CN110194803B

CN110194803B - EpCAM-targeted chimeric antigen receptor and application thereof

Info

Publication number: CN110194803B
Application number: CN201910562513.4A
Authority: CN
Inventors: 郝瑞栋; 李彦涛; 易桥勇; 红丽; 张大挺; 刘根桃; 吴国祥
Original assignee: Shanghai Keqi Pharmaceutical Technology Co ltd
Current assignee: Shanghai Keqi Pharmaceutical Technology Co ltd
Priority date: 2019-06-26
Filing date: 2019-06-26
Publication date: 2021-01-05
Anticipated expiration: 2039-06-26
Also published as: CN110194803A

Abstract

The invention relates to a chimeric antigen receptor targeting EpCAM, which comprises a recognition region, a hinge region, a transmembrane region and an intracellular signal region, wherein the sequence of the extracellular recognition region is a humanized single-chain variable region antibody MOCF for recognizing EpCAM, and the sequence of the extracellular recognition region is shown as SEQ ID NO. 3; specifically, the chimeric antigen receptor comprises a sequence shown as SEQ ID NO. 13. The invention also relates to the application of the chimeric antigen receptor and a preparation method thereof. The invention employs humanized antibodies which are less immunogenic than conventional murine scfvs, and CAR-T cells prepared based thereon are less likely to produce a HAMA effect in vivo, and may have a longer residence time in vivo; the CAR-T cell MOCF-ICOSBBZ background level cytokine release prepared by the humanized antibody is lower, and the safety in clinical application is better; the EpCAM positive tumor can be efficiently and specifically treated by targeting the EpCAM protein on the surface of the tumor cell and activating a signal path downstream of the T cell and endowing the T cell with the capacity of killing tumor cells with EpCAM targets.

Description

EpCAM-targeted chimeric antigen receptor and application thereof

Technical Field

The invention relates to the technical field of cellular immunotherapy, in particular to an EpCAM-targeted chimeric antigen receptor and application thereof.

Background

Epithelial cell adhesion molecule (EpCAM), also known as CD326, is a 40kD transmembrane glycoprotein. It is specifically expressed in a variety of tumor tissues, whereas in normal tissues its expression is restricted to the basal side of epithelial cells, so that its apical side is not accessible. EpCAM is highly expressed in tumors of various epithelial cell origins, including various adenocarcinomas such as colon, stomach, pancreas, lung, ovary, breast, and the like. EpCAM acts as a signaling molecule, downstream of which activates the Wnt signaling pathway, thereby regulating cleavage of intramembrane proteins, leading to tumorigenesis. In tumor tissue, EpCAM expression changes from basal layer to uniform expression on the cell membrane surface, making it easier to target in cell or antibody therapy.

Recently, EpCAM was identified as a surface marker for Circulating Tumor Cells (CTCs) and Cancer Stem Cells (CSCs). CTCs are considered potential precursor cells of primary tumor cells metastasizing to other locations, which can invade the blood circulation system and thus metastasize to other tissues. CTC capture technologies based on EpCAM positive cells have been applied in a variety of tumor types, particularly breast cancer. CSC cells are considered as key target cells for cancer elimination due to the diversity of the phenotype and sternness of cancer cells. EpCAM is expressed on stem cells of a variety of cancers, including breast, colon, pancreatic, and prostate cancers, among others. CSCs are highly resistant to both chemotherapy and radiation therapy, making EpCAM-targeted therapy a hotspot in tumor therapy. And the therapy by targeting EpCAM CAR-T has better curative effect and wider application prospect in the field of malignant solid tumors, but the current reports on the CAR molecule and CAR-T cells targeting EpCAM are rare.

Disclosure of Invention

Based on the shortcomings of the prior art, the present invention provides an EpCAM-specific Chimeric Antigen Receptor (CAR) molecule comprising a humanized single chain antibody sequence, which has the following advantages: on the one hand, the antigen reduces the immunogenicity of scFv sequences in CAR molecules targeting EpCAM antigens, prolongs the survival time of CAR-T cells in vivo, and reduces the risk of allergy; on the other hand, the CAR molecule has a lower background activation level, and thus is safer and more viable during in vivo applications. The invention also verifies the potential application of the compound in the treatment of solid tumors.

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

a first object of the present invention is to provide a chimeric antigen receptor targeting EpCAM comprising an extracellular recognition region which is an anti-EpCAM antibody, a hinge region, a transmembrane region and an intracellular signal region.

In order to further optimize the chimeric antigen receptor, the technical measures adopted by the invention also comprise:

further, the anti-EpCAM antibody is a humanized antibody MOCF, specifically: a humanized single chain variable region antibody MOCF that recognizes EpCAM; wherein the anti-EpCAM humanized antibody MOCF is obtained by humanizing the EpCAM antibody sequence MOC31(SEQ ID NO.1) known in the art. Further, the amino acid sequence of the MOCF is shown as SEQ ID NO. 3.

Further, the hinge region is a CD8hinge region. Furthermore, the amino acid sequence of the CD8hinge region is shown as SEQ ID NO. 4.

Further, the transmembrane region is selected from any one of the CD8 transmembrane region, CD28 transmembrane region, ICOS transmembrane region. Furthermore, the amino acid sequence of the CD8 transmembrane region is shown as SEQ ID NO.5, the amino acid sequence of the CD28 transmembrane region is shown as SEQ ID NO.6, and the amino acid sequence of the ICOS transmembrane region is shown as SEQ ID NO. 7.

Further, the intracellular signaling region comprises at least one of CD28, 4-1BB, ICOS, CD3 ζ. Furthermore, the amino acid sequence of the CD28 is shown as SEQ ID NO.8, the amino acid sequence of the 4-1BB is shown as SEQ ID NO.9, the amino acid sequence of the ICOS is shown as SEQ ID NO.10, and the amino acid sequence of the CD3 zeta is shown as SEQ ID NO. 11.

Further, based on the structure of the above EpCAM-specific CAR molecule, the EpCAM chimeric antigen receptor is MOCF-ICOSBBZ, the extracellular recognition region is MOCF, the hinge region is a CD8hinge region, the transmembrane region is ICOS, and the intracellular signaling region is ICOS/4-1BB/CD3 ζ. Further, the amino acid sequence of MOCF-ICOSBBZ is shown as SEQ ID NO. 13.

It will be appreciated that EpCAM chimeric antigen receptors may also be composed according to other structures suitable for use in the art. Further, in different CAR molecules, their transmembrane regions may be the same or different; the intracellular signaling regions may be the same or different.

It is a second object of the invention to provide a nucleic acid encoding any of the above-described EpCAM-specific chimeric antigen receptors.

Further, the nucleic acid sequence for coding the MOCF-ICOSBBZ is shown as SEQ ID NO. 15.

The invention also relates to a MOCB scFv as a reference antibody sequence, wherein the amino acid sequence of the MOCB scFv is shown as SEQ ID NO.2, the amino acid sequence of the MOCB-ICOSBBZ chimeric antigen receptor constructed by the MOCB-ICOSBBZ chimeric antigen receptor is shown as SEQ ID NO.12, and the nucleic acid sequence of the coded MOCB-ICOSBBZ chimeric antigen receptor is shown as SEQ ID NO. 14.

The amino acid sequence of the CAR molecule and its coding nucleic acid sequence are shown in the following table:

the third object of the present invention is to provide a recombinant expression vector containing the above nucleic acid.

Further, the recombinant expression vector comprises lentivirus, retrovirus, adenovirus, adeno-associated virus or plasmid and the like; further, the original recombinant expression vector is a lentivirus. In one embodiment, the vector used is a lentiviral vector. The CAR lentiviral vector plasmid was co-transfected into HEK293T cells in the presence of the helper packaging plasmid pSPAX2 and the VSV-G envelope plasmid pmd2.G, and packaged as a lentivirus with a CAR molecule.

The fourth object of the present invention is to provide a host cell containing the above recombinant expression vector.

Further, the host cell is a T cell, NK cell, NKT cell or a population of cells comprising T cell, NK cell, NKT cell.

The fifth purpose of the invention is to provide a construction method of the host cell, which comprises the steps of constructing the recombinant expression vector, packaging the recombinant expression vector and transducing the recombinant expression vector into the host cell.

The sixth purpose of the invention is to provide the EpCAM-specific chimeric antigen receptor, the nucleic acid, the recombinant expression vector and the application of the host cell in preparing medicines for treating human solid tumors.

Further, the human solid tumors mainly comprise colorectal cancer, esophageal cancer, bile duct cancer, pancreatic cancer and the like.

Further, the use is an EpCAM-specific CAR-T cell that confers an EpCAM-specific CAR on the T cell by lentivirus-mediated transduction, thereby conferring on the T cell the ability to recognize an EpCAM molecule and targeting an EpCAM-positive human tumor.

In one embodiment, EpCAM CAR-T cells were co-incubated with EpCAM-highly expressing tumor cell line H1650 and EpCAM-lowly expressing tumor cell line a549, and the results showed that EpCAM CAR-T cells were capable of producing IFN- γ and IL2 in large amounts upon stimulation of target cells; but not IFN-gamma and IL2 when co-incubated with EpCAM low expressing cell line A549; at the same time, T cells are unable to produce IFN-gamma and IL2 in large quantities. The example shows that the EpCAM CAR-T cell has good specific targeting property, can activate the killing function of the T cell under the stimulation of EpCAM, and the in-vitro killing activity of MOCF-ICOSBBZ on EpCAM positive tumor cells is superior to that of MOCB-ICOSBBZ; and in the resting state of non-killing target cells, the cytokine production of the MOCF-ICOSBBZ at the background level is lower than that of MOCB-ICOSBBZ, which indicates that the MOCF-ICOSBBZ has better safety and in-vivo survival capability in clinical application.

In another embodiment, EpCAM CAR-T cells demonstrate effective killing of EpCAM positive tumor cells at a certain effective target ratio (CAR-T cells: target cells); but T cells are not able to kill target cells efficiently. This example demonstrates that EpCAM CAR-T is able to effectively lyse EpCAM-positive tumor cells in vitro.

In another embodiment, a model of tumor implantation under the skin of NOD-SCID immunodeficient mice was constructed based on the EpCAM positive lung cancer cell line H1650. The results of treatment of this tumor-bearing mouse with the above EpCAM CAR-T cells MOCF-ICOSBBZ demonstrated that MOCF-ICOSBBZ cells were effective in eliminating EpCAM-positive tumor cells, but tumors grew in the control T cell group.

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

the invention employs humanized antibodies which are less immunogenic than conventional murine scfvs, and CAR-T cells prepared based thereon are less likely to produce a HAMA effect in vivo, and may have a longer residence time in vivo; compared with the previously published MOCB-ICOSBBZ, the CAR-T cell MOCF-ICOSBBZ prepared by the humanized antibody has lower background level cytokine release and better safety in clinical application; the EpCAM positive tumor can be efficiently and specifically treated by targeting the EpCAM protein on the surface of the tumor cell and activating a signal path downstream of the T cell and endowing the T cell with the capacity of killing tumor cells with EpCAM targets.

Drawings

Figure 1 is a schematic diagram illustrating the structure of an EpCAM-specific Chimeric Antigen Receptor (CAR) molecule according to one embodiment of the present invention;

FIG. 2 is a graph showing the results of flow cytometry to determine the transduction efficiency of MOCF/MOCB-CAR transduced T cells in one embodiment of the invention;

FIG. 3 is a graph showing the expression levels of EpCAM in different tumor cell lines according to one embodiment of the present invention;

FIG. 4 is a schematic representation of IFN-. gamma.and IL2 secretion following killing of EpCAM target cells by EpCAM CAR-T cells in one embodiment of the invention;

FIG. 5 is a graph showing the results of in vitro killing of EpCAM target cells by EpCAM CAR-T cells in one embodiment of the invention;

figure 6 is a graphical representation of the results of EpCAM CAR-T cell proliferation in mice in one embodiment of the present invention.

Figure 7 is a graph showing the survival curves of EpCAM CAR-T cell mice after killing of EpCAM positive transplanted tumors in one embodiment of the present invention.

Detailed Description

The invention relates to a chimeric antigen receptor targeting EpCAM, which comprises a recognition region, a hinge region, a transmembrane region and an intracellular signal region, wherein the sequence of the extracellular recognition region is a humanized single-chain variable region antibody MOCF for recognizing EpCAM, and the amino acid sequence of the extracellular recognition region is shown as SEQ ID NO. 3; specifically, the chimeric antigen receptor comprises an amino acid sequence shown as SEQ ID NO. 13. The invention also relates to related application of the chimeric antigen receptor and a preparation method thereof.

The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Example one

This example is the construction of a chimeric antigen receptor molecule targeting EpCAM, comprising the steps of:

humanization was first performed based on the monoclonal antibody MOC31 targeting EpCAM. Humanization is carried out by conventional CDR grafting methods in the art, see the literature (Jones PT, Dear PH, Foote J, et al. the repairing the complementary-determining regions in a human antibody with the human being from a mouse 29-Jun 4; 321(6069): 522-5; Sandhu JS. A rapid procedure for the humanization of monoclonal antibodies. Gene.1994Dec15; 150: 2-10.). The humanized antibody was named MOCF by grafting the CDR regions of MOC31 into the framework regions of the human variable regions with higher homology. And the sequence is transformed into a single chain variable region sequence MOCF scFv; the EPCAM single chain variable region sequence MOCB scFv derived from the article was also used as the reference antibody sequence (Ang WX, Li Z, Chi Z, et al. Intra epithelial immunotherapy with T cells static and transduction expressing anti-EpCAM CAR in xenogram models of epithelial carcinogen carboxytherapy. oncotarget.2017Feb 21; 8(8):13545-13559.doi: 10.18632/oncotarget.14592.). EpCAM-targeting CAR molecules were then constructed based on MOCF or MOCB, and the structure of the third generation CAR is sequentially composed of a signal peptide of CD8, MOCF or MOCB scFv, a CD8hinge region, an ICOS transmembrane region, an ICOS intracellular region, a 4-1BB intracellular region, and a CD3 zeta intracellular region in tandem, and the structural schematic is shown in FIG. 1. Respectively called MOCF-ICOSBBZ or MOCB-ICOSBBZ. MOCF-ICOSBBZ or MOCB-ICOSBBZ molecules are constructed on a lentiviral vector pHAGE-EF1A-MCS, the cloning site is NotI/ClaI, and the molecular structure is constructed as a clone pHAGE-EF1A-MOCF-ICOSBBZ or pHAGE-EF 1A-MOCB-ICOSBBZ.

Example two

This example is the construction of MOCF/MOCB-ICOSBBZ CAR-T cells, which comprises the following steps:

packaging of CAR lentivirus: firstly, the constructed lentiviral plasmids pHAGE-EF1a-MOCF-ICOSBBZ, pHAGE-EF1a-MOCF-ICOSBBZ and lentiviral system auxiliary packaging plasmids pMD2.G and pSPAX2 are respectively extracted by using a QIAGEN endotoxin-free large quality-improving particle kit. 1.8X 10E 7293T was plated into T175 flasks the day before transfection. The 293T cell medium was changed to 30ml serum-free medium 1 hour before transfection. The plasmids were co-transfected into 293T cells using calcium phosphate precipitation and the cell culture medium was changed to 60ml complete medium DMEM + 10% FBS 24 hours after transfection. Cell supernatants were harvested 48 hours post transfection and 60ml of fresh complete medium was added. Cell supernatants were harvested again for 72 hours and discarded. Harvested cell supernatants were centrifuged for 3min at 5000g to remove impurities, followed by filtration using a 0.45um filter followed by centrifugation at 40000g for 4 hours to pellet the virus, resuspended in 0.1ml PBS and tested for virus titer. Standing at-80 deg.C for freezing.

T cell transduction: retronectin, anti-human CD3 and CD28 antibodies were coated one day in advance in 6-well plates overnight at 4 ℃ and washed twice with the previous PBS for use. Blood was collected and PBMC was isolated by a conventional method, T cells were sorted using STEMCELL T cell sorting kit, counted, and the obtained T cells were resuspended at a density of 1X 10E6/ml in X-VIVO15 medium containing 5% human AB serum and 100IU/ml interleukin-2, and cultured on a coated culture plate. 24 hours after the start of the culture, 5ug/ml polybrene was added and the corresponding lentivirus (MOCF-ICOSBBZ, MOCB-ICOSBBZ) was added in accordance with MOI3, mixed well and infected at 37 ℃ for 24 hours. Then, the cell sediment is collected by centrifugation and is cultured by changing the culture medium into X-VIVO15 with 5 percent of human AB serum and 100IU/ml of interleukin-2. Subsequent cultures cells were maintained at a density of 1 × 10E6/ml by supplementing the medium and scFv expression was detected 72 hours later using flow cytometry to test CAR molecule transduction efficiency. CAR-T-MOCF-ICOSBBZ and CAR-T-MOCB-ICOSBBZ were detected using protein L/PE-SA. As shown in FIG. 2, positive rates of CAR-T-MOCF-ICOSBBZ and CAR-T-MOCB-ICOSBBZ were observed at approximately 55% -75%, with T cells as control cells.

EXAMPLE III

This example is the identification of EpCAM target cells and the assay of EpCAM CAR-T cell activity.

Identification of EpCAM target cells: to test whether EpCAM CAR-T can effectively kill tumor cells, the expression of EpCAM on the surface of different tumor cells was first identified, and the results show that the lung cancer cell line H1650 highly expresses EpCAM, while the lung cancer cell line A549 does not express EpCAM, and the results are shown in FIG. 3.

IFN-. gamma.and IL2 secretion experiments: three effector cells, MOCF-ICOSBBZ, MOCB-ICOSBBZ two CAR-T cells and control T cells, were mixed with target cell H1650 and control target cell a549 at A5: 1 effect-to-target ratio in 96-well plates. After incubation at 37 ℃ for 24 hours, the supernatants were assayed for expression of IFN-. gamma.and IL2 using standard ELISA methods. The results are shown in FIG. 4, when two effector cells, MOCF-ICOSBBZ and MOCB-ICOSBBZ, and H1650 target cells are co-cultured, the CAR-T cells secrete a large amount of IFN-gamma and IL2, and the secretion amounts of the two effector cells are equivalent, but the background release amount of the MOCF-ICOSBBZ is only one third or lower than that of the MOCB-ICOSBBZ, which indicates that the MOCF-ICOSBBZ CAR-T cells constructed by the invention have higher safety and stronger survival capability in the in vivo application process; when effector cells are co-cultured with A549 control target cells, MOCF-ICOSBBZ CAR-T cells only secrete a small amount of IFN-gamma and IL2, and the CAR-T cells constructed by the invention have good targeting specificity.

Example four

This example demonstrates the ability of EpCAM CAR-T cells to kill EpCAM target cells in vitro.

CAR-T cells and T cells from example two were co-cultured in 96-well plates with EpCAM-positive target cells H1650 and control target cells a549 in an effective-to-target ratio of 1:1, 5:1, 10:1, while medium blank and target cell control groups were set. After incubation at 37 ℃ for 24 hours, OD450 absorbance was measured using CCK8 method. The results are shown in FIG. 5. MOCF-ICOSBBZ CAR-T and MOCB-ICOSBBZ CAR-T cells can effectively kill EpCAM positive target cells H1650, and the killing activity of the MOCF-ICOSBBZ CAR-T cells is superior to that of the MOCB-ICOSBBZ CAR-T cells, and both the MOCF-ICOSBBZ CAR-T cells cannot effectively kill EpCAM negative control target cells A549 cells, and the control T cells cannot effectively kill H1650 and A549 cells.

This example demonstrates that CAR-T cells constructed from two CAR molecules MOCF-ICOSBBZ and MOCB-ICOSBBZ can effectively kill EpCAM-positive cancer cells and have good EpCAM targeting, and in addition, MOCF-ICOSBBZ has better in vitro killing activity than MOCB-ICOSBBZ.

EXAMPLE five

This example demonstrates the ability of CAR-T cells to inhibit EpCAM-positive tumor growth in a mouse subcutaneous tumor model.

Using 6-week-old NOD-SCID immunodeficient mice, 3 × 10E 6H 1650 cell lines were injected subcutaneously, tumors were allowed to grow for 7-15 days, and tumor size was measured with a caliper and divided by 2 to obtain a tumor size in mm3 units as the longest length of the tumor multiplied by the length perpendicular to the longest length. When the tumor size reached 20-50mm3, no significant CAR-T-induced toxic side effects were found throughout the study period by intravenous injection of 5 × 10E6 control T cells or 5 × 10E6 CAR-positive MOCF-ICOSBBZ CAR-T cells. Tail vein blood was collected 1 week after reinfusion, CAR-T cell levels were measured in mouse blood and tumor size was measured.

Collecting 20ul to 20ul of heparin sodium anticoagulant from mouse tail venous blood, adding 1ul of anti-human CD45 antibody into each sample, incubating for 10 minutes at room temperature, adding 500ul of erythrocyte lysate to split red blood for about 5 minutes, centrifugally collecting cell precipitate, washing once with PBS, and detecting by a flow cytometer. Results as shown in figure 6, both CAR-T cells and T cells can proliferate in the blood of mice. As can be seen from figure 7, the tumors started to become smaller after 12 days of MOCF-ICOSBBZ CAR-T cell injection, and by 23 days the tumors had been essentially completely cleared, the control T cells were unable to reduce the tumor cells.

This example demonstrates that EpCAM-targeted CAR-T cells of the invention are able to effectively eliminate tumor cells in a mouse EpCAM-positive subcutaneous graft tumor model. The tumors of MOCF-ICOSBBZ CAR-T group (6) mice were completely eliminated, and reached the standard of CR, with 100% of effective rate. In summary, MOCF-ICOSBBZ CAR-T cells were able to safely and effectively eliminate EpCAM positive tumors in animals.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. It will be apparent to those skilled in the art that any modifications and substitutions can be made to the present invention in light of the above teachings to achieve the same results. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

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Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Gln Met Ser

65 70 75 80

Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Ser Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

100 105 110

Thr Tyr Tyr Cys Ala Gln Asn Leu Glu Ile Pro Arg Thr Phe Gly Gln

115 120 125

Gly Thr Lys Val Glu Leu Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Pro

145 150 155 160

Gly Leu Val Gln Pro Gly Gly Ser Val Arg Ile Ser Cys Ala Ala Ser

165 170 175

Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro

180 185 190

Gly Lys Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu

195 200 205

Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr Phe Ser Leu Asp

210 215 220

Thr Ser Ala Ser Ala Ala Tyr Leu Gln Ile Asn Ser Leu Arg Ala Glu

225 230 235 240

Asp Thr Ala Val Tyr Tyr Cys Ala Arg Phe Ala Ile Lys Gly Asp Tyr

245 250 255

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

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala

305 310 315 320

Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu

325 330 335

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

340 345 350

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

355 360 365

Val Thr Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

<210> 13

<211> 525

<212> PRT

<213> MOCF-ICOSBBZ(Artificial Sequence)

<400> 13

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Ala Ser Asp Ile Val Met Thr Gln Ser Pro Leu

20 25 30

Ser Leu Pro Val Ser Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser

35 40 45

Thr Lys Ser Leu Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr

50 55 60

Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Gln Met Ser

65 70 75 80

Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

85 90 95

Thr Asp Phe Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly

100 105 110

Val Tyr Tyr Cys Ala Gln Asn Leu Glu Ile Pro Arg Thr Phe Gly Gln

115 120 125

Gly Thr Lys Val Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ser

145 150 155 160

Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

165 170 175

Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro

180 185 190

Gly Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu

195 200 205

Ser Thr Tyr Ala Asp Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu

210 215 220

Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu

225 230 235 240

Asp Thr Ala Thr Tyr Phe Cys Ala Arg Phe Ala Ile Lys Gly Asp Tyr

245 250 255

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

260 265 270

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Leu Pro Ile Gly Cys Ala

305 310 315 320

Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu

325 330 335

Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

340 345 350

Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp

355 360 365

Val Thr Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

<210> 14

<211> 1578

<212> DNA

<213> nucleic acid encoding MOCB-ICOSBBZ (Artificial Sequence)

<400> 14

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggctagcg acattcagat gacccagagc ccttcctctc tcagtgcctc cgtcggcgat 120

agggtcacaa tcacctgccg gtccacaaag agcctgctgc actccaacgg gatcacatac 180

ctgtactggt accagcagaa gcccggcaag gcccccaagc tgctgatcta ccagatgagc 240

aacctggcca gcggggtgcc cagcaggttc agcagcagcg gcagcgggac cgatttcaca 300

ctgacaatca gcagcctgca gcccgaggat ttcgccacct actactgcgc ccagaacctg 360

gagatccccc ggacattcgg ccagggcacc aaggtggagc tgaagcgggg ggggggcggc 420

agcgggggcg gggggagcgg gggcgggggg tccgaggtgc agctggtgca gagcgggccc 480

ggcctggtgc agccaggggg ctccgtgcgg atcagctgcg ccgcctccgg gtacaccttc 540

accaactacg gcatgaactg ggtgaagcag gcccctggga aggggctgga gtggatgggg 600

tggatcaaca catacacagg ggagagcaca tacgccgatt cttttaaggg aaggtttact 660

tttagcctgg atacaagcgc tagtgccgcc tacttgcaga ttaacagcct gagagctgag 720

gatacagccg tgtattactg tgccagattt gccattaagg gagattactg gggacaggga 780

acactgctga cagtgagtag caccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatttct ggttacccat aggatgtgca 960

gcctttgttg tagtctgcat tttgggatgc atacttattt gttggcttac aaaaaagaag 1020

tattcatcca gtgtgcacga ccctaacggt gaatacatgt tcatgagagc agtgaacaca 1080

gccaaaaaat ctagactcac agatgtgacc ctaaaacggg gcagaaagaa actcctgtat 1140

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1200

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1260

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1320

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1380

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1440

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1500

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1560

gccctgcccc ctcgctaa 1578

<210> 15

<211> 1578

<212> DNA

<213> nucleic acid encoding MOCF-ICOSBBZ (Artificial Sequence)

<400> 15

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggctagcg acatcgtgat gacccagtct ccactgagcc tgcccgtgtc ccctggagag 120

ccagcctcta tcagctgcag gtccaccaag tctctgctgc actccaacgg catcacatac 180

ctgtattggt acctgcagaa gcccggccag tctcctcagc tgctgatcta tcagatgagc 240

aatctggcct ccggcgtgcc tgacagattc tccggctctg gcagcggaac cgacttcacc 300

ctgcggatca gcagagtgga ggccgaggat gtgggcgtgt actattgcgc ccagaacctg 360

gagatcccaa ggaccttcgg ccagggcaca aaggtggaga tcaagagggg aggaggaggc 420

tctggaggag gaggcagcgg cggcggcggc tcccaggtgc agctggtgca gtccggctct 480

gagctgaaga agccaggcgc ctctgtgaag gtgagctgta aggcctccgg ctataccttc 540

acaaactacg gcatgaattg ggtgaagcag gcaccaggca agggcctgaa gtggatgggc 600

tggatcaaca cctatacagg cgagtctacc tacgccgacg acttcaaggg ccggttcgcc 660

tttagcctgg agaccagcgc ctccacagcc tacctgcaga tcaacaatct gaagaatgag 720

gacaccgcca catatttctg tgccagattt gccatcaagg gcgattactg gggccagggc 780

accctggtga cagtgagctc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatttct ggttacccat aggatgtgca 960

gcctttgttg tagtctgcat tttgggatgc atacttattt gttggcttac aaaaaagaag 1020

tattcatcca gtgtgcacga ccctaacggt gaatacatgt tcatgagagc agtgaacaca 1080

gccaaaaaat ctagactcac agatgtgacc ctaaaacggg gcagaaagaa actcctgtat 1140

atattcaaac aaccatttat gagaccagta caaactactc aagaggaaga tggctgtagc 1200

tgccgatttc cagaagaaga agaaggagga tgtgaactga gagtgaagtt cagcaggagc 1260

gcagacgccc ccgcgtacaa gcagggccag aaccagctct ataacgagct caatctagga 1320

cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1380

aagccgagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1440

gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1500

ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1560

gccctgcccc ctcgctaa 1578

Claims

1. The chimeric antigen receptor targeting EpCAM comprises an extracellular recognition region, a hinge region, a transmembrane region and an intracellular signal region, and is characterized in that the sequence of the extracellular recognition region is a humanized single-chain variable region antibody MOCF for recognizing EpCAM, the amino acid sequence of the humanized single-chain variable region antibody MOCF is shown as SEQ ID number 3, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 13.

2. A nucleic acid encoding the EpCAM-targeted chimeric antigen receptor of claim 1.

3. The nucleic acid according to claim 2, wherein the nucleotide sequence of the nucleic acid is represented by SEQ ID number 15.

4. A recombinant expression vector comprising the nucleic acid of any one of claims 2 to 3.

5. A host cell comprising the recombinant expression vector of claim 4.

6. A method of constructing the host cell of claim 5, comprising the steps of constructing the recombinant expression vector, packaging the recombinant expression vector, and transducing the recombinant expression vector into the host cell.

7. Use of the chimeric antigen receptor for EpCAM of claim 1, the nucleic acid of any one of claims 2 to 3, the recombinant expression vector of claim 4 and the host cell of claim 5 for the preparation of a medicament for the treatment of a human solid tumor.