CN110194803A

CN110194803A - A kind of Chimeric antigen receptor and its application targeting EpCAM

Info

Publication number: CN110194803A
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: 2019-09-03
Anticipated expiration: 2039-06-26
Also published as: CN110194803B

Abstract

The present invention relates to a kind of Chimeric antigen receptors for targeting EpCAM, including cog region, hinge area, and transmembrane region, intracellular signal area, the extracellular identification region sequence is to identify that the Humanized single chain of EpCAM can be changed domain antibodies MOCF, and sequence is as shown in SEQ ID NO.3；Specifically, the Chimeric antigen receptor includes the sequence as shown in SEQ ID NO.13.The invention further relates to applications for encoding above-mentioned Chimeric antigen receptor and preparation method thereof.A possibility that present invention uses humanized antibody, has lower immunogenicity compared to the scFv of traditional source of mouse, and the CAR-T cell prepared based on this generates HAMA effect in vivo is smaller, and retention time may be longer in vivo；The CAR-T cell MOCF-ICOSBBZ background level cytokine release prepared using above-mentioned humanized antibody is lower, and safety can be more preferable in clinical application；Pass through the EpCAM albumen on targets neoplastic cells surface and activate the signal path in T cell downstream, assigns the ability that T cell killing has EpCAM target tumor cell, treatment EpCAM positive tumor that can be efficiently special.

Description

A kind of Chimeric antigen receptor and its application targeting EpCAM

Technical field

The present invention relates to cellular immunotherapy technical field more particularly to it is a kind of target EpCAM Chimeric antigen receptor and It is applied.

Background technique

Signal transduction factor (epthelial cell adhesion molecule, EpCAM), also known as CD326, It is the transmembrane glycoprotein of 40kD a kind of.It is specific expressed in kinds of tumors tissue, and in the normal tissue, expression only limits In the substrate side of epithelial cell, so that its top side is impalpable.EpCAM high table in the tumour of a variety of epithelial cell origins It reaches, including a variety of gland cancer, such as colon, stomach, pancreas, lung, ovary, mammary gland etc..EpCAM is as signal transduction molecule, downstream Wnt signal path is activated, to regulate and control the cracking of albumen in film, and then leads oncogenic generation.In tumor tissues, EpCAM Expression change into from basal layer and uniformly expressed in cell membrane surface, to make it easier to make in cell or antibody therapy For target spot.

Recently, EpCAM is accredited as the surface marker of circulating tumor cell (CTC) and cancer stem cell (CSC).CTC It is considered as the potential precursor that primary tumor cell is transferred to other positions, blood circulation system can be intruded into, from And it is transferred to other tissues.CTC capture technique based on EpCAM positive cell has been applied in kinds of tumors type, especially It is breast cancer.And CSC cell is due to maintaining the stemness of cancer cell and the diversity of phenotype, it is considered to be remove the key of cancer Target cell.EpCAM is expressed on the stem cell of kinds cancer, including breast cancer, colon cancer, cancer of pancreas and prostate cancer etc..CSC Chemotherapy and radiation is highly resisted, so that EpCAM targeted therapies become hot spot in oncotherapy.And pass through targeting EpCAM CAR-T treatment even more has better curative effect and broader practice prospect in malignant solid tumor field, but at present It is really more rare about the CAR molecule of targeting EpCAM and the report of CAR-T cell.

Summary of the invention

Based on defect existing in the prior art, the present invention provides a kind of EpCAM specific chimeric antigen receptor (CAR), This CAR molecule contains the single chain antibody sequence of humanization, and advantage is as follows: on the one hand its CAR for reducing targeting EpCAM antigen The immunogenicity of scFv sequence in molecule extends the time-to-live of CAR-T cell in vivo, reduces the risk that allergy occurs；Separately The background activation level of outer one side, the CAR molecule is lower, thus its safety is higher in application process in vivo, energy of surviving Power is stronger.The present invention also demonstrates its potential application in treatment of solid tumors.

To achieve the above object, the present invention adopts the following technical scheme:

The first purpose of the invention is to provide a kind of Chimeric antigen receptors for targeting EpCAM, including extracellular cog region, hinge Sequence, transmembrane region and intracellular signal area, the extracellular cog region are anti-EpCAM antibody.

In order to advanced optimize above-mentioned Chimeric antigen receptor, technical measures adopted by the present invention further include:

Further, the anti-EpCAM antibody is humanized antibody MOCF, specifically: identify the humanization list of EpCAM Chain can be changed domain antibodies MOCF；Wherein, the anti-EpCAM humanized antibody MOCF is by by EpCAM antibody sequence known in the art MOC31 (SEQ ID NO.1) is by humanization modified.Further, the amino acid sequence of the MOCF such as SEQ ID Shown in NO.3.

Further, the hinge area is the region CD8 hinge.Further, the amino in the region the CD8 hinge Acid sequence is as shown in SEQ ID NO.4.

Further, the transmembrane region is selected from any of CD8 transmembrane region, CD28 transmembrane region, ICOS transmembrane region.More into One step, the amino acid sequence of the CD8 transmembrane region is as shown in SEQ ID NO.5, the amino acid sequence of the CD28 transmembrane region As shown in SEQ ID NO.6, the amino acid sequence of the ICOS transmembrane region is as shown in SEQ ID NO.7.

Further, the intracellular signal area includes at least one of CD28,4-1BB, ICOS, CD3 ζ.Further Ground, the amino acid sequence of the CD28 is as shown in SEQ ID NO.8, the amino acid sequence of the 4-1BB such as SEQ ID NO.9 institute Show, the amino acid sequence of the ICOS is as shown in SEQ ID NO.10, the amino acid sequence of the CD3 ζ such as SEQ ID NO.11 It is shown.

Further, the structure based on the above EpCAM specific C AR molecule, the EpCAM Chimeric antigen receptor are MOCF-ICOSBBZ, extracellular cog region are MOCF, and hinge area is the region CD8hinge, transmembrane region ICOS, intracellular signal area For ICOS/4-1BB/CD3 ζ.Further, the amino acid sequence of the MOCF-ICOSBBZ is as shown in SEQ ID NO.13.

It is understood that EpCAM Chimeric antigen receptor can also be formed according to the other structures that this field is applicable in.Into one Step ground, in different CAR molecules, transmembrane region can be identical, can also be different；Its intracellular signal area can be identical, can also be different.

A second object of the present invention is to provide a kind of cores for encoding any above-mentioned EpCAM specific chimeric antigen receptor Acid.

Further, the nucleic acid sequence of the MOCF-ICOSBBZ is encoded as shown in SEQ ID NO.15.

The invention further relates to MOCB scFv as reference antibody sequence, the amino acid sequence such as SEQ of the MOCB scFv Shown in ID NO.2, as its construct MOCB-ICOSBBZ Chimeric antigen receptor amino acid sequence as shown in SEQ ID NO.12, The nucleic acid sequence of MOCB-ICOSBBZ Chimeric antigen receptor is encoded as shown in SEQ ID NO.14.

The amino acid sequence and its nucleic acid sequence encoding of above-mentioned CAR molecule are as shown in the table:

Third object of the present invention is to provide a kind of recombinant expression carriers containing above-mentioned nucleic acid.

Further, the recombinant expression carrier includes slow virus, retrovirus, adenovirus, adeno-associated virus or matter Grain etc.；Further, original recombinant expression carrier is slow virus.In a specific embodiment, used carrier is slow Viral vectors.CAR slow virus carrier plasmid assist packaging plasmid pSPAX2 and VSV-G envelope plasmid pMD2.G there are the case where Under, cotransfection HEK293T cell can be packaged as the slow virus with CAR molecule.

Fourth object of the present invention is to provide a kind of host cell containing above-mentioned recombinant expression carrier.

Further, the host cell is T cell, NK cell, NKT cell or contains T cell, NK cell, NKT cell Cell mass.

Fifth object of the present invention is to provide a kind of construction methods of above-mentioned host cell comprising recombinant expression carrier Construction step, recombinant expression carrier packaging step and the step of recombinant expression carrier is transduceed to host cell.

Sixth object of the present invention is to provide above-mentioned EpCAM specific chimeric antigen receptor, above-mentioned nucleic acid, above-mentioned recombinations The application of expression vector and above-mentioned host cell in preparation human entity tumor therapeutic agent.

Further, the human entity tumor mainly includes colorectal cancer, the cancer of the esophagus, cholangiocarcinoma, cancer of pancreas etc..

Further, the application is a kind of CAR-T cell of EpCAM specificity, passes through the transduction of lentivirus mediated So that T cell has EpCAM specific C AR, to impart the ability of T cell identification EpCAM molecule, and EpCAM sun is targeted The human tumor of property.

In a specific embodiment, by the tumor cell line H1650 of EpCAM CAR-T cell and high expression EpCAM And the tumor cell line A549 of low expression EpCAM is incubated for altogether, EpCAM CAR-T cell can be in the stimulation of target cell as the result is shown Lower a large amount of generation IFN-γ and IL2；But IFN- cannot be generated when the cell line A549 with EpCAM low expression is incubated for altogether γ and IL2；T cell can not largely generate IFN-γ and IL2 simultaneously.This embodiment illustrates EpCAM CAR-T cell tools There is good specific target tropism, can activate T cell killing ability under EpCAM stimulation, and MOCF-ICOSBBZ is external The activity for killing EpCAM positive tumor cell is better than MOCB-ICOSBBZ；And MOCF-ICOSBBZ is in the quiet of non-killing target cell Under breath state, the cell factor of background level, which generates, is lower than MOCB-ICOSBBZ, and illustrating it in clinical application has more preferably Safety and internal survival ability.

In another embodiment, it was demonstrated that under certain effect target ratio (CAR-T cell: target cell), EpCAM CAR-T cell can effectively kill the tumour cell of the EpCAM positive；But T cell can not effectively kill target cell.The reality It applies example and illustrates that EpCAM CAR-T can effectively crack the tumour cell of the EpCAM positive in vitro.

In another embodiment, NOD-SCID is constructed based on EpCAM positive lung cancer cells system H1650 to be immunized Defect mouse subcutaneous transplantation tumor model.The tumor-bearing mice is treated using above-mentioned EpCAM CAR-T cell MOCF-ICOSBBZ, as a result Illustrate that MOCF-ICOSBBZ cell can effectively remove EpCAM positive tumor cell, but it is constantly long to compare tumour in T cell group Greatly.

Compared with prior art, technical solutions according to the invention have the advantages that

The present invention uses humanized antibody, has lower immunogenicity compared to the scFv of traditional source of mouse, as A possibility that CAR-T cell of basis preparation generates HAMA effect in vivo is smaller, and retention time may be longer in vivo；It adopts The CAR-T cell MOCF-ICOSBBZ prepared with above-mentioned humanized antibody compared to the MOCB-ICOSBBZ delivered before, The flat cytokine release of bottom water is lower, and safety can be more preferable in clinical application；Pass through the EpCAM albumen on targets neoplastic cells surface And the signal path in T cell downstream is activated, and the ability that T cell killing has EpCAM target tumor cell is assigned, it can be efficiently special Treatment EpCAM positive tumor.

Detailed description of the invention

Fig. 1 is that EpCAM specific chimeric antigen receptor (CAR) molecular structure illustrates to illustrate in one embodiment of the invention Figure；

Fig. 2 is the knot of Flow cytometry MOCF/MOCB-CAR transduction T cell transduction efficiency in one embodiment of the invention Fruit schematic diagram；

Fig. 3 is the schematic diagram of EpCAM expression in different tumor cell lines in one embodiment of the invention；

Fig. 4 is IFN-γ and IL2 secretion after EpCAM CAR-T cell killing EpCAM target cell in one embodiment of the invention The schematic diagram of situation；

Fig. 5 is the result schematic diagram that EpCAM CAR-T cells in vitro kills EpCAM target cell in one embodiment of the invention；

Fig. 6 is the result schematic diagram that EpCAM CAR-T cell is proliferated in Mice Body in one embodiment of the invention.

Fig. 7 is after killing EpCAM positive graft tumour in EpCAM CAR-T cell mouse body in one embodiment of the invention Survivorship curve schematic diagram.

Specific embodiment

The present invention relates to a kind of Chimeric antigen receptor for targeting EpCAM, including cog region, hinge area, transmembrane region, letters intracellular Number area, the extracellular identification region sequence be to identify the variable domain antibodies MOCF of the Humanized single chain of EpCAM, and amino acid sequence is such as Shown in SEQ ID NO.3；Specifically, the Chimeric antigen receptor includes the amino acid sequence as shown in SEQ ID NO.13.This Invention further relates to encode the related application and preparation method thereof of above-mentioned Chimeric antigen receptor.

With reference to the accompanying drawings and examples, further description of the specific embodiments of the present invention.Following embodiment is only For clearly illustrating technical solution of the present invention, and not intended to limit the protection scope of the present invention.

Embodiment one

The present embodiment is the building for targeting the Chimeric antigen receptor molecule of EpCAM comprising following steps:

It is primarily based on the monoclonal antibody MOC31 of targeting EpCAM, is carried out humanization modified.It is humanization modified to pass through ability The conventional CDR region transplantation method in domain carries out, referring to document (Jones PT, Dear PH, Foote J, et al.Replacing the complementarity-determining regions in a human antibody with those from a mouse.Nature.1986May 29-Jun 4；321(6069):522-5.；Sandhu JS.A rapid procedure for the humanization of monoclonal antibodies.Gene.1994Dec15；150(2):409-10.). By entering the CDR region transplanting of MOC31 in the framework region of the higher source of people variable region of homology, humanized antibody name is obtained For MOCF.And by its sequence alterations be single-stranded variable region sequences MOCF scFv；It is mono- using the EPCAM in paper simultaneously Chain variable region sequence MOCB scFv is as reference antibody sequence (Ang WX, Li Z, Chi Z, et al.Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis.Oncotarget.2017Feb 21；8(8): 13545-13559.doi:10.18632/oncotarget.14592.).Targeting is constructed based on MOCF or MOCB later The CAR molecule of EpCAM, structure is third generation CAR structure, successively by the signal peptide of CD8, MOCF MOCB scFv, CD8 hinge area, ICOS transmembrane region and ICOS intracellular region, 4-1BB intracellular region, CD3 ζ intracellular region are sequentially connected in series, and structure is shown It is intended to as shown in Figure 1.It is referred to as MOCF-ICOSBBZ or MOCB-ICOSBBZ.By MOCF-ICOSBBZ or MOCB- On slow virus carrier pHAGE-EF1A-MCS, cloning site NotI/ClaI is configured to clone ICOSBBZ molecule construction PHAGE-EF1A-MOCF-ICOSBBZ or pHAGE-EF1A-MOCB-ICOSBBZ.

Embodiment two

The present embodiment is the building of MOCF/MOCB-ICOSBBZ CAR-T cell comprising following steps:

The packaging of CAR slow virus: plasmid kit is mentioned greatly using QIAGEN endotoxin-free first and extracts the slow of building respectively Virus particle pHAGE-EF1a-MOCF-ICOSBBZ, pHAGE-EF1a-MOCF-ICOSBBZ and slow virus system auxiliary packaging matter Grain pMD2.G and pSPAX2.1.8 × 10E7 293T is spread into T175 culture bottle in the day before transfection.It transfection first 1 hour will 293T cell culture medium is changed to 30ml serum free medium.Using calcium phosphate precipitation by plasmid co-transfection into 293T cell, Cell culture medium was changed to 60ml complete medium DMEM+10%FBS in 24 hours after transfection.It collects on cell within 48 hours after transfection Clearly, and 60ml fresh complete medium is added.Cell conditioned medium is collected again within 72 hours, discard cell.The cell conditioned medium that will be collected 5000g is centrifuged 3min to remove impurity, uses 0.45um membrane filtration later, and subsequent 40000g is centrifuged 4 hours, precipitate virus, Virus is resuspended using 0.1ml PBS, detects virus titer.- 80 DEG C of placement freezes spare.

T cell transduction: mentioning the previous day coating retronectin, anti-human CD3 and CD28 antibody in 6 orifice plates, and 4 DEG C are overnight, It is washed with preceding PBS spare twice.It is taken a blood sample using conventional method and separates PBMC, sorted using STEMCELL T cell sorting kit T cell counts, and will obtain T cell and is resuspended in by 1 × 10E6/ml density containing 5% people AB serum and 100IU/ml leucocyte Jie In the X-VIVO15 culture medium of element -2, it is placed in coated culture plate culture.Start culture 24 hours later, is added 5ug/ml's Simultaneously corresponding slow virus (MOCF-ICOSBBZ, MOCB-ICOSBBZ) is added by MOI3 in polybrene, mixes, 37 DEG C of infection 24 are small When.It is then centrifuged for collecting cell precipitation, is changed to the X-VIVO15 culture of 5% people AB serum and 100IU/ml interleukin 2 Base culture.Cell is maintained at 1 × 10E6/ml density by supplemented medium by follow-up cultivation, and fluidic cell is utilized after 72 hours Art detects scFv expression to detect CAR molecule transduction efficiency.CAR-T-MOCF-ICOSBBZ and CAR-T-MOCB-ICOSBBZ make It is detected with protein L/PE-SA.As shown in Fig. 2, observing CAR-T-MOCF-ICOSBBZ and CAR-T-MOCB-ICOSBBZ Positive rate about in 55%-75%, T cell is as control cell.

Embodiment three

The present embodiment is the identification of EpCAM target cell and the measurement of EpCAM CAR-T cell activity.

The identification of EpCAM target cell: in order to detect EpCAM CAR-T whether can effective killing tumor cell, reflect first The expression of different tumor cell surface EpCAM is determined, the results show that lung cancer cell line H1650 high expresses EpCAM, and lung cancer is thin Born of the same parents system A549 does not express EpCAM in surface, as a result as shown in Figure 3.

IFN-γ and IL2 secretion experiment: in 96 orifice plates that two kinds of CAR-T of MOCF-ICOSBBZ, MOCB-ICOSBBZ are thin Born of the same parents and control three kinds of effector cells of T cell and target cell H1650 and compare target cell A549 mixing is compared with 5:1 effect target.At 37 DEG C Culture 24 hours, later using the expression of IFN-γ and IL2 in standard ELISA assay detection supernatant.As a result as shown in figure 4, working as When two kinds of effector cells of MOCF-ICOSBBZ and MOCB-ICOSBBZ and H1650 target cell co-culture, CAR-T cell can largely divide IFN-γ and IL2 are secreted, and the secretory volume of the two is suitable, but MOCF-ICOSBBZ background burst size only has MOCB-ICOSBBZ sheet The one third of bottom burst size is lower, illustrates that MOCF-ICOSBBZ CAR-T cell constructed by the present invention was applied in vivo There is higher safety, survival ability is stronger in journey；When effector cell, which compares target cell with A549, to be co-cultured, MOCF- ICOSBBZ CAR-T cell only secretes a small amount of IFN-γ and IL2, illustrates that CAR-T cell constructed by the present invention has good target To specificity.

Example IV

The present embodiment verifies the ability of EpCAM CAR-T cells in vitro killing EpCAM target cell.

By the target cell H1650 of CAR-T cell and T cell and the EpCAM positive in embodiment two and right in 96 orifice plates According to target cell A549 with 1:1, the effect target of 5:1,10:1 are co-cultured than mixing, while culture medium blank control group and target cell is arranged Control group.It is cultivated at 37 DEG C 24 hours, uses CCK8 method OD450 light absorption value later.As a result see Fig. 5.MOCF-ICOSBBZ CAR-T and MOCB-ICOSBBZ CAR-T cell can effectively kill EpCAM positive target cell H1650, MOCF-ICOSBBZ The killing activity of CAR-T cell is better than MOCB-ICOSBBZ CAR-T cell, and the two all cannot effectively kill EpCAM negative control Target cell A549 cell, control T cell cannot effectively kill H1650 and A549 cell.

This example demonstrates that the CAR-T cell of two kinds of CAR molecule constructions of MOCF-ICOSBBZ and MOCB-ICOSBBZ can It effectively kills the cancer cell of the EpCAM positive and there is good EpCAM targeting, in addition the Cytotoxicity in vitro of MOCF-ICOSBBZ Activity is better than MOCB-ICOSBBZ.

Embodiment five

The present embodiment verifying CAR-T cell inhibits the ability of EpCAM positive tumor growth in mouse subcutaneous tumors model.

Using 6 week old NOD-SCID Immune deficient mices, 3 × 10E6 H1650 cell line is subcutaneously injected, allows tumour growth 7-15 days, tumor size is measured with caliper, obtains one divided by 2 with the length that tumour extreme length multiplies extreme length vertical direction Mm3 is the tumor size of unit.When tumor size reaches 20-50mm3, by intravenous injection 5 × 10E6 control T cell or The MOCF-ICOSBBZ CAR-T cell of the 5 × 10E6 CAR positive does not find that apparent CAR-T causes during entire research Toxic side effect.Tail vein is acquired after feeding back 1 week, and detection is in mouse blood CAR-T cellular level and to measure tumour big It is small.

Mouse tail vein blood 20ul is acquired into 20ul sodium heparin anticoagulant, the anti-human CD45 antibody of 1ul is added in each sample, Incubation at room temperature 10 minutes is added 500ul erythrocyte cracked liquid and splits red 5 minutes or so, cell precipitation is collected by centrifugation, and PBS is washed once, Flow cytomery.As a result as shown in fig. 6, CAR-T cell and T cell can be all proliferated in mouse blood.It can from Fig. 7 To find out, tumour starts to become smaller after injection MOCF-ICOSBBZ CAR-T cell 12 days, has substantially completely removed by 23 days Tumour, control T cell can not be such that tumour cell reduces.

This example demonstrates that the CAR-T cell of targeting EpCAM of the invention can effectively remove mouse EpCAM positive skin Tumour cell in lower Transplanted tumor model.The tumour of MOCF-ICOSBBZ CAR-T group (6) mouse is all removed, and CR is reached Standard, effective percentage 100%.In conclusion MOCF-ICOSBBZ CAR-T cell can be safely and effectively clear in animal body Except EpCAM positive tumor.

Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention is not intended to limit In particular embodiments described above.To those skilled in the art, any principle according to the present invention is modified and is replaced In generation, also can achieve effect same.Therefore, made equal transformation and modification without departing from the spirit and scope of the invention, all It should cover within the scope of the invention.

Sequence table

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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 Gln Met Thr Gln Ser Pro Ser

20 25 30

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

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 (Artificial Sequence) of MOCB-ICOSBBZ is encoded

<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 (Artificial Sequence) of MOCF-ICOSBBZ is encoded

<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. a kind of Chimeric antigen receptor for targeting EpCAM, including cog region, hinge area, transmembrane region, intracellular signal area, feature It is, the extracellular identification region sequence is to identify that the Humanized single chain of EpCAM can be changed domain antibodies MOCF, and amino acid sequence is such as Shown in SEQ ID NO.3.

2. a kind of Chimeric antigen receptor for targeting EpCAM according to claim 1, which is characterized in that the hinge area is The region CD8hinge.

3. a kind of Chimeric antigen receptor for targeting EpCAM according to claim 2, which is characterized in that the CD8hinge The amino acid sequence in region is as shown in SEQ ID NO.4.

4. a kind of Chimeric antigen receptor for targeting EpCAM according to claim 1, which is characterized in that the transmembrane region is Any of CD8 transmembrane region, CD28 transmembrane region, ICOS transmembrane region.

5. a kind of Chimeric antigen receptor for targeting EpCAM according to claim 4, which is characterized in that the CD8 transmembrane region Amino acid sequence as shown in SEQ ID NO.5, the amino acid sequence of the CD28 transmembrane region is as shown in SEQ ID NO.6, institute The amino acid sequence of ICOS transmembrane region is stated as shown in SEQ ID NO.7.

6. a kind of Chimeric antigen receptor for targeting EpCAM according to claim 1, which is characterized in that the intracellular signal Area includes at least one of CD28,4-1BB, ICOS, CD3 ζ.

7. a kind of Chimeric antigen receptor for targeting EpCAM according to claim 6, which is characterized in that the ammonia of the CD28 Base acid sequence is as shown in SEQ ID NO.8, and the amino acid sequence of the 4-1BB is as shown in SEQ ID NO.9, the ammonia of the ICOS Base acid sequence is as shown in SEQ ID NO.10, and the amino acid sequence of the CD3 ζ is as shown in SEQ ID NO.11.

8. a kind of Chimeric antigen receptor for targeting EpCAM according to claim 1, which is characterized in that the chimeric antigen Receptor includes the amino acid sequence as shown in SEQ ID NO.13.

9. a kind of coding such as the nucleic acid of the Chimeric antigen receptor of targeting EpCAM according to any one of claims 1 to 8.

10. nucleic acid according to claim 9, which is characterized in that the nucleotide sequence of the nucleic acid includes SEQ I D Nucleotide sequence shown in NO.15.

11. a kind of recombinant expression carrier containing the nucleic acid as described in any one of claim 9~10.

12. a kind of host cell containing recombinant expression carrier as claimed in claim 11.

13. a kind of construction method of host cell as claimed in claim 12, which is characterized in that including recombinant expression carrier Construction step, the packaging step of recombinant expression carrier and the step of recombinant expression carrier is transduceed to host cell.

14. the Chimeric antigen receptor of EpCAM of any of claims 1-8, described in any one of claim 9-10 Nucleic acid, host cell described in recombinant expression carrier and claim 12 described in claim 11 is in preparation human entity Application in tumor therapeutic agent.