug is improved. In addition, the anti-epcammscfv gene used in the present patent is based on the previous research results of the applicant team, that is, the patent "light chain and heavy chain variable regions of FMU-EPCAM-2a9 monoclonal antibody" (national invention patent, patent No. ZL200910218795.2, granted on 26/10/2011), codon optimization is performed on the murine scFv gene fragment according to codon degeneracy and human codon preference, and the rare codon of the gene is synonymously replaced to be closer to the scFv gene fragment obtained by using the codon of the human cell, so that the normal expression of the gene in the human cell is ensured.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Sequence listing

<110> China people liberation military and military medical university

<120> recombinant CAR gene targeting human EpCAM, vector thereof, CAR-T cell, preparation method and application thereof

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ctgtttcaaa ggcctggaca atcccctaaa agactcattt atgtcgtctc caaactggat 180

tccggcgtgc ccgatagatt cacaggcagc ggcagcggaa cagattttac actgaaaatt 240

tccagagtgg aagctgaaga tctgggagtg tattattgtt ggcaaggaac acattttcct 300

tggacatttg gcggcggaac aaagctggaa atcggtggcg gtggctcggg cggtggtggg 360

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aatagcgcta caagctataa acaaaagttc aaagacaaag ctaccctcac agccgtgacc 600

agcgctagca ccgcttatat ggaactgtcc agcctgacaa atgaagattc cgctgtctac 660

tattgtatta gaggaggaaa ttattgggga caaggcacca ccgtgaccgt gtcctccacc 720

acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 780

ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 840

ttcgcctgtg atatctacat ctgggcgccc ttggccggga cttgtggggt ccttctcctg 900

tcactggtta tcacccttta ctgcaaacgg ggcagaaaga aactcctgta tatattcaaa 960

caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1020

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aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1260

tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1320

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gacgtcgtgc tgacccaaag ccccctgacc ctgagcgtca caattggcca acccgccagc 60

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ctgtttcaaa ggcctggaca atcccctaaa agactcattt atgtcgtctc caaactggat 180

tccggcgtgc ccgatagatt cacaggcagc ggcagcggaa cagattttac actgaaaatt 240

tccagagtgg aagctgaaga tctgggagtg tattattgtt ggcaaggaac acattttcct 300

tggacatttg gcggcggaac aaagctggaa atcggtggcg gtggctcggg cggtggtggg 360

tcgggtggcg gcggatctga agtgaagctg cagcagtccg gcaccgtgct cgccagacct 420

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cattggatta aacaaagacc tggacaagga ctggaatggg tgggaggaat ttatcctgga 540

aatagcgcta caagctataa acaaaagttc aaagacaaag ctaccctcac agccgtgacc 600

agcgctagca ccgcttatat ggaactgtcc agcctgacaa atgaagattc cgctgtctac 660

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<213> Artificial Sequence

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aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

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gaactg 126

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agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

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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

<210> 6

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<213> Artificial Sequence

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Asp Val Val Leu Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Val Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

115 120 125

Lys Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Thr Ser Val

130 135 140

Lys Met Ser Cys Arg Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Leu

145 150 155 160

His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Val Gly Gly

165 170 175

Ile Tyr Pro Gly Asn Ser Ala Thr Ser Tyr Lys Gln Lys Phe Lys Asp

180 185 190

Lys Ala Thr Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr Met Glu

195 200 205

Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Ile Arg

210 215 220

Gly Gly Asn Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Thr

225 230 235 240

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

245 250 255

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

260 265 270

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

275 280 285

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

290 295 300

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

305 310 315 320

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

325 330 335

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

340 345 350

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn

355 360 365

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

370 375 380

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

385 390 395 400

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

405 410 415

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

420 425 430

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

435 440 445

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

450 455 460

<210> 7

<211> 239

<212> PRT

<213> Artificial Sequence

<400> 7

Asp Val Val Leu Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Val Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

115 120 125

Lys Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Thr Ser Val

130 135 140

Lys Met Ser Cys Arg Ala Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Leu

145 150 155 160

His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Val Gly Gly

165 170 175

Ile Tyr Pro Gly Asn Ser Ala Thr Ser Tyr Lys Gln Lys Phe Lys Asp

180 185 190

Lys Ala Thr Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr Met Glu

195 200 205

Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Ile Arg

210 215 220

Gly Gly Asn Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

225 230 235

<210> 8

<211> 69

<212> PRT

<213> Artificial Sequence

<400> 8

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 9

<211> 42

<212> PRT

<213> Artificial Sequence

<400> 9

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 10

<211> 112

<212> PRT

<213> Artificial Sequence

<400> 10

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 11

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 11

Glu Val Lys Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Met Ser Cys Arg Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Leu His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Val

35 40 45

Gly Gly Ile Tyr Pro Gly Asn Ser Ala Thr Ser Tyr Lys Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ile Arg Gly Gly Asn Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

100 105 110

Ser

<210> 12

<211> 15

<212> PRT

<213> Artificial Sequence

<400> 12

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 13

<211> 111

<212> PRT

<213> Artificial Sequence

<400> 13

Asp Val Val Leu Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Leu Phe Gln Arg Pro Gly Gln Ser

35 40 45

Pro Lys Arg Leu Ile Tyr Val Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Claims (8)

1. A recombinant CAR gene targeting human EpCAM is characterized in that the recombinant CAR gene is a fusion gene consisting of EpCAMhScFv, CD8, 4-1BB costimulatory factor and CD3 zeta T cell activation domain, and the nucleotide sequence of the recombinant CAR gene is shown as SEQ ID NO. 1; wherein:

the nucleotide sequence of the EpCAMhScFv is shown in SEQ ID NO. 2;

the nucleotide sequence of the CD8 is shown as SEQ ID NO. 3;

the nucleotide sequence of the 4-1BB co-stimulatory factor is shown as SEQ ID NO. 4;

the nucleotide sequence of the CD3 zeta T cell activation domain is shown as SEQ ID NO. 5.

2. A recombinant CAR gene vector, wherein the recombinant CAR gene vector is a nucleic acid construct, plasmid, lentivirus or retrovirus incorporating the recombinant CAR gene targeting human EpCAM of claim 1.

3. The recombinant CAR gene vector of claim 2, which comprises EpCAMhScFv, CD8, 4-1BB costimulatory factor and CD3 ζ T cell activation domain, the amino acid sequence of which is set forth in SEQ ID No. 6; wherein:

the amino acid sequence of the EpCAMhScFv is shown in SEQ ID NO. 7;

the amino acid sequence of the CD8 is shown as SEQ ID NO. 8;

the amino acid sequence of the 4-1BB co-stimulation factor is shown in SEQ ID NO. 9;

the amino acid sequence of the CD3 zeta T cell activation domain is shown in SEQ ID NO. 10.

4. The recombinant CAR gene vector of claim 2, which is an Anti-EpCAMhScFv-CD8-4-1BB-CD3 ζ chimeric antigen receptor consisting of Anti-EpCAM single chain antibody heavy chain VH, Linker, Anti-EpCAM single chain antibody light chain VL, CD8 Hinge chimeric receptor Hinge region and transmembrane region, 4-1BB co-stimulatory factor and CD3 ζ T cell activation domain; wherein:

the amino acid sequence of the Anti-EpCAM single-chain antibody heavy chain VH is shown in SEQ ID NO. 11;

the amino acid sequence of the Linker is shown as SEQ ID NO. 12;

the amino acid sequence of the Anti-EpCAM single-chain antibody light chain VL is shown in SEQ ID NO. 13.

5. A CAR-T cell targeting human EpCAM, wherein the CAR-T cell is made by transforming a T cell with the recombinant CAR gene targeting human EpCAM of claim 1 or with the recombinant CAR gene vector of claims 2-4.

6. The method of making human EpCAM-targeted CAR-T cells of claim 5, comprising the steps of:

1) synthesizing Anti-EpCAMhScFv-CD8-4-1BB-CD3 zeta chimeric antigen receptor gene;

2) constructing a lentivirus expression vector which is provided with an Anti-EpCAMhScFv-CD8-4-1BB-CD3 zeta chimeric antigen receptor gene;

3) transfecting 293T cells by using the lentivirus expression vector constructed in the step 2) to obtain high-titer lentiviruses;

4) infecting the T cells by using the high-titer lentiviruses obtained in the step 3) to obtain CAR-T cells targeting human EpCAM.

7. Use of a human EpCAM-targeted recombinant CAR gene of claim 1, a recombinant CAR gene vector of claims 2-4, or a human EpCAM-targeted CAR-T cell of claim 5 in the manufacture of a medicament or kit for the treatment of a disease in which an EpCAM molecule is highly expressed.

8. An anti-cancer agent comprising the human EpCAM-targeted CAR-T cell of claim 5 and a pharmaceutically acceptable additive.

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