CN110526974A

CN110526974A - It is a kind of to target the single-chain antibody of MUC16, Chimeric antigen receptor T cell and its preparation method and application

Info

Publication number: CN110526974A
Application number: CN201810513872.6A
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 single-chain antibody for targeting MUC16, the amino acid sequence of the single-chain antibody of the targeting MUC16 includes the amino acid sequence as shown in SEQ ID NO:1.The present invention also provides a kind of Chimeric antigen receptor T cells of single-chain antibody including the targeting MUC16, the Chimeric antigen receptor of the targeting MUC16 can be with the targeting MUC16 of specificity, 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 MUC16.

Description

A kind of single-chain antibody, Chimeric antigen receptor T cell and its preparation side targeting MUC16 Method and application

Technical field

The present invention relates to field of medical biotechnology, in particular to a kind of single-chain antibody, Chimeric antigen receptor T for targeting MUC16 Cell and its preparation method and application.

Background technique

Oophoroma is the highest gynecologic malignant tumor of lethality in the world, due to not apparent symptom and shortage section Believable detection means is learned, most of patient has been cancer of late stage when making a definite diagnosis.Therefore, the cure rate of oophoroma is generally lower. MUC16 is a kind of transmembrane protein, is one of the member of mucoprotein (mucin, MUC) family.MUC16 is in cervical carcinoma, cancer of pancreas, ovum Expression quantity in nest cancerous tissue increases, and is oophoroma important marker, can be to oophoroma early diagnosis, detection treatment effect Whether fruit and evaluation tumour recur.Studies have shown that MUC16 can pass through the dead induction of adjusting relevant to tumor necrosis factor Ligand interaction, inhibits Apoptosis, and the high expression of MUC16 can also induce tumor cell invasion ability to enhance.

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 MUC16 target spot, as new Tumor therapeutic agent, be of great importance to the healing of tumour.

Summary of the invention

In view of this, the present invention provides a kind of single-chain antibodies for targeting MUC16, and including the targeting MUC16's The Chimeric antigen receptor T cell of single-chain antibody.It is described targeting MUC16 Chimeric antigen receptor can with the targeting MUC16 of specificity, Promote T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific.The present invention also provides a kind of targets To the preparation method and application of the Chimeric antigen receptor T cell of MUC16.

In a first aspect, the present invention provides a kind of single-chain antibody for targeting MUC16, the single-chain antibody of the targeting MUC16 Amino acid sequence include the amino acid sequence as shown in SEQ ID NO:1.

Optionally, the encoding gene of the single-chain antibody of the targeting MUC16 includes the nucleotide as shown in SEQ ID NO:2 Sequence.

Optionally, the encoding gene of the single-chain antibody of the targeting MUC16 should consider degeneracy base, i.e., such as SEQ ID The encoding gene of amino acid sequence shown in NO:1 includes the nucleotide sequence as shown in SEQ ID NO:2, and protection scope is also answered The protection and SEQ ID NO:2 have the nucleotide sequence of base degeneracy matter, the corresponding amino acid sequence of these nucleotide sequences Column remain as SEQ ID NO:1.

With MUC16 albumen specificity can occur for the single-chain antibody for the targeting MUC16 that first aspect present invention provides In conjunction with to the tumour cell of expression MUC16, especially solid tumor cell with stronger affine activity.

Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells for targeting MUC16, including targeting MUC16's Chimeric antigen receptor CAR-MUC16, the amino acid sequence of the CAR-MUC16 include sequentially connected from aminoterminal to c-terminus Target the single-chain antibody of MUC16, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein the targeting The amino acid sequence of the single-chain antibody of MUC16 includes the amino acid sequence as shown in SEQ ID NO:1.

Wherein, described " being sequentially connected with from aminoterminal to c-terminus " specifically: the ammonia of the single-chain antibody of the targeting MUC16 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 in the intracellular signal area.

The extracellular hinge area described in the present invention is used to promote on the single-chain antibody and tumour of the targeting MUC16 MUC16 is combined.

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:5.

Optionally, the encoding gene of the CD8 α hinge area includes the nucleotide sequence as shown in SEQ ID NO:6.

Optionally, the encoding gene of the CD8 α hinge area should consider degeneracy base, i.e., as shown in SEQ ID NO:5 The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:6, and protection scope should also protect and SEQ ID NO:6 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:5。

The transmembrane region is used to fix the Chimeric antigen receptor CAR-MUC16 of the targeting MUC16 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:7.

Optionally, the encoding gene of the CD8 transmembrane region includes the nucleotide sequence as shown in SEQ ID NO:8.

Optionally, the encoding gene of the CD8 transmembrane region should consider degeneracy base, i.e., as shown in SEQ ID NO:7 The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:8, and protection scope should also protect and SEQ ID NO:8 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:7。

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.

Optionally, the intracellular signal area includes 4-1BB signaling zone, CD3 ζ signaling zone, ICOS signaling zone, OX40 signal One of area, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRSF19L signaling zone or a variety of combinations.

Optionally, the intracellular signal area is 4-1BB signaling zone and CD3 ζ signaling zone.CD3 ζ signaling zone is intracellular signal biography Transduction domain, 4-1BB signaling zone are costimulation structural domain, collective effect activating T cell.It is further alternative, the transmembrane region The c-terminus of amino acid sequence be connected with the aminoterminal of the amino acid sequence of the 4-1BB signaling zone, the 4-1BB signaling zone The c-terminus of amino acid sequence be connected with the aminoterminal of the amino acid sequence of the CD3 ζ signaling zone.

Optionally, the amino acid sequence of the 4-1BB signaling zone includes the amino acid sequence as shown in SEQ ID NO:9.

Optionally, the encoding gene of the 4-1BB signaling zone includes the nucleotide sequence as shown in SEQ ID NO:10.

Optionally, the encoding gene of the 4-1BB signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:9 Amino acid sequence encoding gene 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:9。

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。

Optionally, the amino acid sequence of the CAR-MUC16 includes the amino acid sequence as shown in SEQ ID NO:3.

Optionally, the encoding gene of the CAR-MUC16 includes the nucleotide sequence as shown in SEQ ID NO:4.

Optionally, the encoding gene of the CAR-MUC16 should consider degeneracy base, i.e., as shown in SEQ ID NO:3 The encoding gene of amino 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。

The Chimeric antigen receptor T cell for the targeting MUC16 that second aspect of the present invention provides, including targeting MUC16's Chimeric antigen receptor CAR-MUC16, tumour cell of this receptor for T cell targeted expression MUC16 in specific manner, In After CAR-MUC16 is in conjunction with MUC16, the intracellular signal area of the T cell is activated, promote T cell patient's body amplification, 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 second aspect The encoding gene of the CAR-MUC16 of the Chimeric antigen receptor T cell of the targeting MUC16.

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, the CAR-MUC16 of the Chimeric antigen receptor T cell of MUC16 will be targeted described in second aspect Encoding gene insertion pWPXLD carrier in I restriction enzyme site of I restriction enzyme site of BamH and EcoR between.

The recombinant viral vector that third aspect present invention provides can be used for targeting the Chimeric antigen receptor T of MUC16 The preparation of cell, 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, and the heredity of the CAR-MUC16 is carried by virus prepared by host cell Information has strong infectivity.

5th aspect, the present invention provides a kind of preparation methods of Chimeric antigen receptor T cell for targeting MUC16, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-MUC16 of targeting MUC16 is provided, including suitable from 5 ' ends to 3 ' ends The encoding gene of the signal peptide of secondary connection, the encoding gene of single-chain antibody for targeting MUC16, extracellular hinge area encoding gene, The encoding gene of transmembrane region and the encoding gene in intracellular signal area, wherein the encoding gene of the single-chain antibody of the targeting MUC16 Including the corresponding nucleotide sequence of amino acid sequence as shown in SEQ ID NO:1；

(2) encoding gene of the CAR-MUC16 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-MUC16 weight Group plasmid；

(3) it by the pWPXLD-CAR-MUC16 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 MUC16 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 MUC16 are connected, and the 3 ' of the encoding gene of the single-chain antibody of the targeting MUC16 End is connected with the 5 ' of the encoding gene of extracellular hinge area ends, the 3 ' of the encoding gene of the extracellular hinge area hold and it is described across 5 ' ends of the encoding gene in film area are connected, the encoding gene at 3 ' ends and the intracellular signal area of the encoding gene of the transmembrane region 5 ' end be connected.

The signal peptide is used to that the Chimeric antigen receptor CAR-MUC16 to be instructed to express to cell surface in the present invention, The signal peptide is 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 second aspect part, which is not described herein again.

Optionally, the amino acid sequence of the CAR-MUC16 includes the amino acid sequence as shown in SEQ ID NO:15.

Optionally, the coding gene sequence of the CAR-MUC16 includes the nucleotide sequence as shown in SEQ ID NO:16.

Optionally, the encoding gene of the CAR-MUC16 should consider degeneracy base, i.e., as shown in SEQ IDNO: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-MUC16 is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR, and After the EF1 α of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CAR-MUC16 is inserted into pWPXLD load When body, BamH I in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-MUC16 Restriction enzyme 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, the CD3 positive t lymphocytes are to separate to obtain from source of people peripheral blood mononuclear cells.Further Optionally, the source of people peripheral blood mononuclear cells is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..More Fresh peripheral blood or marrow further alternative, acquired after cancer patient's operation one month, after chemicotherapy one month.

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 single-chain antibodies of targeting MUC16 as described in relation to the first aspect a kind of, such as second party Described in face or the Chimeric antigen receptor T cell of targeting MUC16, such as third party made from the preparation method as described in terms of the 5th Recombinant viral vector described in face or the host cell as described in fourth aspect are preparation prevention, diagnosing and treating malignant tumour Application in drug.

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 single-chain antibody of MUC16, as described in second aspect targeting MUC16 Chimeric antigen receptor T cell, as described in the third aspect One of recombinant viral vector, host cell as described in fourth aspect are a variety of.

Beneficial effects of the present invention:

The single-chain antibody and its Chimeric antigen receptor T cell of targeting MUC16 provided by the invention can be with the targeting of specificity The malignant cell of MUC16, efficient and specific combination malignant cell are generated, and promotes T cell in patient's body Amplification, to malignant cell generate fragmentation effect.

Detailed description of the invention

Fig. 1 is the plasmid map of pWPXLd-CAR-MUC16 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 MUC16, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-MUC16 of preparation targeting MUC16

Prepare respectively signal peptide, target the single-chain antibody of MUC16, CD8 α hinge area, CD8 transmembrane region, 4-1BB 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 MUC16 The encoding gene of chain antibody as shown in SEQ ID NO:2, the encoding gene of the CD8 α hinge area as shown in SEQ ID NO:6, The encoding gene of the CD8 transmembrane region is as shown in SEQ IDNO:8, the encoding gene of the 4-1BB 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, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, 4- of MUC16 1BB 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 MUC16 The encoding gene of antigen receptor CAR-MUC16, the encoding gene of the CAR-MUC16 is as shown in SEQ ID NO:16.

(2) pWPXLd-CAR-MUC16 recombinant plasmid is constructed

The encoding gene of CAR-MUC16 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-MUC16 is inserted into pWPXLD carrier, The I digestion position BamH in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-MUC16 Point is connected, and 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 turn Enter competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.It is examined by PCR product gel electrophoresis It surveys and sequencing identification meets target fragment size and sequence, successfully construct pWPXLd-CAR-MUC16 recombinant plasmid, as shown in Figure 1 For pWPXLd-CAR-MUC16 recombinant plasmid.

(3) recombinant slow virus constructs

PWPXLd-CAR-MUC16 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 MUC16 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 MUC16, 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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Asn Thr Leu His Leu Gln Met Gly Ser Leu Arg Ser Gly Asp Thr Ala

210 215 220

Met Tyr Tyr Cys Ala Arg Gln Gly Phe Gly Asn Tyr Gly Asp Tyr Tyr

225 230 235 240

Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Thr

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475

<210> 4

<211> 1434

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gacattgagc tcacccagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60

atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ccagttggct 120

tggtaccagc aaaaaccagg acagtctcct gaactgctga tctactgggc atccactagg 180

caatctggag tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gccagcaatc ttataatcta 300

ctcacgttcg gtcctgggac caagctggag gtcaaacggg gaggcggagg atctggcggc 360

ggaggaagtg gcggaggggg atctggggga ggcggaagcg tgaagctgca ggagtcaggg 420

ggaggcttcg tgaagcctgg agggtccctc aaagtctcct gtgcagcctc tggattcact 480

ttcagtagct atgccatgtc ctgggttcgc ctgagtccgg agatgaggct ggagtgggtc 540

gcaaccatta gcagtgctgg tggttacatc ttctattctg acagtgtgca gggacgattc 600

accatttcca gagacaatgc caagaacacc ctgcacctgc aaatgggcag tctgaggtct 660

ggggacacgg ccatgtatta ctgtgcaagg cagggatttg gtaactacgg tgattactat 720

gctatggact actggggcca agggaccacg gtcaccgtct cctcaaccac gacgccagcg 780

ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 840

gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 900

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 960

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1020

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1080

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 1140

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1200

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1260

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1320

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1380

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 1434

<210> 5

<211> 45

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

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> 6

<211> 135

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 7

<211> 24

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

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> 8

<211> 72

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 9

<211> 42

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 9

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

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 10

<211> 126

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<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> 498

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

20 25 30

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

35 40 45

Leu Leu Asn Ser Arg Thr Arg Lys Asn Gln Leu Ala Trp Tyr Gln Gln

50 55 60

Lys Pro Gly Gln Ser Pro Glu Leu Leu Ile Tyr Trp Ala Ser Thr Arg

65 70 75 80

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Leu Ser

180 185 190

Pro Glu Met Arg Leu Glu Trp Val Ala Thr Ile Ser Ser Ala Gly Gly

195 200 205

Tyr Ile Phe Tyr Ser Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg

210 215 220

Asp Asn Ala Lys Asn Thr Leu His Leu Gln Met Gly Ser Leu Arg Ser

225 230 235 240

Gly Asp Thr Ala Met Tyr Tyr Cys Ala Arg Gln Gly Phe Gly Asn Tyr

245 250 255

Gly Asp Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr

260 265 270

Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu

340 345 350

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu

355 360 365

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

370 375 380

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

465 470 475 480

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

485 490 495

Pro Arg

<210> 16

<211> 1494

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

gacattgagc tcacccagtc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 120

atgagctgca aatccagtca gagtctgctc aacagtagaa cccgaaagaa ccagttggct 180

tggtaccagc aaaaaccagg acagtctcct gaactgctga tctactgggc atccactagg 240

caatctggag tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 300

atcagcagtg tgcaggctga agacctggca gtttattact gccagcaatc ttataatcta 360

ctcacgttcg gtcctgggac caagctggag gtcaaacggg gaggcggagg atctggcggc 420

ggaggaagtg gcggaggggg atctggggga ggcggaagcg tgaagctgca ggagtcaggg 480

ggaggcttcg tgaagcctgg agggtccctc aaagtctcct gtgcagcctc tggattcact 540

ttcagtagct atgccatgtc ctgggttcgc ctgagtccgg agatgaggct ggagtgggtc 600

gcaaccatta gcagtgctgg tggttacatc ttctattctg acagtgtgca gggacgattc 660

accatttcca gagacaatgc caagaacacc ctgcacctgc aaatgggcag tctgaggtct 720

ggggacacgg ccatgtatta ctgtgcaagg cagggatttg gtaactacgg tgattactat 780

gctatggact actggggcca agggaccacg gtcaccgtct cctcaaccac gacgccagcg 840

ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 900

gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 960

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1020

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 1200

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgc 1494

Claims

1. a kind of single-chain antibody for targeting MUC16, which is characterized in that the amino acid sequence of the single-chain antibody of the targeting MUC16 Including the amino acid sequence as shown in SEQ ID NO:1.

2. the single-chain antibody of targeting MUC16 as described in claim 1, which is characterized in that the single-chain antibody of the targeting MUC16 Encoding gene include the nucleotide sequence as shown in SEQ ID NO:2.

3. a kind of Chimeric antigen receptor T cell for targeting MUC16, which is characterized in that the Chimeric antigen receptor including targeting MUC16 CAR-MUC16, the amino acid sequence of the CAR-MUC16 include the sequentially connected targeting MUC16 from aminoterminal to c-terminus Single-chain antibody, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein the targeting MUC16's is single-stranded anti- The amino acid sequence of body includes the amino acid sequence as shown in SEQ ID NO:1.

4. the Chimeric antigen receptor T cell of targeting MUC16 as claimed in claim 3, which is characterized in that the CAR-MUC16 Amino acid sequence include the amino acid sequence as shown in SEQ ID NO:3.

5. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes such as any one of claim 3 or 4 institute The encoding gene of the CAR-MUC16 of the Chimeric antigen receptor T cell of the targeting MUC16 stated.

6. recombinant viral vector as claimed in claim 5, which is characterized in that the encoding gene of the CAR-MUC16 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 MUC16 characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-MUC16 of targeting MUC16 is provided, including is sequentially connected from 5 ' ends to 3 ' ends The encoding gene of the signal peptide connect, the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, cross-film for targeting MUC16 The encoding gene in area and the encoding gene in intracellular signal area, wherein the encoding gene of single-chain antibody of the targeting MUC16 includes The corresponding nucleotide sequence of amino acid sequence as shown in SEQ ID NO:1；

(2) encoding gene of the CAR-MUC16 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-MUC16 recombination matter Grain；

(3) by the pWPXLD-CAR-MUC16 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 of targeting MUC16 is obtained through separation T cell.

9. the preparation method of the Chimeric antigen receptor T cell of targeting MUC16 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 target the single-chain antibody of MUC16, such as any one of claim 3-4 institute as claim 1-2 is described in any item The Chimeric antigen receptor T cell of MUC16 is targeted made from preparation method state or as described in claim 8-9 or such as right It is required that the described in any item recombinant viral vectors of 5-6 or host cell as claimed in claim 7 prevent, diagnose and control in preparation Treat the application in the drug of malignant tumour.