CN107033248A

CN107033248A - Recognize Chimeric antigen receptor and its application of carcinomebryonic antigen

Info

Publication number: CN107033248A
Application number: CN201710305078.8A
Authority: CN
Inventors: 不公告发明人
Original assignee: Chongqing Precision Biological Technology Co Ltd
Current assignee: Chongqing Precision Biological Technology Co Ltd
Priority date: 2017-05-03
Filing date: 2017-05-03
Publication date: 2017-08-11
Anticipated expiration: 2037-05-03
Also published as: CN107033248B

Abstract

The invention belongs to genetic engineering field, and in particular to a kind of Chimeric antigen receptor of identification carcinomebryonic antigen and its application.The Chimeric antigen receptor of the anti-carcinoembryonic antigen of the present invention, by the single-chain antibody (scFV) of identification carcinomebryonic antigen, hinge area, transmembrane region and intracellular signal domain are sequentially connected composition；The amino acid sequence that the amino acid sequence of the single-chain antibody of the identification carcinomebryonic antigen contains M5A scFV or the amino acid sequence by being obtained to M5A scFV polypeptides progress random point mutation.The Chimeric antigen receptor recognizes carcinomebryonic antigen, can be more stable be expressed in T lymphocytes, it can maintain to target CEA positive rate of the Chimeric antigen receptor in patient's cell cultivation process and CAR T propagation can be strengthened and the ability of tumour is killed, and to the cytotoxic side effect of antigen negative, it can be used in the targeted therapy of tumour；And humanization degree is high, CAR immunogenicity, the lasting and securities of enhancing CAR T in vivo can be effectively reduced.

Description

Recognize Chimeric antigen receptor and its application of carcinomebryonic antigen

Technical field

The invention belongs to genetic engineering field, it is related to Chimeric antigen receptor and its application of identification carcinomebryonic antigen, further relates to The slow virus carrier of Chimeric antigen receptor comprising identification carcinomebryonic antigen (CEA) scFV and application.

Background technology

The generation technology of monoclonal antibody antibody experienced three phases：The heterologous Anti-TNF-α that classical immunization method is produced Body；The human monoclonal antibody that the mouse resource monoclonal antibody and genetic engineering that cell engineering is produced are produced.With mouse source Dan Ke Grand antibody is used for disease treatment, when directly carrying out human body therapy using mouse antibodies, because the anti-heterogeneity of mouse can cause people to resist Mouse antibody response (Human anti-mouse antibody reaction, HAMA), causes antibody half life short, in cyclic system Removed quickly in system, lose curative effect.Therefore, treatment is needed to carry out humanization modification to improve the people source of antibody with mouse source monoclonal antibody Change degree, decrease HAMA.

Single-chain antibody (Single-chain variable fragment (scFv)) is variable region (VH and the VL by antibody Area) it is formed by connecting by the small peptide of 15-20 amino acid, scFv can preferably retain its affine activity to antigen, and have Molecular weight is small, penetration power is strong and it is antigenic weak the features such as.

Humanized antibody refers to the variable region portion (i.e. VH and VL areas) or antibody of antibody all by human antibody gene institute Coding.Humanized antibody can greatly reduce the immune side reaction that heterologous antibody is caused to human body, the shape of humanized antibody Formula also progressively develops into humanized antibody from initial chimeric antibody, reshaping antibody etc..

Transforming mouse source antibody as humanized antibody has following several principal modes：Mouse variable region (Viarable Region, VRs)+human constant region (Constant region, CRs), mouse complementary determining region (Complementarity Determining region, CDRs)+people's framework region (Fragment regions, FRs), mouse CDRs+ people FRs etc., especially So that " mouse CDRs+ people's FR " patterns are most commonly seen, and this humanized antibody form only remains the mouse source residue in CDRs, and incites somebody to action It is the anti-residue of people that remainder, which is replaced, the heterologous reaction that mouse resists can be dropped into reduced levels.But directly move into the anti-CDRs of mouse The anti-FRs of people normally results in antibody and loses original antigen-binding activity, it is therefore desirable to which what influence antigen-antibody was combined is crucial residual Base is changed repeatedly, is then detected by a large amount of antigen-antibody binding specificities, affinity, is obtained active so as to screen Antibody sequence.

Adoptive cellular treatment (adoptive cell therapy, ACT) is one kind of biological therapy technology, is exempted to autologous Epidemic disease cell (mainly T cell) carries out amplification in vitro, is then fed back to tumor patient to reach the method for therapeutic purposes, quilt It is considered after the 4th kind of therapeutic modality after operation, Radiotherapy chemotherapy, by extensive use in clinical treatment.Adoptive cellular treatment is wide General application mainly：Lymphatic circulation (lymphokine activated killer cell, LAK) with IL-2 therapeutic alliance late malignant tumours achieve certain curative effect；Tumor infiltrating lymphocyte (tumor infiltrating Lymphocytes, TIL) treatment metastasis melanin tumor achieves preferable effect in clinical test；Cytokine induction is killed Hinder cell (cytokine induced killer cell, CIK), it is domestic at present to carry out more clinical test, to liver cancer, lung The tumours such as cancer achieve significant effect.But above-mentioned three kinds for the treatment of methods are both needed to activating T cell at present, T cell activation needs The molecule of TCR-CD3 and MHC- I of two kinds of activation signalses, i.e. T cell surface is combined into the first signal of activation, determines T cell pair The killing activity of tumour cell；The costimulatory molecules on T cell surface is combined into the secondary signal of activation with respective ligand, determines T Cell is bred.But tumour cell, tumor microenvironment can lower MHC, ligand molecular, so as to suppress killing for T cells against tumor cells Wound activity.Therefore need that genetic modification can be carried out, mainly there is two ways：Gene transfer TCR (T cell receptor, TCR) With Chimeric antigen receptor (chimeric antigen receptor, CAR).Chimeric antigen receptor (chimeric antigen Receptor, CAR) it is the artificial receptors for simulating TCR functions, by antigen recognition domain, hinge area and transmembrane region and intracellular signal domain Composition is sequentially connected, intracellular signal domain is usually CD3 ζ chains or FcR γ, or is connected with one or more costimulatory moleculeses, such as 4- 1BB (CD137), CD28, ICOS (CD278).The antigen (acceptor) and the antibody of Chimeric antigen receptor of tumor cell surface (are matched somebody with somebody Body) when combining, intracellular is transmitted signals to by hinge area and transmembrane region, intracellular signal domain converts the signal to activation signals, Activation effect cell, effector cell's propagation, generation cell factor are so as to killing tumor cell.Chimeric antigen receptor is transformed compared with TCR More advantage：(1) it is specific：Antibody (part) specific recognition antigen (acceptor)；(2) efficiency high：Be not in transgenosis TCR Occurs mispairing with patient's endogenous TCR；(3) non-MHC- I is restricted：It need not be combined with the molecules of MHC- I, tumour can be overcome thin Born of the same parents, tumor microenvironment lower the immunologic escape that the molecules of MHC- I are caused；(4) antigen range of choice is wide：Antigen can be carbohydrate, fat Class, albumen.

In recent years, T lymphocytes Chimeric antigen receptor (CAR-T) treatment is presented in oncotherapy significantly controls curative effect In terms of the evil or angry countenance tumour or leukaemia of the fruit especially treatment CD19 positives；The research for carrying out solid tumor using CAR-T is also continuous Paid attention to, but the effect of most of CAR-T treatment solid tumors is but and unsatisfactory, CAR-T is for treating solid tumor also Need more explorations.ScFV (single-chain antibody) plays decisive for CAR antigen-binding affinity and CAR function Effect, optimal scFV and CAR combination can cause CAR-T treatments more effective safer, therefore scFV in CAR structures It is very necessary and significant to optimize for the CAR-T applications treated.

Relative to the ability of killing tumour in CAR-T treatment, security and CAR-T can validity identification tumour Cell is the factor of more important consideration.A series of to monoclonal antibody progress amino acid mutation generation have to same epitope The antibody of different affinity, builds CAR-T cells, and wherein scFV presents more effective antitumous effect and preferably safety Property.Different effects are had for different epitopes or the scFV structures CAR for coming from different hybridomas, but it is high close There is higher tumor-killing effect with the scFV-CAR of power.

CEA is also known as carcinomebryonic antigen, its non-discovery table in most of normal structure of the mark detected as colorectal cancer Reach, there is CEA preferable tumour-specific can be treated as a good antigenic targets applied to CAR-T.We are The trial of anti-CEA tumours is being carried out using CAR-T cells, but is not finding the alleviation effective to tumour, and it is thin in patient Find that some patient's cell CAR positive rates are notable as the extension of incubation time declines after dysuria with lower abdominal colic dye CAR during culture. Because some scFV can assemble or carry out independent antigen signals conduction, it can weaken after being combined with CAR or even interference CAR-T The function of cell, it is presumed that be due to the scFV BW431/26 not most suitable combination CAR of utilization scFV, therefore we The scFV for wishing to search out most suitable combination CEA-CAR is applied to express the treatment of CEA tumour patient, is the clinic of solid tumor Using the new thinking of offer.

The scFV in mouse source can cause HAMA (human anti-mouse antibodies) or HACA in human body (cellular anti-CAR immune responses) reacts, and the scFV of humanization can be effectively reduced the immune of CAR Originality, the lasting and securities of enhancing CAR-T in vivo.Therefore select scFV when we have selected humanization anti-CEA resist Body design prepares scFV.

Our antibody design personnel are when arranging CEA humanized antibody data, for being mixed in a crystalline substance therein The article of body structure elucidation generates keen interest, and the crystal structure is perceived by its abundant antibody design experience acumen Corresponding scFV perfect and CAR can be combined very much, and according to the antibody structure, we devise its scFV and inserted it into Tested in three generations's CAR structures.By being contrasted with a variety of scFV, the dimension of CAR positive rates after different scFV-CAR transfections is detected Hold, CAR-T cell phenotypes, CAR-T cells propagation and the killing ability to tumour cell determine the Anti-CEA-scFV for most The suitable scFV combined with CAR, the scFV derive from anti-CEA monoclonal antibodies M5A, and it can not only maintain CAR thin in patient Positive rate in born of the same parents' incubation and CAR-T propagation can be strengthened and the ability of tumour is killed；Pacify for clinical treatment The consideration of full property, our technical staff is further to M5A-scFV to have carried out humanization improved construction SDR grafted antibodies, really Security of the CAR-T cells in human body is protected and its sustainable survival in human body can be strengthened.

Currently for the expression CEA also no therapeutic scheme well of solid tumor, antigen Chimerical receptor is transfected in T cell Stability also without good solution, do not report display M5A-scFV combination antigen Chimerical receptors to chimeric antigen yet The stability of recipient cell and the influence to CEA solid tumor curative effects.

The content of the invention

In view of this, an object of the present invention is to provide a kind of Chimeric antigen receptor for recognizing carcinomebryonic antigen and its answered With the Chimeric antigen receptor of, identification carcinomebryonic antigen of the invention can be more stable be expressed in T lymphocytes, with preferably clear Except the ability of tumour cell.

To achieve the above object, the technical scheme is that：

The Chimeric antigen receptor of anti-carcinoembryonic antigen, by the single-chain antibody (scFV) of identification carcinomebryonic antigen, hinge area, transmembrane region Composition is sequentially connected with intracellular signal domain；The amino acid sequence of the single-chain antibody (scFV) of the identification carcinomebryonic antigen contains M5A- ScFV amino acid sequence or the amino acid sequence by being obtained to M5A-scFV polypeptides progress random point mutation.

M5A-scFV amino acid sequence such as SEQ ID NO:Shown in 12.

Further, the amino acid sequence such as SEQ ID NO of the single-chain antibody (scFV) of the identification carcinomebryonic antigen:12 or SEQ ID NO:13 or SEQ ID NO:14 shown or SEQ ID NO:Shown in 15.

Described Chimeric antigen receptor needs to go beyond two technology barriers, and one is to find more stable effective identification cancer embryo to resist Former scFV, two be that optimal scFV polypeptides are carried out into humanization processing.

Why to be transformedBecause the Chimeric antigen receptor for the identification carcinomebryonic antigen that we use at present is when applying The T lymphocytes for being expressed in T lymphocytes especially patient source can not be stablized, with the extension T of incubation time after transfection The Chimeric antigen receptor expression of cell surface is significantly reduced.The higher scFV of humanization degree can be effectively reduced exempting from for CAR Epidemic focus, the lasting and securities of enhancing CAR-T in vivo.

The factor of influence Chimeric antigen receptor expression stability essentially consists in scFV and built used during scFV Hinge sequences.When selecting scFV, we have selected known CEA antibodie design and prepare scFV.Mouse source antibody is transform as Humanized antibody has following several principal modes：1) mouse variable region (Viarable region, VRs)+human constant region (Constant region, CRs)；2) mouse complementary determining region (Complementarity determining region, CDRs) + people framework region (Fragment regions, FRs)；3) mouse CDRs+ people FRs etc..How the antibody of identification carcinomebryonic antigen is transformed, Enable preferably to retain its affine activity to antigen, and be combined as optimal identification CEA (carcinomebryonic antigen) inosculating antibody Original receptor, is a problem.Inventor team will at present by exclusive method in the case where not there is more effective technique prompting 4 anti-CEA monoclonal antibodies of report all prepare scFV and build CAR (Chimeric antigen receptor) by different combinations Screened.

The method for transforming polypeptide, is to retain in CDR region without mutation.There is randomness in the result of method, only with Put it is proper in the case of, its affinity is possible to suitable with the affinity of original mouse antibodies.And the side that the present invention is protected Method, is the method for the anti-method pushed away, i.e. " wise afterwards " formula after preferable result is known.

In the Chimeric antigen receptor described in protection, we also eliminated chimeric antigen under three groups of other target spot thinkings by Body, but other three groups are unqualified because of affinity test experiments, suffer from eliminating.And this only protected by the present invention Group, with unexpected technique effect.

The CAR of this part protection combination, after test, can play the stable T for being expressed in patient source Lymphocyte, and with the ability for preferably removing tumour cell, for the adoptive cellular treatment for solid tumor.

Further, the amino acid sequence of hinge area such as SEQ ID NO:Shown in 17.

Further, such as SEQ ID NO of the amino acid sequence of transmembrane region described in the transmembrane region:18 or SEQ ID NO:19 institutes Show.

Further, the intracellular signal domain be CD3 ζ chains and/or FcR γ and/or CD137 and/or CD28 and/or CD278.Intracellular signal domain is usually CD3 ζ chains or FcR γ, or is connected with one or more costimulatory moleculeses, such as 4-1BB (CD137), CD28, ICOS (CD278).CD137 nucleotide sequence such as SEQ ID NO:Shown in 7, CD28 nucleotide sequence Such as SEQ ID NO:Shown in 6, CD3 ζ nucleotide sequence such as SEQ ID NO:Shown in 8.

As a preferred embodiment, the intracellular signal domain is followed successively by CD28, CD137 and CD3 ζ.

When the antigen (acceptor) of tumor cell surface is combined with the antibody (part) of described Chimeric antigen receptor, pass through hinge Sequence and transmembrane region transmit signals to intracellular, and intracellular signal domain converts the signal to activation signals, activation effect cell, effect Cell propagation, generation cell factor are so as to killing tumor cell.Chimeric antigen receptor has more advantage compared with TCR transformations：(1) it is special Property：Antibody (part) specific recognition antigen (acceptor)；(2) efficiency high：Be not in transgenosis TCR and patient's endogenous TCR Generation mispairing；(3) non-MHC- I is restricted：It need not be combined with the molecules of MHC- I, tumour cell, tumor microenvironment can be overcome to lower The immunologic escape that the molecules of MHC- I are caused；(4) antigen range of choice is wide：Antigen can be carbohydrate, lipid, albumen.

Further, the nucleotide sequence of the Chimeric antigen receptor such as SEQ ID NO:1、SEQ ID NO:2、 SEQ ID NO:3、SEQ ID NO:Shown in 4.

The Chimeric antigen receptor of the present invention, it can be used for treating for the adoptive cellular of solid tumor.

Many reports are mentioned：Adoptive cellular treatment effect in leukaemia is notable, but the CAR-T clinics of solid tumor are controlled Therapeutic effect is limited.We confirm that not engineered single-chain antibody causes existing anti-CEA CAR positive rates by previous experiments It is low, it is very unstable in patient's cell cultivation process, and CAR positive rates can be caused to decline over time, Jin Erying Ring clinical therapeutic efficacies of the CAR to colorectal cancer.And after we are transformed the amino acid sequence of single-chain antibody, overcome State problem.

The second object of the present invention is the preparation method of the slow virus carrier of described Chimeric antigen receptor, specifically includes Following steps：

1) gene order of the Chimeric antigen receptor of single-chain antibody (scFV) of the synthesis containing identification carcinomebryonic antigen：Synthesis according to Secondary different single-chain antibody ScFv, hinge area, transmembrane region and intracellular signal domain containing leader peptide, anti-human CEA antigens；The hinge Area's nucleotide sequence such as SEQ ID NO:5th, transmembrane region nucleotide sequence such as SEQ ID NO:9th, intracellular signal domain nucleotide sequence such as SEQ ID NO:6、SEQ ID NO:7 and SEQ ID NO:8；

2) slow virus carrier of construction expression Chimeric antigen receptor：Design primer, the nucleotide sequence such as SEQ of forward primer ID NO:Shown in 20, the nucleotide sequence such as SEQ ID NO of reverse primer:Shown in 21, with the sequence of the Chimeric antigen receptor Enter performing PCR amplification for template, obtain DNA fragmentation；

By the gene order of DNA fragmentation restriction enzyme NheI and SalI double digestion, while with restricted interior Enzyme cutting NheI and SalI digestion Lentiviral pCDH-EF1, purpose fragment and slow virus expression after then plum is cut are carried Body fragment is attached by T4 ligases, obtains the slow virus carrier of expression Chimeric antigen receptor.

Further, in step 2) after, pack and purify the slow virus carrier.

The third object of the present invention is to provide the slow virus carrier obtained by a kind of described preparation method.

Described slow virus carrier is obtained under described method, the positive expression rate of such slow virus carrier is high, It is very stable in patient's cell cultivation process, and CAR positive rates will not can be caused to decline over time.Then, institute is used The T cell of slow virus carrier infection is stated, such T cell possesses the function of killing target cell.

The present invention also aims to provide a kind of T cell of described slow virus carrier infection.

The present invention also aims to provide a kind of described T cell answering in the medicine for preparing treatment solid tumor With.

Further, the cell of described solid tumor can express CEA.CEA is also known as carcinomebryonic antigen, is detected as colorectal cancer Mark its non-discovery table in most of normal structure reach, CEA has preferable tumour-specific can be as one very Good antigenic targets are treated applied to CAR-T.

Further, the solid tumor is colorectal cancer tumour.

In addition, returning to a source for invention, the present invention also aims to provide a kind of described identification carcinomebryonic antigen Application of the amino acid sequence of single-chain antibody (scFV) in the medicine for preparing treatment solid tumor.The identification carcinomebryonic antigen Single-chain antibody (scFV) amino acid sequence such as SEQ ID NO:14 shown or SEQ ID NO:Shown in 15.The transformation it is many Fragments of peptides, before it is relative to being modified, with more the characteristic of humanized antibody.

Further, the cell of described solid tumor can express CEA.

The present invention also aims to provide a kind of amino acid of the single-chain antibody (scFV) of described identification carcinomebryonic antigen Sequence is for preparing the application precisely captured in can expressing the carrier of CEA solid tumor cells.Except cell therapy, also may be used For radiation treatment.The amino acid sequence such as SEQ ID NO of the single-chain antibody (scFV) of the identification carcinomebryonic antigen:12 Or SEQ ID NO:13 or SEQ ID NO:14 shown or SEQ ID NO:Shown in 15.The ammonia is the polypeptide fragment of transformation, its Before being modified, with more the characteristic of humanized antibody.

It is a kind of such as SEQ ID NO the present invention also aims to provide:12 or SEQ ID NO:Amino acid sequence shown in 13 It is listed in the application for preparing the antigen recognition domain in CAR-T skeletons.

Generally speaking, not only can be with after scFV-CAR Chimeric antigen receptors of the present invention are expressed in immunocyte Targeting CEA Chimeric antigen receptor (chimeric antigen receptor, CAR) is maintained in patient's cell cultivation process Positive rate and CAR-T propagation can be strengthened and the ability of tumour is killed, and the cytotoxic of antigen negative pair is made With can be used in the targeted therapy of tumour.

The beneficial effects of the present invention are：

1) the Chimeric antigen receptor identification carcinomebryonic antigen that the present invention is provided, can be more stable be expressed in T lymphocytes, tool There is the ability for preferably removing tumour cell, can not only maintain the Chimeric antigen receptor for targetting CEA in patient's cell culture Positive rate in journey and CAR-T propagation can be strengthened and the ability of tumour is killed, the cytotoxic pair to antigen negative is made With can be used in the targeted therapy of tumour.

2) the Chimeric antigen receptor humanization degree by transformation that the present invention is provided is high, can be effectively reduced CAR's Immunogenicity, the lasting and securities of enhancing CAR-T in vivo.

3) Chimeric antigen receptor that the present invention is provided can stablize the T pouring for being expressed in T lymphocytes especially patient source Bar cell, the adoptive cellular that can be used for preparing in the medicine for treating solid tumor for solid tumor is treated.

Brief description of the drawings

Fig. 1 is four groups of different scFv-CAR-T structure charts.

Fig. 2 is affinity measurement results of four groups of difference scFV to CEA target spots, and figure A, B, C and D are respectively M5A, hMN- 14th, the affinity result of BW431/26 and C2-45 and different CEA concentration gradients (KD values more low-affinity is stronger).

With the number of days chimeric antigen of cell culture after the Chimeric antigen receptor transfecting T cells that Fig. 3 merges for four groups of scFV The change of expression of receptor positive rate, ordinate represents the percentage of Chimeric antigen receptor positive expression, and abscissa represents to cultivate day Number.

Fig. 4 is the testing result of the 14th day CAR positive rate of four groups of CEA-CAR-T cells the 8th day.

Fig. 5 is the CAR positive rates detection in the 4th day, the 8th day, the 14th day and the 18th day of CEA-CAR-3 transfecting T cells.

Fig. 6 is four groups of different scFV-CAR-T cells in vitro killing activity detections；Figure A represent killing 24 hours it is enzyme-linked Immunization (ELISA) detects the release of each scFV-CAR-T IFN-γs；Figure B represents that 8 hours CAR-T cells imitate target with target cell Than 6:1 is killing ability of the CAR-T cells to target cell；

Fig. 7 is four groups of different scFV-CAR-T cells design sketch of 8 hours, Fig. 7-A represent 8 hours different effect targets than CAR-T killing ability；Fig. 7-B represent that effect target compares 3:Each CAR-T of 1 different time points killing ability.

Fig. 8 is killing experiments in four groups of different scFV-CAR-T animal bodies；LoVo- is subcutaneously injected in every NOD/SCID (the height expression of LoVo cells CEA, Luc are that firefly luciferase can by small animal imaging instrument in the presence of substrate to Luc cells With the growth in vivo of real-time monitored tumour and distribution situation) 1*10^6 is grouped every group of 4 mouse, every group of difference at random after 6 days Tail vein injection CEA-CAR-1, CEA-CAR-2, CEA-CAR-4, UTD (control T cell), are observed using small animal imaging instrument Growing state of 6th day, the 13rd day, the 20th day and the 27th day tumour cell in Mice Body

Fig. 9 is tumour fluorescence intensity statistics figure.

Figure 10 discharges for IFN-g after the CAR-T cell killings of humanization modified rear difference hscFV compositions.

Figure 11 contrasts for the Scavenging activity of the CAR-T cells against tumor cells of humanization modified rear difference hscFV compositions.Knot Fruit display, which carries out improved CEA-CAR-1-2, the ability for preferably removing tumour cell.

Embodiment

Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.Unreceipted tool in preferred embodiment The experimental method of concrete conditions in the establishment of a specific crime, generally according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. Write) described in condition, or according to the condition proposed by manufacturer.Illustrated embodiment is in order to preferably in the present invention Appearance is illustrated, but is not that present disclosure is only limitted to illustrated embodiment.So those skilled in the art according to Foregoing invention content carries out nonessential modifications and adaptations to embodiment, still falls within protection scope of the present invention.

Part I

This part experiment is obtained by the single-chain antibody to four kinds of different monoclonal antibodies (epitope peptide is different), and The mutation of humanization purpose is carried out to single-chain antibody, the single-chain antibody of mutation is used to prepare Chimeric antigen receptor, and to money and Antigen receptor carries out functional verification experiment.Drawn a conclusion by the experiment of this part：Although " single-chain antibody " can be used in theory In preparation Chimeric antigen receptor, but it is not actually that each single-chain antibody may be used to prepare Chimeric antigen receptor.This needs Inventor pays performing creative labour, and the production with unexpected effect could be found in the single-chain antibody of numerous transformations Product.

The structure of scFV of the embodiment 1 containing M5A, hMN-14, C2-45 and BW431/26 Chimeric antigen receptor virus

In order to prove anti-CEA CAR and other anti-CEA scFV that the CAR ratios for the anti-CEA that we screen are used at present CAR is advantageously, it is therefore desirable to while the scFV-CAR names CEA-CAR-1 of the different anti-CEA Humanized monoclonal antibodies of structure, CEA-CAR-2, CEA-CAR-3 and CEA-CAR-4, it is as follows respectively：

The gene order of the Chimeric antigen receptor of targeting carcinomebryonic antigen of 1 synthesis containing different scFV

Synthesis is successively containing leader peptide (also known as signal peptide) (abbreviation LP), the different single-chain antibodies of anti-human CEA antigens ScFv, IgG4 hinge area (abbreviation IgG4), CD28 transmembrane regions or CD8 transmembrane regions (referred to as TM), its structure is as shown in Figure 1.

The slow virus carrier of 2 construction expression Chimeric antigen receptors

Following primer is designed, and it is synthesized by Nanjing Jin Sirui biotechnologies company, specific primer is as follows：

Primer 1：5’-aggctagcaTgggatggagctgtatcat-3 ', underscore is NheI restriction enzymes position Point；

Primer 2：5’-gattgtcgacTtagcgagggggcagggcctgcatgtga-3 ', underscore is that SalI is restricted Restriction enzyme site.

Then using above-mentioned shown sequence as primer, each Chimeric antigen receptor sequence of above-mentioned synthesis enters performing PCR expansion for template Increase, reaction system is by KOD FX NEO archaeal dna polymerases (being purchased from TOYOBO companies) specification sample-adding, and PCR reaction conditions are 95 DEG C pre-degeneration 5min；95 DEG C of denaturation 10s, 55 DEG C of annealing 15s, 68 DEG C of extension 30s, 30 circulations.By 1% fine jade of amplified production Sepharose is identified.As a result show, amplification obtains about 2000bp DNA fragmentation, then with QIAquick Gel Extraction Kit (Promega Company) DNA fragmentation recovery is carried out, specific method is shown in specification, reclaims and obtains Chimeric antigen receptor, and south is sent by DNA recovery fragment Jing Jinsirui biotechnologies company is sequenced.

The gene order restriction enzyme NheI and SalI that clone the encoding chimeric antigen acceptor obtained (are purchased from Thermo companies) double digestion, while (purchase is extremely with restriction enzyme NheI and SalI digestion Lentiviral pCDH-EF1 Addgene Plasmid#72266), endonuclease reaction by specification is carried out.Digestion products are used after being separated through agarose gel electrophoresis Agarose gel DNA fragment QIAquick Gel Extraction Kit carry out DNA fragmentation recovery, then by purpose fragment and carrier segments connected by T4 Connect enzyme (being purchased from Promega companies) to be attached, obtain the slow virus carrier of expression Chimeric antigen receptor, be named as Lv-scFv (CEA).5 μ l are taken to convert picking monoclonal, the monoclonal of picking after Escherichia coli TOP10,30 DEG C of culture 16h slow virus carrier Plasmid is extracted with plasmid extraction kit (Invitrogen companies) after culture 12h under the conditions of 30 DEG C, specific method is shown in explanation Book.

Four slow virus carriers are built respectively according to as above method (underscore refers to the single-chain antibody of transformation)：

“M5A-scFv-IgG4hinge-CD28TM-CD28-CD137-CD3Z”；

“hMN-14-scFv-IgG4hinge-CD28TM-CD28-CD137-CD3Z”；

“C2-45-scFv-IgG4hinge-CD28TM-CD28-CD137-CD3Z”；

“BW431/26-scFv-IgG4hinge-CD28TM-CD28-CD137-CD3Z”。

The packaging of 3 slow virus

The present embodiment packaging slow virus uses calcium phosphate method, comprises the following steps that：With the DMEM trainings containing 10% (wt) FBS Base culture 293T cells are supported, to preferable states；Then 293T cells are reached to 1 75cm2 training with 1 × 105/cm2 density Support in bottle and cultivate 22h, it is ensured that cell confluency degree is 70-80% during transfection；Again with the cultures of the DMEM containing 2% (wt) FBS of preheating Base changes liquid, cultivates 2h, standby；By 680 μ l ddH2O, 20 μ g slow virus carriers, 20 μ g pMDLg/pRRE plasmids, 20 μ g PRSV-Rev and 10 μ g pMD2.G plasmids, 100 μ l 2.5mM CaCl2 are added in 15ml centrifuge tubes, are well mixed, after mixing 2 × HBS is added dropwise into mixed liquor with pipettor, mixes, is stored at room temperature after mixing while being blown and beaten with 10ml pipettors 15min, then mixed liquor is added dropwise in the cell of above-mentioned preparation, continues to cultivate after 12-16h, with containing 10%FBS (wt) DMEM culture mediums change training liquid, continue to cultivate and collect cell conditioned medium after 48h, 72h respectively and for viral purification.

The purifying of 4 slow virus

Viral supernatants are collected in 50ml centrifuge tubes, 3000r/min centrifugations 10min；Then with 0.45 μm of filter membrane mistake Filter, filtrate centrifuges 10min to new 50ml centrifuge tubes in 3000r/min；Then according to viral supernatants amount, it is separately added into quality point Number is 50% PEG6000 and 4M NaCl, then is settled to 35mL with medical salt solution, makes PEG6000 final concentration of 8.5%, The final concentration of 0.3M of NaCl, it is fixed molten after 4 DEG C of refrigerators standing 90min, 30min/ reverse mixing；Then in 4 DEG C, 5000r/ 30min is centrifuged under the conditions of min, most supernatant is abandoned, virus, 1.5mlEP pipes point is resuspended with DMEM culture mediums of the 200 μ l containing 10%FBS Fill, often the μ l of pipe 40, -80 DEG C save backup.Obtained virus is respectively designated as：

CEA-CAR-1, CEA-CAR-2, CEA-CAR-3 and CEA-CAR-4, sequence are respectively SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4。

5 slow virus titer determinations

Step 1:Virus infection 293T cells

293T cells are taken into purified viral 1 μ l to spread in 24 orifice plates before infection, 10 are diluted with medical saline Times, then 1 μ l polybrenes (Polybrene) solution is added into every hole cell, then by the virus liquid point after 1 μ l and 9 μ l dilutions It is not added in 293T cells, changes liquid after 24h with the DMEM culture mediums containing 10%FBS (wt), 72h is after 1000r/min bars for infection 5min is centrifuged under part to collect cell, extracts genome.

Step 2:Extract genome

Genome extraction agent box is that QIAamp DNA Blood Mini Kit are purchased from Qiagen companies (article No. 511004), operated by kit specification, it is specific as follows：The cell of collection is resuspended with PBS, 1000r/min centrifugation 5min, Most supernatant is abandoned, is repeated 1 times；Cell is resuspended with 200 μ L PBS again, and adds 20 μ l Proteinase Ks, 200 μ l AL lysates, piping and druming Mix, 56 DEG C of incubation 10min；Then the ethanol of 200 μ l precoolings is added, solution is transferred in Filter column by mixing of turning upside down, 1min, 8000r/min centrifugation 1min are stored at room temperature, most supernatant is abandoned；It is subsequently added into 700 μ l AW1 solution, 8000r/min centrifugations 1min, abandons most supernatant；500 μ lAW2 solution are added, 8000r/min centrifugation 1min abandon most supernatant, Filter column is transferred to newly 1.5mlEP pipes in, uncap standing 1min with the ethanol that volatilizees；50 μ l sterilizings ddH2O (60 DEG C of preheatings) is added, 2min is stood, 12000g/min centrifuges 1min.

Step 3:QRT-PCR determines virus titer

Reaction system is as follows：Premix Ex TaqTM II (2 ×) 10 μ l, the μ l of sense primer (GAG up) 1, anti-sense primer (GAG dn) 1 μ l, genome 1 μ l, the RNase-Free dH of extracting₂The μ l of O 7, each sample, standard items at least three are repeated Hole.Then expanded by following program：95 DEG C of pre-degenerations 30s, 95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, 72 DEG C of extension 30s, instead After should terminating, analysis software data are used, virus titer is calculated according to standard curve.Result of calculation shows that virus titer is 1 ×10⁸TU/ml。

Embodiment 2scFV is determined with target antigen (CEA) affinity

Foregoing 4 scFV are marked with His label rear clones and enter plasmid vector and transfected HEK 293, the cell training of transfection Support 6 days and collect all supernatants, supernatant by affinity chromatography after 0.22um membrane filtrations by purifying the scFV- in supernatant His, after purification scFV-His purity reach 95%, scFV-His concentration pass through Bradford hair determine；Different scFV with CEA affinity passes through ForteBioRED96 transactional analysis instrument (Pall, CA, U.S.) is checked that experiment is walked It is rapid to be carried out according to specification.Anti-human igg-Fc (AHC) biology sensor is bought in Pall Fort é according to specification requirement Bio,1512121；Recombinant C EA-Fc concentration is 15ug/ml, and it is 11077- to buy in Sino Biological Inc article No.s H03H-50, CEA-Fc is incubated in advance in biology sensor, scFV and negative control dilute 5 gradients with PBS, with reference to power Student movement is calculated and different scFV is analyzed in CEA affinity using Fort é Bio DAS.Experiment has carried out three It is independent to repeat, as a result as shown in Fig. 2 M5A-scFV has preferable affinity with CEA antigens.

The detection that the Chimeric antigen receptor of embodiment 3 is maintained in T cell

The separation of 1 human peripheral blood mononuclear cell

Peripheral blood about 60ml is gathered with the heparin tube added with anti-coagulants, each 30ml of 50ml centrifuge tubes is sub-packed in, adds 7.5ml HESs dilute；Room temperature (18~25 DEG C) natural subsidence about 30min, collects upper plasma, by the upper strata blood of collection Starch and 15min is centrifuged under the conditions of 1400rpm/min；Then it is resuspended and is precipitated with physiological saline, is by volume 1:1 to be added to lymph thin In born of the same parents' separating liquid, gradient centrifugation, 400g/min, centrifuge reduction of speed is set to 1, centrifuges 20min；After centrifugation, centrifuge tube is by up to Under be divided into：First layer：Plasma layer；The second layer：Ring-type milky buffy coat；Third layer：Transparent separation liquid layer；4th layer： Red blood cell layer；Take the white buffy coat of the second layer, and with brine 2 times, first time 400g/min, centrifugation 10min, Second of 1100rpm/min, centrifuges 5min, and cell is resuspended in physiological saline, adds the RPMI 1640 containing 10%FBS and trains completely Base culture is supported, human peripheral blood mononuclear cell is obtained.

2 slow virus carriers infect T lymphocytes

With the complete medium cultures of the RPMI 1640 freshly prepd mononuclearcell PBMC containing 10% hyclone, the 1st day Add CD 3-resisting monoclonal antibody activation；Carry out slow-virus infection within first 3 days；The corresponding slow virus carriers of 5MOI are added, are uninfected by T lymphocytes are used as blank control；Culture medium is replaced by the RPMI 1640 containing 500IU/ml recombinant human il-2s after 24h complete Full culture medium, continues to cultivate 10-20 days.

Respectively to cultivating to the T cells for having infected virus of 4 days, 8 days, 14 days and 18 days in incubation, 300g/min, centrifuges 5min, abandons most supernatant to collect cell；It is resuspended with the PBS solution containing the hyclone of volume fraction 1% thin Born of the same parents, and be 1 × 106/ml by cell adjustment density；The cell of collection is packed as 6 pipes, often the μ l of pipe 100, into every solencyte Add the recombined human CEACAM5 albumen (Yi Qiao Divine Land, Beijing company) that the His that 5 μ l concentration are 0.3 μ g/ml is marked, 4 DEG C of incubations 30min, PBS solution is washed 2 times, adds the anti-His antibody of mouse (Nanjing Jin Sirui companies) of AF647 marks, 4 DEG C of incubations 30min, PBS solution wash 2 times, flow cytometer is detected, respectively detection cultivate the 4th day, the 8th day, the 14th day and 18th day CAR the positive expression rate, as a result such as Fig. 3, Fig. 4, shown in Fig. 5, is counted as statistical chart as shown in the table, CEA-CAR-1 What can be stablized after transfection T lymphocytes is expressed in T cell surface, and CEA-CAR-3 can not then transfect T cell.

The 4th day, the 8th day, the 14th day and the 18th day CAR the positive expression rate of table

The virus infection CAR-T cells against tumor cells of embodiment 4 kill the influence of ability

Different scFV Chimeric antigen receptor passes through ACEA xCELLigence RTCA MP to the killing ability of target cell Instrument is completed, and experimental procedure is carried out according to instrument specification；First day by target cell (expression CEA tumour cell) with 2-5*10^{^4}It is plated on per hole in 96 orifice plates of apparatus preparation, the tumour cell for being attached to bottom hole is remembered for every 15 minutes using index of resistance as data Record once, is spread into corresponding CAR-T cells after 24 hours according to the effect target that is pre-designed than every hole, record CAR-T cells spread into The index of resistance of 24 hours, collects each group supernatant and carries out enzyme linked immunological (ELISA) detection and pass through Instrumental Analysis after 24 hours Each group index of resistance analysis result, CAR-T cell killings rate=baseline electrical resistance index-real time resistance index, ELISA detections pass through ELISA kits detect IFN-γ, and kit is purchased from BD Biosciences article No.s 4316955.Mortaility results such as Fig. 6 B and Shown in Fig. 7, as a result show that CEA-CAR-1 has the ability for preferably removing tumour cell.

Cytokines measurement

The method that 1 cytokines measurement uses Elisa, is carried out using BD companies kit.

The dilution of 2 standard items：Prepare small test tube 6, number is finished successively, standard items dilution is first added in each small test tube The μ l of liquid 100, then take the μ l of original content standard items 100 to add one in the test tube for the number of finishing, fully mix；Again in the test tube Take 100 μ l to add in second test tube, fully mix；100 μ l are taken to add in the 3rd test tube in the test tube again, it is fully mixed It is even；Take 100 μ l to add in the 4th test tube in the test tube again, fully mix；100 μ l are taken to add the 5th in the test tube again In test tube, fully mix；Then 100 μ l are taken in the test tube, are discarded；6th test tube is used as No. 0 standard items.

3 are marked with quasi- sample wells on enzyme mark coating plate, sequentially add the μ l of standard items 50 of various concentrations, each concentration does 2 Parallel hole.

4 sample-addings：Set respectively blank well (blank control wells are not added with the antibody of sample, enzyme marking reagent and biotin labeling, remaining Each step operation is identical), testing sample hole；The μ l of product 40 are first loaded in testing sample hole on enzyme mark coating plate, it is then again plus biological The μ l of antibody 10 of element mark.Sample is added on ELISA Plate bottom hole portion by sample-adding, is not touched hole wall as far as possible, is gently rocked mixing.

5 incubate：With shrouding film shrouding, rearmounted 37 DEG C incubate 30 minutes.

6 match somebody with somebody liquid：By 30 times of concentrated cleaning solutions with standby after the dilution of 30 times of distilled water.

7 washings：Carefully take shrouding film off, discard liquid, dry, cleaning solution is filled it up with per hole, discarded after standing 30 seconds, so It is repeated 5 times, pats dry；

8 is enzyme-added：Added per hole except the μ l of enzyme marking reagent 50, blank well；

9 incubate：Operation is with 3；

10 washings：Operation is with 5；

11 terminate：Add the μ l of terminate liquid 50, terminating reaction per hole；

12 determine：With blank air-conditioning zero, 450nm wavelength sequentially measures the absorbance (OD values) in each hole, and measure should add end Only carried out after liquid within 15 minutes.

As a result as Fig. 6 A show that the IFN-γ that CEA-CAR-1 and CEA-CAR-2CAR-T cells discharge after killing is higher than CEA-CAR-4。

The CAR-T cells that example 5 infects are straight to the knot of mouse xenografts in NOD-SCID γ -/- (NSG) Mice Body Killing/Scavenging activity of intestinal cancer tumour

NOD-SCID γ -/- (NSG) mouse is bought in BeiJing, China Vital River Laboratory Animal The mouse purchase of Technology Co., Ltd, 6-8 week old every sub-cutaneous injections after SPF grades of Animal Houses are fed one week LoVo-Luc cell 1*10^6, random packet difference tail vein injection CEA-CAR-1, CEA-CAR-2, CEA- after feeding 6 days CAR-4, UTD (control T cell) 1*10^7 cells, the size of measurement tumour per 3-4 days utilizes weekly the observation of small animal imaging instrument Distribution of the tumour in Mice Body, is as a result shown in Fig. 8 Fig. 9, and the T cell of CEA-CAR-1 transfections can preferably suppress or even remove Mouse interior tumor cell.

Part II

This part, which is employed, is not used in another single-chain antibody of Part I as transformation target, obtains several and new changes The polypeptide made, the single-chain antibody of mutation is used to prepare Chimeric antigen receptor, and carry out functional verification examination to Chimeric antigen receptor Test.We are protected the successful data of functional verification by claim.Side of this part with reference to Part I embodiment Method and step are carried out, then this does not just repeat to record.Experimental result such as Figure 10, Figure 11, as a result show the polypeptide scFV- that we transform The CAR that 1-2 is constituted：CEA-CAR-1-1T cells have the ability of more preferable killing tumor cell.

In summary, the as shown by data that we provide is because improved M5A-scFV addition CEA-CAR-1 is in expression There is more preferable effect in stability, the removing and relevant cell factor secretion to tumour cell；We utilize M5A-scFV Mouse source antibody carry out humanization modified acquisition scFV-1-1 (SEQ ID NO:14), scFV-1-2 (SEQ ID NO:15) and scFV-1-3(SEQ ID NO:16) the scFV sequences after three humanizations, further prepare CAR-T cells and are tested, its core Acid sequence such as SEQ ID NO:11 or SEQ ID NO:10 find scFV-1-2 (SEQ ID NO:15) the CAR transfections constituted T cells (SEQ ID NO:10) M5A-scFV is better than to the elimination effect of tumour cell.

Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to skill of the invention Art scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover at this Among the right of invention.

<110>The accurate Bioisystech Co., Ltd in Chongqing

<120>Recognize Chimeric antigen receptor and its application of carcinomebryonic antigen

<160> 21

<170> PatentIn version 3.3

<210> 1

<211> 899

<212> DNA

<213> Artificial

<220>

<223> CEA-CAR-1

<400> 1

aagcttgcta gcctagcatg gccttaccag tgaccgcctt gctcctgccg ctggccttgc 60

tgctccacgc cgccaggccg gacatccagc tgacccagag ccctagctcc ctgtccgcct 120

ctgtgggcga tagggtgacc atcacatgca gagccggcga gtccgtggac atcttcggcg 180

tgggctttct gcactggtat cagcagaagc ccggcaaggc ccctaagctg ctgatctata 240

gggcaagcaa cctggagtcc ggcgtgccct ctaggttcag cggctccggc tctcgcacag 300

actttaccct gaccatcagc agcctccagc cagaggattt cgccacctac tattgtcagc 360

agacaaatga ggacccctac acctttggcc agggcacaaa ggtggagatc aagggatcca 420

cttccggttc aggaaagccc gggagtggtg aaggtagcac taaaggcgag gtgcagctgg 480

tggagtctgg aggaggactg gtgcagccag gaggctctct gcggctgagc tgcgcagcat 540

ccggcttcaa catcaaggac acctacatgc actgggtgcg gcaggcacca ggcaagggcc 600

tggagtgggt ggccagaatc gaccctgcca acggcaattc caagtacgcc gattctgtga 660

agggcaggtt caccatctct gccgatacca gcaagaacac agcctacctc cagatgaata 720

gcctgcgcgc cgaggacaca gccgtgtact attgtgcccc ctttggctac tacgtgagcg 780

attacgccat ggcctattgg ggccagggca ccctggtgac agtgagctcc ctcgaggact 840

acaaggacga cgatgacaag gaattccacc accaccatca ccattaagtc gactctaga 899

<210> 2

<211> 878

<212> DNA

<213> Artificial

<220>

<223> CEA-CAR-2

<400> 2

aagcttgcta gcctagcatg gccttaccag tgaccgcctt gctcctgccg ctggccttgc 60

tgctccacgc cgccaggccg gacatccagc tgacccagtc ccccagctcc ctgtccgcct 120

ctgtgggcga cagggtgacc atcacatgca aggcctctca ggatgtgggc acaagcgtgg 180

cctggtatca gcagaagccc ggcaaggccc ctaagctgct gatctattgg acctccacac 240

ggcacaccgg cgtgcccagc cggttcagcg gctccggctc tggcacagac ttcaccttca 300

ccatcagcag cctccagccc gaggacatcg ccacctacta ttgtcagcag tacagcctgt 360

atcggtcctt tggccagggc acaaaggtgg agatcaaggg atccacttcc ggttcaggaa 420

agcccgggag tggtgaaggt agcactaaag gcgaggtgca gctggtggag agcggaggag 480

gagtggtgca gcctggccgg tccctgagac tgtcctgctc tgccagcggc ttcgacttta 540

ccacatactg gatgtcctgg gtgcggcagg caccaggcaa gggcctggag tggatcggcg 600

agatccaccc cgatagctcc accatcaact atgccccttc cctgaaggac aggttcacca 660

tctctcgcga taacgccaag aatacactgt tcctccagat ggactctctg aggcccgagg 720

atacaggcgt gtacttctgt gccagcctgt atttcggctt tccatggttt gcatactggg 780

gacagggcac cccagtgaca gtgtctagcc tcgaggacta caaggacgac gatgacaagg 840

aattccacca ccaccatcac cattaagtcg actctaga 878

<210> 3

<211> 908

<212> DNA

<213> Artificial

<220>

<223> CEA-CAR-3

<400> 3

aagcttgcta gcctagcatg gccctgccag tgaccgccct gctgctgccc ctggccctgc 60

tgctgcacgc agcacggccc caggtgcagc tgcagcagtc cggcggcggc gtggtgcagc 120

ctggcaggtc tctgaggctg agctgcgcag cctccggctt caccctgagc tcctatggaa 180

tgtactgggt gaggcaggca ccaggcaagg gactggagtg ggtggccgtg atctggtatg 240

acggcagcaa caagtactat gccgattccg tgaagggccg gtttaccatc tctagagaca 300

acagcaagaa tacactgtac ctgcagatga atagcctgag ggcagaggac accgccgtgt 360

actattgtgc ccgggataga ctgacaggcg ccccttacta ttactattac ggcatggacg 420

tgtggggcag aggcaccctg gtgacagtgt ctagcggatc cacctctgga agcggcaagc 480

caggatccgg agagggatct accaagggca cagacgtggt catgacccag tctcctggca 540

cactgtccct gtctccagga gagagggcca cactgagctg cagggccagc cagtccgtgt 600

cctctaggta tctggcatgg taccagcaga agccaggaca ggcacctagg ctgctgatct 660

atggagccag ctcccgggcc accggaatcc ctgacagatt ctctggcagc ggctccggca 720

cagacttcac cctgacaatc tccaggctgg agccagagga tttcgccgtg tattactgtc 780

agcagtacgg ctctagccca ctgacctttg gcggaggcac aaaggtggag atcaagcgcc 840

tcgaggacta caaggacgac gatgacaagg aattccacca ccaccatcac cattaagtcg 900

actctaga 908

<210> 4

<211> 810

<212> DNA

<213> Artificial

<220>

<223> CEA-CAR-4

<400> 4

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgaccca gagcccaagc agcctgagcg ccagcgtggg tgacagagtg 120

accatcacct gtagtaccag ttcgagtgta agttacatgc actggtacca gcagaagcca 180

ggtaaggctc caaggctgct gatctacagc acatccaacc tggcttctgg tgtgccaagc 240

agattcagcg gtagcggtag cggtaccgac ttcaccttca ccatcagcag cctccagcca 300

gaggacatcg ccacctacta ctgccatcag tggagtagtt atcccacgtt cggccaaggg 360

accaaggtgg aaatcaaagg atccacttcc ggttcaggaa agcccgggag tggtgaaggt 420

agcactaaag gccaggtcca gctgcaggag agcggtccag gtcttgtgag acctagccag 480

accctgagcc tgacctgcac cgtgtctggc ttcaccatca gcagtggtta tagctggcac 540

tgggtgagac agccacctgg acgaggtctt gagtggattg gatacataca gtacagtggt 600

atcactaact acaacccctc tctcaaaagt agagtgacaa tgctggtaga caccagcaag 660

aaccagttca gcctgagact cagcagcgtg acagccgccg acaccgcggt ctattattgt 720

gcaagagaag actatgatta ccactggtac ttcgatgtct ggggtcaagg cagcacggtc 780

accgtctcct caggtgcggc cgccctcgag 810

<210> 5

<211> 687

<212> DNA

<213>

<220>

<223> Ig4

<400> 5

gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcct gggcggaccc 60

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg gacccccgag 120

gtgacctgtg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt caactggtac 180

gtggacggcg tggaggtgca caacgccaag accaagcccc gggaggagca gttcaatagc 240

acctaccggg tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 300

tacaagtgta aggtgtccaa caagggcctg cccagcagca tcgagaaaac catcagcaag 360

gccaagggcc agcctcggga gccccaggtg tacaccctgc cccctagcca agaggagatg 420

accaagaatc aggtgtccct gacctgcctg gtgaagggct tctaccccag cgacatcgcc 480

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 540

gacagcgacg gcagcttctt cctgtacagc aggctgaccg tggacaagag ccggtggcag 600

gagggcaacg tctttagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctgt ccctgagcct gggcaag 687

<210> 6

<211> 204

<212> DNA

<213>

<220>

<223> CD28

<400> 6

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180

cgcgacttcg cagcctatcg ctcc 204

<210> 7

<211> 132

<212> DNA

<213>

<220>

<223> CD137

<400> 7

gttaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 60

caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 120

tgtgaactga ga 132

<210> 8

<211> 333

<212> DNA

<213>

<220>

<223> CD3

<400> 8

gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 60

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 120

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 180

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 240

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 300

gacgcccttc acatgcaggc cctgccccct cgc 333

<210> 9

<211> 87

<212> DNA

<213> Artificial

<220>

<223> CD28TM

<400> 9

Gaattcttct gggtgctggt cgtggtgggt ggcgtgctgg cctgctacag cctgctggtg 60

acagtggcct tcatcatctt ttgggtg 87

<210> 10

<211> 750

<212> DNA

<213> Artificial

<220>

<223> Anti-CEA-humanized-scFV-1-2

<400> 10

gatattgtgc tgacccagag cccggcgagc ctggcggtga gcctgggcca gcgcgcgacc 60

attacctgcc gcgcgggcga aagcgtggat atttttggcg tgggctttct gcattggtat 120

cagcagaaac cgggccagcc gccgaaactg ctgatttatc gcgcgagcaa cctggaaagc 180

ggcgtgccgg cgcgctttag cggcagcggc agcggcaccg attttaccct gaccattaac 240

ccggtggaag cggaagatgt gggcgtgtat tattgccagc agaccaacga agatccgtat 300

acctttggcg gcggcaccaa actggaaatt aaaggcagca ccagcggcag cggcaaaccg 360

ggcagcggcg aaggcagcac caaaggccag gtgcagctgg tgcagagcgg cgcggaagtg 420

aaaaaaccgg gcgcgagcgt gaaagtgagc tgcaaagcga gcggctttaa cattaaagat 480

acctatatgc attgggtgcg ccaggcgccg ggccagggcc tggaatggat gggccgcatt 540

gatccggcga acggcaacag caaatatgtg ccgaaatttc agggccgcgt gaccattacc 600

cgcgatacca gcgcgagcac cgcgtatatg gaactgagca gcctgcgcag cgaagatacc 660

gcggtgtatt attgcgcgcc gtttggctat tatgtgagcg attatgcgat ggcgtattgg 720

ggccagggca ccagcgtgac cgtgagcagc 750

<210> 11

<211> 750

<212> DNA

<213> Artificial

<220>

<223> Anti-CEA-humanized-scFV-1-1

<400> 11

gatattgtgc tgacccagag cccggcgagc ctggcggtga gcctgggcca gcgcgcgacc 60

atgacctgcc gcgcgggcga aagcgtggat atttttggcg tgggctttct gcattggtat 120

cagcagaaac cgggccagcc gccgaaactg ctgatttatc gcgcgagcaa cctggaaagc 180

ggcgtgccgg tgcgctttag cggcaccggc agcggcaccg attttaccct gattattgat 240

ccggtggaag cggaagatgt gggcgtgtat tattgccagc agaccaacga agatccgtat 300

acctttggcg gcggcaccaa actggaaatt aaaggcagca ccagcggcag cggcaaaccg 360

ggcagcggcg aaggcagcac caaaggccag gtgcagctgg tgcagagcgg cgcggaagtg 420

aaagaaccgg gcgcgagcgt gaaagtgagc tgcaaagcga gcggctttaa cattaaagat 480

acctatatgc attgggtgcg ccaggcgccg ggccagggcc tggaatggat gggccgcatt 540

gatccggcga acggcaacag caaatatgtg ccgaaatttc agggccgcgt gaccattacc 600

gcggatacca gcgcgagcac cgcgtatatg gaactgacca gcctgcgcag cgaagatacc 660

gcggtgtatt attgcgcgcc gtttggctat tatgtgagcg attatgcgat ggcgtattgg 720

ggccagggca ccagcgtgac cgtgagcagc 750

<210> 12

<211> 250

<212> PRT

<213> Artificial

<220>

<223> M5A-scFV

<400> 12

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

5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Glu Ser Val Asp

20 25 30

Ile Phe Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Lys Ala Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

50 55 60

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe

65 70 75

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

80 85 90

Tyr Cys Gln Gln Thr Asn Glu Asp Pro Tyr Thr Phe Gly Gln Gly

95 100 105

Thr Lys Val Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro

110 115 120

Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Gln Leu Val Glu

125 130 135

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser

140 145 150

Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Met His Trp

155 160 165

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

170 175 180

Asp Pro Ala Asn Gly Asn Ser Lys Tyr Ala Asp Ser Val Lys Gly

185 190 195

Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu

200 205 210

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

215 220 225

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp

230 235 240

Gly Gln Gly Thr Leu Val Thr Val Ser Ser

245 250

<210> 13

<211> 243

<212> PRT

<213> Artificial

<220>

<223> hMN-scFV

<400> 13

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

5 10 15

Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly

20 25 30

Thr Ser Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys

35 40 45

Leu Leu Ile Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile

65 70 75

Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln

80 85 90

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

95 100 105

Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly

110 115 120

Ser Thr Lys Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val

125 130 135

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

140 145 150

Phe Asp Phe Thr Thr Tyr Trp Met Ser Trp Val Arg Gln Ala Pro

155 160 165

Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile His Pro Asp Ser Ser

170 175 180

Thr Ile Asn Tyr Ala Pro Ser Leu Lys Asp Arg Phe Thr Ile Ser

185 190 195

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

200 205 210

Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Ser Leu Tyr Phe

215 220 225

Gly Phe Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Pro Val Thr

230 235 240

Val Ser Ser

243

<210> 14

<211> 250

<212> PRT

<213> Artificial

<220>

<223> Anti-CEA-humanized-scFV-1-1

<400> 14

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

5 10 15

Gly Gln Arg Ala Thr Met Thr Cys Arg Ala Gly Glu Ser Val Asp

20 25 30

Ile Phe Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

50 55 60

Gly Val Pro Val Arg Phe Ser Gly Thr Gly Ser Gly Thr Asp Phe

65 70 75

Thr Leu Ile Ile Asp Pro Val Glu Ala Glu Asp Val Gly Val Tyr

80 85 90

Tyr Cys Gln Gln Thr Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly

95 100 105

Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro

110 115 120

Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln

125 130 135

Ser Gly Ala Glu Val Lys Glu Pro Gly Ala Ser Val Lys Val Ser

140 145 150

Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Met His Trp

155 160 165

Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile

170 175 180

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

185 190 195

Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr Met

200 205 210

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

215 220 225

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp

230 235 240

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

245 250

<210> 15

<211> 250

<212> PRT

<213> Artificial

<220>

<223> Anti-CEA-humanized-scFV-1-2

<400> 15

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

5 10 15

Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Gly Glu Ser Val Asp

20 25 30

Ile Phe Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser

50 55 60

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

65 70 75

Thr Leu Thr Ile Asn Pro Val Glu Ala Glu Asp Val Gly Val Tyr

80 85 90

Tyr Cys Gln Gln Thr Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly

95 100 105

Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro

110 115 120

Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln

125 130 135

Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser

140 145 150

Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Met His Trp

155 160 165

Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile

170 175 180

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

185 190 195

Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr Met

200 205 210

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

215 220 225

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp

230 235 240

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

245 250

<210> 16

<211> 250

<212> PRT

<213> Artificial

<220>

<223> Anti-CEA-humanized-scFV-1-3

<400> 16

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

5 10 15

Gly Gln Arg Ala Thr Ile Thr Cys Arg Ala Gly Glu Ser Val Ser

20 25 30

Ile Phe Gly Val Gly Leu Leu His Trp Tyr Gln Gln Lys Pro Gly

35 40 45

Gln Pro Pro Lys Leu Leu Ile Tyr Gln Ala Ser Asn Lys Asp Thr

50 55 60

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

65 70 75

Thr Leu Thr Ile Asn Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr

80 85 90

Tyr Cys Leu Gln Thr Lys Asn Phe Pro Tyr Thr Phe Gly Gly Gly

95 100 105

Thr Lys Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro

110 115 120

Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln

125 130 135

Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser

140 145 150

Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr Tyr Met His Trp

155 160 165

Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile

170 175 180

Asn Pro Asn Asn Gly Asn Thr Lys Tyr Val Gln Lys Phe Gln Gly

185 190 195

Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr Met

200 205 210

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

215 220 225

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp

230 235 240

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

245 250

<210> 17

<211> 229

<212> PRT

<213> Artificial

<220>

<223>Hinge area

<400> 17

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65 70 75

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

80 85 90

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

95 100 105

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

110 115 120

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

125 130 135

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

140 145 150

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

155 160 165

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

170 175 180

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

185 190 195

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

200 205 210

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

215 220 225

Ser Leu Gly Lys

229

<210> 18

<211> 27

<212> PRT

<213> Artificial

<220>

<223> CD28TM

<400> 18

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

5 10 15

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 19

<211> 24

<212> PRT

<213> Artificial

<220>

<223> CD8TM

<400> 19

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

5 10 15

Leu Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 20

<211>

<212> DNA

<213> Artificial

<220>

<223>Forward primer

<400> 20

aggctagcat gggatggagc tgtatcat 28

<210> 21

<211>

<212> DNA

<213> Artificial

<220>

<223>Reverse primer

<400> 21

gattgtcgac ttagcgaggg ggcagggcct gcatgtga 38

Claims

1. the Chimeric antigen receptor of anti-carcinoembryonic antigen, it is characterised in that by the single-chain antibody (scFV) of identification carcinomebryonic antigen, hinge Area, transmembrane region and intracellular signal domain are sequentially connected composition；The amino acid sequence of the single-chain antibody of the identification carcinomebryonic antigen contains M5A-scFV amino acid sequence or the amino acid sequence by being obtained to M5A-scFV polypeptides progress random point mutation.

2. Chimeric antigen receptor according to claim 1, it is characterised in that the single-chain antibody of the identification carcinomebryonic antigen (scFV) amino acid sequence such as SEQ ID NO:12 or SEQ ID NO:13 or SEQ ID NO:14 shown or SEQ ID NO: Shown in 15.

3. Chimeric antigen receptor according to claim 1, it is characterised in that the amino acid sequence of hinge area such as SEQ ID NO:Shown in 17.

4. Chimeric antigen receptor according to claim 1, it is characterised in that the amino acid sequence of the transmembrane region such as SEQ ID NO:18 or SEQ ID NO:Shown in 19.

5. Chimeric antigen receptor according to claim 1, it is characterised in that the intracellular signal domain be CD3 ζ chains and/or FcR γ and/or CD137 and/or CD28 and/or CD278.

6. Chimeric antigen receptor according to claim 1, it is characterised in that the nucleotide sequence of the Chimeric antigen receptor Such as SEQ ID NO:1 or SEQ ID NO:Shown in 2.

7. the preparation method of the slow virus carrier of the Chimeric antigen receptor described in right any one of 1-5, it is characterised in that specific bag Include following steps：

1) gene order of the Chimeric antigen receptor of single-chain antibody (scFV) of the synthesis containing identification carcinomebryonic antigen：Synthesis contains successively Different single-chain antibody ScFv, hinge area, transmembrane region and the intracellular signal domain of leader peptide, anti-human CEA antigens；

The hinge area nucleotide sequence such as SEQ ID NO:5th, transmembrane region nucleotide sequence such as SEQ ID NO:9th, intracellular signal domain core Acid sequence such as SEQ ID NO:6、SEQ ID NO:7 and SEQ ID NO:8；

2) slow virus carrier of construction expression Chimeric antigen receptor：Design primer, the nucleotide sequence such as SEQ ID of forward primer NO:Shown in 20, the nucleotide sequence such as SEQ ID NO of reverse primer:Shown in 21, using the sequence of the Chimeric antigen receptor as mould Plate enters performing PCR amplification, obtains DNA fragmentation；

By the gene order of DNA fragmentation restriction enzyme NheI and SalI double digestion, while using restriction enzyme NheI and SalI digestion Lentiviral pCDH-EF1, purpose fragment and Lentiviral piece after then plum is cut Section is attached by T4 ligases, obtains the slow virus carrier of expression Chimeric antigen receptor.

8. preparation method according to claim 7, it is characterised in that in step 2) after, pack and purify the slow disease Poisonous carrier.

9. the slow virus carrier obtained by preparation method described in claim 8.

10. the T cell of the slow virus carrier infection described in claim 9.

11. application of the T cell in the medicine for preparing treatment solid tumor described in claim 10.

12. application according to claim 11, it is characterised in that the cell of described solid tumor can express CEA.

13. application according to claim 11, it is characterised in that the solid tumor is colorectal cancer tumour.

14. the amino acid sequence of the single-chain antibody (scFV) of the identification carcinomebryonic antigen described in claim 2 is for preparing treatment Application in the medicine of solid tumor.

15. application according to claim 14, it is characterised in that the cell of described solid tumor can express CEA.

16. the amino acid sequence of the single-chain antibody (scFV) of the identification carcinomebryonic antigen described in claim 2 is for preparing essence Quasi- capture can express the application in the carrier of CEA solid tumor cells.

17. such as SEQ ID NO:The application of antigen recognition domain of the amino acid sequence in CAR-T skeletons are prepared shown in 12.