CN110526988A

CN110526988A - A kind of Chimeric antigen receptor and Chimeric antigen receptor T cell and its preparation method and application targeting MUC1

Info

Publication number: CN110526988A
Application number: CN201810515282.7A
Authority: CN
Inventors: 万晓春; 李欣; 刘绿艳; 唐超
Original assignee: Shenzhen Benta Biological Technology Co Ltd
Current assignee: Shenzhen Benta Biological Technology Co Ltd
Priority date: 2018-05-25
Filing date: 2018-05-25
Publication date: 2019-12-03

Abstract

The present invention provides a kind of Chimeric antigen receptor for targeting MUC1 and the Chimeric antigen receptor T cells of targeting MUC1, the Chimeric antigen receptor of the targeting MUC1 can be with the targeting MUC1 of specificity, and using CD27 signaling zone as costimulatory signal area, promote T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific preferably maintains the vigor and lethality of cell.The present invention also provides a kind of preparation method and application of Chimeric antigen receptor T cell for targeting MUC1.

Description

A kind of Chimeric antigen receptor and Chimeric antigen receptor T cell and its system targeting MUC1 Preparation Method and application

Technical field

The present invention relates to field of medical biotechnology, in particular to a kind of Chimeric antigen receptor for targeting MUC1 and chimeric antigen by Body T cell and its preparation method and application.

Background technique

Mucoprotein (mucin, MUC) is a kind of glycoprotein of high molecular weight, and wherein MUC1 is the mucoprotein man studied extensively One of family member.The study found that MUC1 increases in the expression of kinds of tumors, the grade malignancy of increase degree and tumour is at just Than, and polarity distribution is lost, and occur glycosylating incomplete situation, so that the antigen on MUC1 polypeptide backbone determines Cluster is exposed, and becomes the target spot of immunization therapy.Meanwhile MUC1 can be identified by a variety of MUC1 antibody, it can also be by cytotoxic T Lymphocyte (CTL) identification and killing, and do not limited by MHC.Therefore, MUC1 is ideal anti-tumor target, becomes one The new tumor marker of kind.

Immune cell therapy be it is existing science and technology in be uniquely possible to the method for thoroughly removing cancer cell, wherein chimeric antigen by Body T cell technology (CAR-T) is one of current adoptive newest immunocyte technology of cell adoptive therapy technology, because it can Self immune system can be activated in vivo, and routinely targets neoplastic cells are killed, and it is thin to be finally reached removing malignant tumour The purpose of born of the same parents and widely paid close attention to and studied.Therefore, in conjunction with CAR-T means, targeting and in conjunction with MUC1 target spot, as new Tumor therapeutic agent is of great importance to the healing of tumour.

Summary of the invention

In view of this, the present invention provides a kind of Chimeric antigen receptor for targeting MUC1 and the chimeric antigens of targeting MUC1 Recipient T cells.It is described targeting MUC1 Chimeric antigen receptor can with the targeting MUC1 of specificity, and using CD27 signaling zone as be total to Stimulus signal area, promotes T cell in the amplification of patient's body, and killing tumor cell that can be efficient and specific is preferably tieed up Hold the vigor and lethality of Chimeric antigen receptor T cell.The present invention also provides a kind of Chimeric antigen receptor T for targeting MUC1 is thin The preparation method and application of born of the same parents.

In a first aspect, the present invention provides a kind of Chimeric antigen receptor for targeting MUC1, the inosculating antibody of the targeting MUC1 Original receptor CAR-MUC1 include from aminoterminal to c-terminus the sequentially connected targeting single-chain antibody of MUC1, extracellular hinge area, across The amino acid sequence in film area and intracellular signal area, wherein the single-chain antibody of the targeting MUC1 includes as shown in SEQ ID NO:1 Amino acid sequence, the intracellular signal area includes costimulatory signal area and the first signaling zone, and the costimulatory signal area includes CD27 signaling zone, the CD27 signaling zone include the amino acid sequence as shown in SEQ ID NO:2.

Wherein, described " being sequentially connected with from aminoterminal to c-terminus " specifically: the ammonia of the single-chain antibody of the targeting MUC1 The c-terminus of base acid sequence is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, the amino of the extracellular hinge area The c-terminus of acid sequence is connected with the aminoterminal of the amino acid sequence of the transmembrane region, the carboxylic of the amino acid sequence of the transmembrane region Cardinal extremity is connected with the aminoterminal of the amino acid sequence of the CD27 signaling zone, the carboxyl of the amino acid sequence of the CD27 signaling zone End is connected with the aminoterminal of the amino acid sequence of first signaling zone.

In the present invention, the Chimeric antigen receptor of the targeting MUC1 can be specifically bound with MUC1 albumen, right Expressing the tumour cell of MUC1, especially solid tumor cell has stronger affine activity.

Optionally, the encoding gene of the single-chain antibody of the targeting MUC1 includes the nucleotide as shown in SEQ ID NO:5 Sequence.

Optionally, the single-chain antibody encoding gene of the targeting MUC1 should consider degeneracy base, i.e., such as SEQ ID NO:1 Shown in the encoding gene of amino acid sequence include the nucleotide sequence as shown in SEQ ID NO:5, protection scope should also protect Shield has the nucleotide sequence of base degeneracy matter with SEQ ID NO:5, and the corresponding amino acid sequence of these nucleotide sequences is still It is so SEQ ID NO:1.

The extracellular hinge area described in the present invention is used to promote the MUC1 on the single-chain antibody and tumour of the targeting MUC1 In conjunction with.

Optionally, the extracellular hinge area include CD8 α hinge area, CD28 hinge area, CD4 hinge area, CD5 hinge area, One of CD134 hinge area, CD137 hinge area, ICOS hinge area or a variety of combinations.

Further alternative, the extracellular hinge area is CD8 α hinge area.

Optionally, the amino acid sequence of the CD8 α hinge area includes the amino acid sequence as shown in SEQ ID NO:6.

Optionally, the encoding gene of the CD8 α hinge area includes the nucleotide sequence as shown in SEQ ID NO:7.

Optionally, the encoding gene of the CD8 α hinge area should consider degeneracy base, i.e., as shown in SEQ ID NO:6 The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:7, and protection scope should also protect and SEQ ID NO:7 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:6。

The transmembrane region is used to fix the Chimeric antigen receptor CAR-MUC1 of the targeting MUC1 in the present invention.

Optionally, the transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region Or a variety of combination.

Further alternative, the transmembrane region is CD8 transmembrane region.

Optionally, the amino acid sequence of the CD8 transmembrane region includes the amino acid sequence as shown in SEQ ID NO:8.

Optionally, the encoding gene of the CD8 transmembrane region includes the nucleotide sequence as shown in SEQ ID NO:9.

Optionally, the encoding gene of the CD8 transmembrane region should consider degeneracy base, i.e., as shown in SEQ ID NO:8 The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:9, and protection scope should also protect and SEQ ID NO:9 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:8。

The intracellular signal area is for providing the signal of T cell activation in the present invention, maintain T cell life span and Activate T cell proliferation signal access.

The intracellular signal area includes costimulatory signal area and the first signaling zone, and the costimulatory signal area includes CD27 letter Number area, the CD27 signaling zone include the amino acid sequence as shown in SEQ ID NO:2.

Optionally, the encoding gene of the CD27 signaling zone includes the nucleotide sequence as shown in SEQ ID NO:10.

Optionally, the encoding gene of the CD27 signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:2 The encoding gene of amino acid sequence include the nucleotide sequence such as SEQ ID NO:10 shown in, protection scope should also protect and SEQ ID NO:10 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:2。

Optionally, first signaling zone includes at least one of CD3 ζ signaling zone, Fc ε RI γ signaling zone.

Further alternative, first signaling zone is CD3 ζ signaling zone.

Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:11.

Optionally, the encoding gene of the CD3 ζ signaling zone includes the nucleotide sequence as shown in SEQ ID NO:12.

Optionally, the encoding gene of the CD3 ζ signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:11 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:12 shown in, protection scope should also protect and SEQ ID NO:12 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:11。

In the present invention, CD3 ζ signaling zone is intracellular signal transduction structural domain (the first signaling zone), and CD27 signaling zone is total Stimulus structure domain (costimulatory signal area), collective effect activating T cell, particularly, using CD27 signaling zone as costimulation structure Domain can preferably promote the vigor and lethality of T cell.

Optionally, the amino acid sequence of the CAR-MUC1 includes the amino acid sequence as shown in SEQ ID NO:3.

Optionally, the encoding gene of the CAR-MUC1 includes the nucleotide sequence as shown in SEQ ID NO:4.

Optionally, the encoding gene of the CAR-MUC1 should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:3 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:4, and protection scope should also protect and SEQ ID NO:4 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:3。

Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells for targeting MUC1, including such as first aspect institute The Chimeric antigen receptor of the targeting MUC1 stated.

The targeting that the Chimeric antigen receptor and second aspect for the targeting MUC1 that first aspect present invention provides provide The Chimeric antigen receptor T cell of MUC1, can targeted expression MUC1 in specific manner tumour cell, using CD27 signaling zone as altogether Stimulus signal area, activating T cell promote T cell in the amplification of patient's body, and efficient and specific killing tumor cell.

The third aspect, the present invention provides a kind of recombinant viral vector, the recombinant viral vector includes such as first aspect The encoding gene of the Chimeric antigen receptor CAR-MUC1 of the targeting MUC1.

Optionally, the viral vectors in the recombinant viral vector includes slow virus carrier, adenovirus vector or reverse transcription Viral vectors.

Further alternative, the viral vectors is slow virus carrier.It is further optional, the slow virus carrier packet Include at least one of pWPXLD carrier, pLEX-MCS carrier, pSico carrier and pCgpV carrier.Specifically, working as the slow disease When poisonous carrier is pWPXLD carrier, it will target the CAR-MUC1's of the Chimeric antigen receptor T cell of MUC1 described in second aspect Encoding gene is inserted between I restriction enzyme site of I restriction enzyme site of BamH and EcoR in pWPXLD carrier.

The Chimeric antigen receptor T that the recombinant viral vector that third aspect present invention provides can be used for targeting MUC1 is thin The preparation of born of the same parents, may advantageously facilitate T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific.

Fourth aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in the third aspect Group viral vectors.

Optionally, the host cell may include HEK293T cell, 293 cells, 293T cell, 293FT cell, SW480 cell, u87MG cell, HOS cell, COS1 cell or COS7 cell etc., but not limited to this.

Further alternative, the host cell is HEK293T cell.

Fourth aspect present invention provide host cell for provide by the recombinant viral vector as described in the third aspect into Row assembles and prepares the place for generating corresponding virus, is believed by the heredity that virus prepared by host cell carries the CAR-MUC1 Breath has strong infectivity.

5th aspect, the present invention provides a kind of preparation methods of Chimeric antigen receptor T cell for targeting MUC1, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-MUC1 of targeting MUC1 is provided, including sequentially from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of connection, the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, cross-film for targeting MUC1 The encoding gene in area and the encoding gene in intracellular signal area, wherein the encoding gene of single-chain antibody of the targeting MUC1 includes The corresponding nucleotide sequence of amino acid sequence as shown in SEQ ID NO:1, the intracellular signal area include costimulatory signal area With the first signaling zone, the costimulatory signal area includes CD27 signaling zone, and the encoding gene of the CD27 signaling zone includes such as SEQ The corresponding nucleotide sequence of amino acid sequence shown in ID NO:2；

(2) encoding gene of the CAR-MUC1 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-MUC1 recombination Plasmid；

(3) it by the pWPXLD-CAR-MUC1 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains To recombinant slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, the chimeric antigen of targeting MUC1 is obtained through separation Recipient T cells.

It is above-mentioned " from 5 ' end to 3 ' end be sequentially connected with " specifically: the coding gene sequence of the signal peptide 3 ' end with it is described 5 ' the ends for targeting the encoding gene of the single-chain antibody of MUC1 are connected, 3 ' ends of the encoding gene of the single-chain antibody of the targeting MUC1 It is connected with 5 ' ends of the encoding gene of the extracellular hinge area, 3 ' ends of the encoding gene of the extracellular hinge area and the cross-film 5 ' ends of the encoding gene in area are connected, 3 ' ends and the encoding gene of the CD27 signaling zone of the encoding gene of the transmembrane region 5 ' ends are connected, and 3 ' ends of the encoding gene of the CD27 signaling zone are connected with 5 ' ends of the encoding gene of first signaling zone.

The signal peptide is for instructing the Chimeric antigen receptor CAR-MUC1 expression to cell surface, institute in the present invention Signal peptide is stated to be cut in protein translation maturation by signal peptidase.

Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:13.

Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:14.

Optionally, the encoding gene of the signal peptide should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:13 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:14, and protection scope should also protect and SEQ ID NO:14 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:13。

The extracellular hinge area, transmembrane region, the specific choice in intracellular signal area and corresponding coding gene sequence such as this hair Described in bright first aspect part, which is not described herein again.

Optionally, the amino acid sequence of the CAR-MUC1 includes the amino acid sequence as shown in SEQ ID NO:15.

Optionally, the coding gene sequence of the CAR-MUC1 includes the nucleotide sequence as shown in SEQ ID NO:16.

Optionally, the encoding gene of the CAR-MUC1 should consider degeneracy base, i.e., as shown in SEQ ID NO:15 The encoding gene of amino acid sequence include the nucleotide sequence such as SEQ ID NO:16 shown in, protection scope should also protect and SEQ ID NO:16 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:15。

The encoding gene of the CAR-MUC1 is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR, and position After the EF1 α of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CAR-MUC1 is inserted into pWPXLD carrier When, I digestion of BamH in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-MUC1 Site is connected, and 3 ' ends can be added terminator codon (such as TAA) and be connected with I restriction enzyme site of EcoR in pWPXLD carrier.

Optionally, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T Cell.

The envelope plasmid PMD2G encodes vesicular stomatitis virus glycoprotein capsid, the vesicular stomatitis virus sugar egg White capsid assists recombinant slow virus to adhere to cell membrane, and keeps the infectivity of recombinant slow virus.

Recombinant slow virus of the present invention can further contain the envelope protein from other viruses.For example, as this Kind protein, is preferably the virus enveloped protein for carrying out self-infection human cell.To this protein, there is no particular limitation, can example The amphophilic virus hand epithelium albumen etc. for enumerating retrovirus, can be used for example from mouse leukemia virus (MuMLV) 4070A plants of envelope protein.Alternatively, it is also possible to use the envelope protein from MuMLV 10Al.In addition, as herpetoviridae Albumen, it can be cited for example that, gB, gD, gH, gp85 albumen of herpes simplex virus, gp350, gp220 albumen of Epstein-Barr virus Deng.As the albumen of Hepadna Virus section, the S protein etc. of hepatitis B virus can be included.The envelope protein can also be morbilli It is formed after viral glycoprotein and other single chain antibody fusions.

The packaging of recombinant slow virus is generallyd use transient transfection or is packed using cell line.It may be used as wrapping when transient transfection Human cell's strain that dress cell uses, for example including 293 cells, 293T cell, 293FT cell, 293LTV cell, 293EBNA Cell and other clones separated from 293 cells；SW480 cell, u87MG cell, HOS cell, C8166 cell, MT-4 are thin Born of the same parents, Molt-4 cell, HeLa cell, HT1080 cell, TE671 cell etc..It can also be using the cell strain from monkey, example Such as, COS1 cell, COS7 cell, CV-1 cell, BMT10 cell etc..Moreover, the calcium phosphate and PEI transfection reagent that generally use, It is also well used there are also some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin.

The packaging of recombinant slow virus also uses some slow virus package cell lines, such as most common Env glycoprotein of use, Stable cell lines caused by VSVG albumen or HIV-1gag-pol albumen.

For the sake of security, the slow virus carrier system of large-scale use is all the method using split gene group, i.e., will The assignment of genes gene mapping of different miscellaneous functions is played in different plasmids.There are four pUC pUCs (encoding gag-pol gene, Rev base at present Cause, VSVG gene, SIN metastatic gene be located at four different plasmids), three pUC pUCs (eliminate coding Rev gene Plasmid, gag-pol gene uses the codon of the preferences in people's cell in gag-pol plasmid) and two pUC pUCs are (slowly Auxiliary gene necessary to viral vectors is packed is located on the same plasmid, these auxiliary genes are single gene orders；Separately One is then transgenosis plasmid).Also the slow virus packaging system for having more than four pUC pUCs is using.

Optionally, in step (4), the CD3 positive t lymphocytes are to separate to obtain from source of people peripheral blood mononuclear cells .

Optionally, the source of people peripheral blood mononuclear cells derives from autologous vein blood, autologous bone marrow, Cord blood and placenta Blood etc..

Fresh peripheral that is further alternative, being acquired after cancer patient's operation one month, after chemicotherapy one month Blood or marrow.

Specifically, the acquisition process of the CD3 positive t lymphocytes is as follows: into peripheral blood mononuclear cells by certain CD3/CD28 immunomagnetic beads are added in ratio, after being incubated for a period of time, are put into magnet and are screened, it is coated to obtain immunomagnetic beads CD3 positive t lymphocytes after removing magnetic bead, obtain CD3 positive t lymphocytes.

6th aspect, the present invention provides the Chimeric antigen receptor of targeting MUC1 as described in relation to the first aspect a kind of or such as Described in second aspect or the Chimeric antigen receptor T cell of targeting MUC1 made from the preparation method as described in terms of the 5th or Recombinant viral vector as described in the third aspect or the host cell as described in fourth aspect are disliked in preparation prevention, diagnosing and treating Application in the drug of property tumour

Wherein, the malignant tumour include in oophoroma, lung cancer, cancer of pancreas, prostate cancer, gastric cancer and colorectal cancer extremely Few one kind, the especially application in the drug for preparing prevention, diagnosing and treating oophoroma.

The application specifically: provide a kind of kit, the kit includes targeting as described in relation to the first aspect The Chimeric antigen receptor of MUC1, as described in second aspect targeting MUC1 Chimeric antigen receptor T cell, as described in the third aspect Recombinant viral vector, one of host cell as described in fourth aspect or a variety of.

Beneficial effects of the present invention:

The Chimeric antigen receptor of targeting MUC1 provided by the invention can be with the targeting MUC1 of specificity, especially generation MUC1 Malignant cell, target the Chimeric antigen receptor T cell of MUC1 using CD27 signaling zone as costimulatory signal area, promote T Cell is in the amplification of patient's body, combination tumour cell that can be efficient and specific, generates killing effect to malignant cell Fruit.

Detailed description of the invention

Fig. 1 is the plasmid map of pWPXLd-CAR-MUC1 recombinant plasmid provided in an embodiment of the present invention.

Specific embodiment

The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as Protection scope of the present invention.

Embodiment one

A kind of preparation method for the Chimeric antigen receptor T cell targeting MUC1, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-MUC1 of preparation targeting MUC1

Prepare respectively signal peptide, target the single-chain antibody of MUC1, CD8 α hinge area, CD8 transmembrane region, CD27 signaling zone and The encoding gene of CD3 ζ signaling zone, the encoding gene of the signal peptide is as shown in SEQ ID NO:14, the list of the targeting MUC1 The encoding gene of chain antibody as shown in SEQ ID NO:5, the encoding gene of the CD8 α hinge area as shown in SEQ ID NO:7, The encoding gene of the CD8 transmembrane region is as shown in SEQ ID NO:9, the encoding gene of the CD27 signaling zone such as SEQ ID NO: Shown in 10, the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:12.

By the method for PCR by above-mentioned signal peptide, the single-chain antibody for targeting MUC1, CD8 α hinge area, CD8 transmembrane region, CD27 signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the chimeric of targeting MUC1 The encoding gene of antigen receptor CAR-MUC1, the encoding gene of the CAR-MUC1 is as shown in SEQ ID NO:16.

(2) pWPXLd-CAR-MUC1 recombinant plasmid is constructed

The encoding gene of CAR-MUC1 is inserted between I restriction enzyme site of BamH I and EcoR of pWPXLD carrier, and After pWPXLD carrier EF1 α, using EF1 α as promoter.When the encoding gene of the CAR-MUC1 is inserted into pWPXLD carrier, institute I restriction enzyme site of BamH in initiation codon (such as ATG) and pWPXLD carrier can be added in the 5 ' ends for stating the encoding gene of CAR-MUC1 It is connected, 3 ' ends can also be added terminator codon (such as TAA) and be connected with I restriction enzyme site of EcoR in pWPXLD carrier.Then it is transferred to Competent escherichia coli cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis Meet target fragment size and sequence with sequencing identification, successfully constructs pWPXLd-CAR-MUC1 recombinant plasmid, be as shown in Figure 1 PWPXLd-CAR-MUC1 recombinant plasmid.

(3) recombinant slow virus constructs

PWPXLd-CAR-MUC1 recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into training The HEK293T cell supported.48h harvest is protected in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration It deposits；Supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration merge with the viral supernatants of 48h harvest and are added together It in ultracentrifugation pipe, is put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, and centrifugation time is 2h, centrifuging temperature are controlled at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and virus is added Liquid is saved, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence method after being centrifuged 5min Measuring titre, virus is according to 100 μ l, and 2 × 10⁸A/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains recombinant slow virus.

(4) preparation of the Chimeric antigen receptor T cell of MUC1 is targeted

A) separation of PBMC (peripheral blood mononuclear cells)

PBMC is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..Preferably derive from cancer patient's hand The fresh peripheral blood or marrow acquired after art one month, after chemicotherapy one month.

Extract patient blood, sample presentation to blood separating chamber；Peripheral blood mononuclear cells is acquired, is taken after Ficoll centrifuge separation Intermediate layer cell；After PBS is washed, PBMC is obtained.

B) immunomagnetic beads antigenspecific T lymphocyte

Above-mentioned PBMC is taken, the basal medium for being free of serum is added, is made into cell suspension；Ratio in magnetic bead and cell is 3:1, is added CD3/CD28 immunomagnetic beads, and room temperature incubates 1-2h；The cell for being incubated for magnetic bead is screened using magnet；PBS is washed It washs, after removing immunomagnetic beads, obtains CD3 positive t lymphocytes.

C) virus transfection method prepares antigenspecific T lymphocyte

The above-mentioned CD3 positive t lymphocytes obtained by magnetic activated cell seperation are taken, are added and CD3 positive cell number phase The recombinant slow virus for the virus titer answered is cultivated.

The 3rd day of culture carries out cell count and changes liquid, and adjustment cell concentration is 1 × 10⁶A/mL is inoculated with, culture；Training Cell state is observed in feeding the 5th day, if cell density increases, diluting cells concentration is 1 × 10⁶A/mL detects cell Activity continues to cultivate.Amplification cultivation collected cell by the 9-11 days, obtained the Chimeric antigen receptor T cell of targeting MUC1, and It is stored in and feeds back in dedicated cells frozen storing liquid.

The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.

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370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475

<210> 4

<211> 1428

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gaactcgtga tgacccagag ccccagctct ctgacagtga cagccggcga gaaagtgacc 60

atgatctgca agtcctccca gagcctgctg aactccggcg accagaagaa ctacctgacc 120

tggtatcagc agaaacccgg ccagcccccc aagctgctga tcttttgggc cagcacccgg 180

gaaagcggcg tgcccgatag attcacaggc agcggctccg gcaccgactt taccctgacc 240

atcagctccg tgcaggccga ggacctggcc gtgtattact gccagaacga ctacagctac 300

cccctgacct tcggagccgg caccaagctg gaactgaagg gaggcggagg atctggcggc 360

ggaggaagtg gcggaggggg atctggggga ggcggaagcc aggtgcagct gcagcagtct 420

gatgccgagc tcgtgaagcc tggcagcagc gtgaagatca gctgcaaggc cagcggctac 480

accttcaccg accacgccat ccactgggtc aagcagaagc ctgagcaggg cctggaatgg 540

atcggccact tcagccccgg caacaccgac atcaagtaca acgacaagtt caagggcaag 600

gccaccctga ccgtggacag aagcagcagc accgcctaca tgcagctgaa cagcctgacc 660

agcgaggaca gcgccgtgta cttctgcaag accagcacct tctttttcga ctactggggc 720

cagggcacaa ccctgacagt gtctagcacc acgacgccag cgccgcgacc accaacaccg 780

gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 840

gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 900

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaggaaa 960

tatagatcaa acaaaggaga aagtcctgtg gagcctgcag agccttgtcg ttacagctgc 1020

cccagggagg aggagggcag caccatcccc atccaggagg attaccgaaa accggagcct 1080

gcctgctccc ccagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1140

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1200

aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1260

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1320

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1380

accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgc 1428

<210> 5

<211> 747

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gaactcgtga tgacccagag ccccagctct ctgacagtga cagccggcga gaaagtgacc 60

atgatctgca agtcctccca gagcctgctg aactccggcg accagaagaa ctacctgacc 120

tggtatcagc agaaacccgg ccagcccccc aagctgctga tcttttgggc cagcacccgg 180

gaaagcggcg tgcccgatag attcacaggc agcggctccg gcaccgactt taccctgacc 240

atcagctccg tgcaggccga ggacctggcc gtgtattact gccagaacga ctacagctac 300

cccctgacct tcggagccgg caccaagctg gaactgaagg gaggcggagg atctggcggc 360

ggaggaagtg gcggaggggg atctggggga ggcggaagcc aggtgcagct gcagcagtct 420

gatgccgagc tcgtgaagcc tggcagcagc gtgaagatca gctgcaaggc cagcggctac 480

accttcaccg accacgccat ccactgggtc aagcagaagc ctgagcaggg cctggaatgg 540

atcggccact tcagccccgg caacaccgac atcaagtaca acgacaagtt caagggcaag 600

gccaccctga ccgtggacag aagcagcagc accgcctaca tgcagctgaa cagcctgacc 660

agcgaggaca gcgccgtgta cttctgcaag accagcacct tctttttcga ctactggggc 720

cagggcacaa ccctgacagt gtctagc 747

<210> 6

<211> 45

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

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

1 5 10 15

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

20 25 30

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

35 40 45

<210> 7

<211> 135

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 8

<211> 24

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 9

<211> 72

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 10

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

aggaaatata gatcaaacaa aggagaaagt cctgtggagc ctgcagagcc ttgtcgttac 60

agctgcccca gggaggagga gggcagcacc atccccatcc aggaggatta ccgaaaaccg 120

gagcctgcct gctccccc 138

<210> 11

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 12

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210> 13

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His

1 5 10 15

Ala Ala Arg Pro

20

<210> 14

<211> 60

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

<210> 15

<211> 496

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His

1 5 10 15

Ala Ala Arg Pro Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Thr

20 25 30

Val Thr Ala Gly Glu Lys Val Thr Met Ile Cys Lys Ser Ser Gln Ser

35 40 45

Leu Leu Asn Ser Gly Asp Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Phe Trp Ala Ser Thr Arg

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr

115 120 125

Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

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

145 150 155 160

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

165 170 175

Ala Ser Gly Tyr Thr Phe Thr Asp His Ala Ile His Trp Val Lys Gln

180 185 190

Lys Pro Glu Gln Gly Leu Glu Trp Ile Gly His Phe Ser Pro Gly Asn

195 200 205

Thr Asp Ile Lys Tyr Asn Asp Lys Phe Lys Gly Lys Ala Thr Leu Thr

210 215 220

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

225 230 235 240

Ser Glu Asp Ser Ala Val Tyr Phe Cys Lys Thr Ser Thr Phe Phe Phe

245 250 255

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Thr Thr Thr

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Tyr Cys Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro

340 345 350

Ala Glu Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr

355 360 365

Ile Pro Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

<210> 16

<211> 1488

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

gaactcgtga tgacccagag ccccagctct ctgacagtga cagccggcga gaaagtgacc 120

atgatctgca agtcctccca gagcctgctg aactccggcg accagaagaa ctacctgacc 180

tggtatcagc agaaacccgg ccagcccccc aagctgctga tcttttgggc cagcacccgg 240

gaaagcggcg tgcccgatag attcacaggc agcggctccg gcaccgactt taccctgacc 300

atcagctccg tgcaggccga ggacctggcc gtgtattact gccagaacga ctacagctac 360

cccctgacct tcggagccgg caccaagctg gaactgaagg gaggcggagg atctggcggc 420

ggaggaagtg gcggaggggg atctggggga ggcggaagcc aggtgcagct gcagcagtct 480

gatgccgagc tcgtgaagcc tggcagcagc gtgaagatca gctgcaaggc cagcggctac 540

accttcaccg accacgccat ccactgggtc aagcagaagc ctgagcaggg cctggaatgg 600

atcggccact tcagccccgg caacaccgac atcaagtaca acgacaagtt caagggcaag 660

gccaccctga ccgtggacag aagcagcagc accgcctaca tgcagctgaa cagcctgacc 720

agcgaggaca gcgccgtgta cttctgcaag accagcacct tctttttcga ctactggggc 780

cagggcacaa ccctgacagt gtctagcacc acgacgccag cgccgcgacc accaacaccg 840

gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 900

gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 960

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaggaaa 1020

tatagatcaa acaaaggaga aagtcctgtg gagcctgcag agccttgtcg ttacagctgc 1080

cccagggagg aggagggcag caccatcccc atccaggagg attaccgaaa accggagcct 1140

gcctgctccc ccagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1200

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260

aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440

accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgc 1488

Claims

1. a kind of Chimeric antigen receptor for targeting MUC1, which is characterized in that the Chimeric antigen receptor CAR- of the targeting MUC1 MUC1 includes the sequentially connected targeting single-chain antibody of MUC1, extracellular hinge area, transmembrane region and intracellular from aminoterminal to c-terminus The amino acid sequence of signaling zone, wherein the single-chain antibody of the targeting MUC1 includes the amino acid sequence as shown in SEQ ID NO:1 Column, the intracellular signal area includes costimulatory signal area and the first signaling zone, and the costimulatory signal area includes CD27 signaling zone, The CD27 signaling zone includes the amino acid sequence as shown in SEQ ID NO:2.

2. the Chimeric antigen receptor of targeting MUC1 as described in claim 1, which is characterized in that the extracellular hinge area includes CD8 α hinge area, the transmembrane region include CD8 transmembrane region, and first signaling zone includes CD3 ζ signaling zone.

3. the Chimeric antigen receptor of targeting MUC1 as claimed in claim 2, which is characterized in that the amino acid of the CAR-MUC1 Sequence includes the amino acid sequence as shown in SEQ ID NO:3.

4. a kind of Chimeric antigen receptor T cell for targeting MUC1, which is characterized in that including as described in claim any one of 1-3 Targeting MUC1 Chimeric antigen receptor.

5. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes as described in claim any one of 1-3 Targeting MUC1 Chimeric antigen receptor CAR-MUC1 encoding gene.

6. recombinant viral vector as claimed in claim 5, which is characterized in that the encoding gene of the CAR-MUC1 includes such as Nucleotide sequence shown in SEQ ID NO:4.

7. a kind of host cell, which is characterized in that the host cell includes such as the described in any item recombinations of claim 5 or 6 Viral vectors.

8. a kind of preparation method for the Chimeric antigen receptor T cell for targeting MUC1 characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-MUC1 of targeting MUC1 is provided, including is sequentially connected with from 5 ' ends to 3 ' ends Signal peptide encoding gene, target the encoding gene of single-chain antibody of MUC1, the encoding gene of extracellular hinge area, transmembrane region The encoding gene of encoding gene and intracellular signal area, wherein the encoding gene of the single-chain antibody of the targeting MUC1 includes such as SEQ The corresponding nucleotide sequence of amino acid sequence shown in ID NO:1, the intracellular signal area include costimulatory signal area and first Signaling zone, the costimulatory signal area include CD27 signaling zone, and the encoding gene of the CD27 signaling zone includes such as SEQ ID The corresponding nucleotide sequence of amino acid sequence shown in NO:2；

(2) encoding gene of the CAR-MUC1 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-MUC1 recombination matter Grain；

(3) by the pWPXLD-CAR-MUC1 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, weight is obtained Group slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, the Chimeric antigen receptor T of targeting MUC1 is obtained through separation Cell.

9. the preparation method of the Chimeric antigen receptor T cell of targeting MUC1 as claimed in claim 8, which is characterized in that described Envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T cell.

10. a kind of Chimeric antigen receptor or as claimed in claim 4 of targeting MUC1 as described in any one of claims 1-3 Or as made from the described in any item preparation methods of claim 8-9 target MUC1 Chimeric antigen receptor T cell or as weigh Benefit require the described in any item recombinant viral vectors of 5-6 or host cell as claimed in claim 7 preparation prevention, diagnosis and Treat the application in the drug of malignant tumour.