CN109957025A

CN109957025A - It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and its preparation method and application

Info

Publication number: CN109957025A
Application number: CN201711423229.6A
Authority: CN
Inventors: 张宏玲; 龙丽梅; 钟春颖
Original assignee: Shenzhen Benta Biological Technology Co Ltd
Current assignee: Shenzhen Benta Biological Technology Co Ltd
Priority date: 2017-12-25
Filing date: 2017-12-25
Publication date: 2019-07-02

Abstract

The present invention provides a kind of single-chain antibody for targeting DR5, the single-chain antibody of the targeting DR5 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 DR5, the Chimeric antigen receptor of the targeting DR5 can be with the targeting DR5 of specificity, promote T cell in the amplification of patient's body, killing tumor cell that can be efficient and specific, the vigor and lethality of cell are preferably maintained, and not will cause damage to normal cell.The present invention also provides a kind of preparation method and application of Chimeric antigen receptor T cell for targeting DR5.

Description

It is a kind of to target the single-chain antibody of DR5, Chimeric antigen receptor T cell and preparation method thereof 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 DR5 is thin Born of the same parents and its preparation method and application.

Background technique

Cancer has become the arch-criminal for threatening human life, and for disease incidence at ascendant trend, people cause tumour from induction is found Apoptosis or the angle for overcoming tumour cell to be resistant to apoptosis are started with, it was found that tumor necrosin relative death inducing is matched Body (TRAIL) is the tumor necrosis factor superfamily member that can be induced cell apoptosis.Death receptor 5 (death receptor 5；DR5) belong to A member of the TNF receptor family (TN FRSF10B), cytoplasmic region death domain-containing is main to be distributed In tumour cell.TRAIL can induce Apoptosis in conjunction with cell surface DR5.DR5 is in a variety of solid tumor cells such as liver cancer Wide expression, and very faint cell surface antigen is expressed in ordinary cells.

Immune cell therapy is that the method for thoroughly removing cancer cell is uniquely possible in existing science and technology, and treatment tumour has High specificity, almost non-toxic side effect huge advantage the drawbacks of compensating for traditional remedies, at home and abroad have been used to clinic and control Malignant tumour is treated, Chimeric antigen receptor T cell technology (CAR-T) is that current adoptive cell adoptive therapy technology is newest immune One of cell technology, because it can activate self immune system in vivo, routinely targets neoplastic cells are killed, most Achieve the purpose that remove malignant cell and widely paid close attention to and studied eventually.There is not the inosculating antibody of targeting DR5 also at present The preparation and research of original receptor T cell.

Summary of the invention

In view of this, the present invention provides a kind of single-chain antibodies for targeting DR5, and including the single-stranded of the targeting DR5 The Chimeric antigen receptor T cell of the targeting DR5 of antibody.The Chimeric antigen receptor of the targeting DR5 can be with the targeting of specificity DR5, promotes T cell in the amplification of patient's body, and killing tumor cell that can be efficient and specific preferably maintains inosculating antibody The vigor and lethality of original receptor T cell, and not will cause damage to normal cell.The present invention also provides a kind of targetings The preparation method and application of the Chimeric antigen receptor T cell of DR5.

In a first aspect, the present invention provides a kind of single-chain antibody for targeting DR5, the single-chain antibody of the targeting DR5 includes The amino acid sequence as shown in SEQ ID NO:1.

Optionally, the encoding gene of the single-chain antibody of the targeting DR5 includes the nucleotides sequence as shown in SEQ ID NO:2 Column.

Optionally, the single-chain antibody encoding gene of the targeting DR5 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:2, protection scope should also protect Shield has the nucleotide sequence of base degeneracy matter with SEQ ID NO:2, and the corresponding amino acid sequence of these nucleotide sequences is still It is so SEQ ID NO:1.

The single-chain antibody for the targeting DR5 that first aspect present invention provides can be specifically bound with DR5 albumen, The solid tumor cell for especially expressing tumour cell DR5 has stronger affine activity.

Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells for targeting DR5, including the chimeric of targeting DR5 Antigen receptor CAR-DR5, the CAR-DR5 include the single-chain antibody of sequentially connected targeting DR5, born of the same parents from aminoterminal to c-terminus The amino acid sequence of outer hinge area, transmembrane region and intracellular signal area, wherein the single-chain antibody of the targeting DR5 includes such as SEQ ID Amino acid sequence shown in NO:1.

Wherein, described " being sequentially connected with from aminoterminal to c-terminus " specifically: the amino of the single-chain antibody of the targeting DR5 The c-terminus of acid sequence is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, the amino acid of the extracellular hinge area The c-terminus of sequence is connected with the aminoterminal of the amino acid sequence of the transmembrane region, the carboxyl of the amino acid sequence of the transmembrane region End is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.

In the present invention, the extracellular hinge area is used to promote the DR5 knot on the single-chain antibody and tumour of the targeting DR5 It closes.Optionally, the extracellular hinge area includes CD8 α hinge area, CD28 hinge area, CD4 hinge area, CD5 hinge area, CD134 hinge One of sequence, 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。

In the present invention, the transmembrane region is used to fix the Chimeric antigen receptor CAR-DR5 of the targeting DR5.Optionally, institute Stating transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region or a variety of combinations.

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。

In the present invention, the intracellular signal area for providing the signal of T cell activation, 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 letter Number area, CD27 signaling zone, OX40 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRSF19L signaling zone One of or a variety of combinations.

Optionally, the intracellular signal Qu Weicong aminoterminal is to the sequentially connected CD27 signaling zone of c-terminus and CD3 ζ signal Area.Correspondingly, the encoding gene in the intracellular signal area includes the coding from 5 ' ends to 3 ' the sequentially connected CD27 signaling zones in end The encoding gene of gene and CD3 ζ signaling zone.

Optionally, the amino acid sequence of the CD27 signaling zone includes the amino acid sequence as shown in SEQ ID NO:10.

Optionally, the encoding gene of the CD27 signaling zone includes the nucleotide sequence as shown in SEQ ID NO:11.

Optionally, the encoding gene of the CD27 signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:10 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:11 shown in, protection scope should also protect and SEQ ID NO:11 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:10。

Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:12.

Optionally, the encoding gene of the CD3 ζ signaling zone includes the nucleotide sequence as shown in SEQ ID NO:13.

Optionally, the encoding gene of the CD3 ζ signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:12 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:13 shown in, protection scope should also protect and SEQ ID NO:13 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:12。

Optionally, the amino acid sequence of the CAR-DR5 includes the amino acid sequence as shown in SEQ ID NO:3.At this point, The CD3 ζ signaling zone as shown in the amino acid sequence of SEQ ID NO:12 as the T cell antigentic specificity signal (that is, One signal), costimulatory signal of the CD27 signaling zone as the T cell as shown in the amino acid sequence of SEQ ID NO:10, Under their collective effect, T cell is activated completely after identifying antigen.Particularly, it selects such as SEQ ID NO:10 CD27 signaling zone can preferably promote amplification ability, the killing vigor of subsequent T cell as costimulatory signal.

Optionally, the encoding gene of the CAR-DR5 includes the nucleotide sequence as shown in SEQ ID NO:4.

Optionally, the encoding gene of the CAR-DR5 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。

Further, the encoding gene of the CAR-CD22 includes the nucleotide sequence as shown in SEQ ID NO:5.

The Chimeric antigen receptor T cell for the targeting DR5 that second aspect of the present invention provides, including the chimeric of targeting DR5 Antigen receptor CAR-DR5, this receptor for T cell targeted expression DR5 in specific manner tumour cell, CAR-DR5 with After DR5 is combined, the intracellular signal area of the T cell is activated, and promotes T cell in the amplification of patient's body, efficient and specificity Killing tumor cell, and normal cell is hardly caused to damage.

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-DR5 of the Chimeric antigen receptor T cell of the targeting DR5.

Optionally, the encoding gene of the CAR-DR5 includes the nucleotide sequence as shown in SEQ ID NO:5.Such as SEQ Nucleotide sequence shown in ID NO:5 is compared with the nucleotide sequence as shown in SEQ ID NO:4, more coding bases of link peptide Cause.The encoding gene of the signal peptide can be expressed with Chimeric antigen receptor CAR-CD22 described in guide to cell surface.

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.

The Chimeric antigen receptor T that the recombinant viral vector that third aspect present invention provides can be used for targeting DR5 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.

The host cell is used to assemble the recombinant viral vector as described in the third aspect, makes it have infectivity.

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.

5th aspect, the present invention provides a kind of preparation methods of Chimeric antigen receptor T cell for targeting DR5, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-DR5 of targeting DR5 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, transmembrane region for targeting DR5 Encoding gene and intracellular signal area encoding gene, wherein it is described targeting DR5 single-chain antibody be Humanized single chain antibody, The encoding gene of the single-chain antibody of the targeting DR5 includes the nucleotide sequence as shown in SEQ ID NO:2；

(2) encoding gene of the CAR-DR5 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-DR5 recombination matter Grain；

(3) it by the pWPXLD-CAR-DR5 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains Recombinant slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes；

(5) separate and obtain the Chimeric antigen receptor T cell of targeting DR5.

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 Target DR5 single-chain antibody encoding gene 5 ' end be connected, it is described targeting DR5 single-chain antibody encoding gene 3 ' end with 5 ' ends of the encoding gene of the extracellular hinge area are connected, 3 ' ends of the encoding gene of the extracellular hinge area and the transmembrane region 5 ' ends of encoding gene be connected, the 3 ' ends and the 5 ' of the encoding gene in the intracellular signal area of the encoding gene of the transmembrane region End is connected.

The signal peptide is for instructing the Chimeric antigen receptor CAR-DR5 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:14.

Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:15.

Optionally, the encoding gene of the signal peptide should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:14 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:15, and protection scope should also protect and SEQ ID NO:15 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:14。

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 coding gene sequence of the CAR-DR5 is as shown in SEQ ID NO:5.As shown in SEQ ID NO:5 Nucleotide sequence is compared with the nucleotide sequence as shown in SEQ ID NO:4, more encoding genes of the link peptide, but in institute When stating Chimeric antigen receptor CAR-DR5 expression to cell surface, the signal peptide is in protein translation maturation by signal peptide Digestion is cut.Therefore, in the amino acid sequence of the Chimeric antigen receptor CAR-DR5 translated into and not with such as SEQ ID Amino acid sequence shown in NO:14.

The encoding gene of the CAR-DR5 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-DR5 is inserted into pWPXLD carrier When, BamH1 digestion in initiation codon (such as ATG) and pWPXLD carrier can be added in 5 ' ends of the encoding gene of the CAR-DR5 Site is connected, and 3 ' ends can be added terminator codon (such as TAA) and be connected with EcoR1 restriction enzyme site 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 single-chain antibodies of targeting DR5 as described in relation to the first aspect a kind of, such as second aspect The Chimeric antigen receptor T cell of targeting DR5 made from described or as described in terms of the 5th preparation method, such as third aspect institute The recombinant viral vector stated or the host cell as described in fourth aspect are in preparation prevention, the drug of diagnosing and treating malignant tumour In application.

The application specifically: provide a kind of kit, the kit includes targeting DR5 described in first aspect Single-chain antibody, the Chimeric antigen receptor T cell for targeting DR5 as described in second aspect, the recombinant virus as described in the third aspect One of carrier, host cell as described in fourth aspect are a variety of.

Beneficial effects of the present invention:

The Chimeric antigen receptor T cell of targeting DR5 provided by the invention can promote T cell to exist with the targeting DR5 of specificity The amplification of patient's body, killing tumor cell that can be efficient and specific, while DR5 wide expression in tumour cell, and Expressed in ordinary cells it is very faint, therefore target DR5 Chimeric antigen receptor T cell can specificity combination tumour it is thin Born of the same parents generate fragmentation effect to tumour cell, not will cause damage to normal cell.

Detailed description of the invention

Fig. 1 is the plasmid map of pWPXLd-CAR-DR5 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 DR5, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-DR5 of preparation targeting DR5

Single-chain antibody, CD8 α hinge area, CD8 transmembrane region, CD27 signaling zone and the CD3 for preparing signal peptide respectively, targeting DR5 The encoding gene of ζ signaling zone, for the encoding gene of the signal peptide as shown in SEQ ID NO:15, the targeting DR5's is single-stranded anti- For the encoding gene of body as shown in SEQ ID NO:2, the encoding gene of the CD8 α hinge area is described as shown in SEQ ID NO:7 The encoding gene of CD8 transmembrane region is as shown in SEQ ID NO:9, the encoding gene of the CD27 signaling zone such as SEQ ID NO:11 institute Show, the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:13.

By the method for PCR by above-mentioned signal peptide, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, CD27 of DR5 Signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the chimeric antigen of targeting DR5 The encoding gene of receptor CAR-DR5, the encoding gene of the CAR-DR5 is as shown in SEQ ID NO:5.

(2) pWPXLd-CAR-DR5 recombinant plasmid is constructed

The encoding gene of CAR-DR5 is inserted between BamH1 the and EcoR1 restriction enzyme site of pWPXLD carrier, and After pWPXLD carrier EF1 α, using EF1 α as promoter.When the encoding gene of the CAR-DR5 is inserted into pWPXLD carrier, institute Initiation codon (such as ATG) and BamH1 restriction enzyme site phase in pWPXLD carrier can be added in the 5 ' ends for stating the encoding gene of CAR-DR5 Even, 3 ' ends can also be added terminator codon (such as TAA) and be connected with EcoR1 restriction enzyme site in pWPXLD carrier.Then it is transferred to big Enterobacteria competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and Sequencing identification meets target fragment size and sequence, successfully constructs pWPXLd-CAR-DR5 recombinant plasmid, is as shown in Figure 1 PWPXLd-CAR-DR5 recombinant plasmid.

(3) recombinant slow virus constructs

PWPXLd-CAR-DR5 recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into culture Good HEK293T cell.48h harvest saves in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration； It is super to merge addition together with the viral supernatants of 48h harvest for supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration In fast centrifuge tube, be put into Beckman ultracentrifuge one by one, setting parameter of noncentricity be 25000rpm, centrifugation time 2h, Centrifuging temperature is 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 and saves Liquid, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence spectrometry after being centrifuged 5min 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 DR5 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 DR5, and protect In the presence of in the dedicated cells frozen storing liquid of feedback.

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.

Sequence table

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

Cys Pro Arg Glu Glu Glu Gly Ser Thr Ile Pro Ile Gln Glu Asp Tyr

340 345 350

Arg Lys Pro Glu Pro Ala Cys Ser Pro Arg Val Lys Phe Ser Arg Ser

355 360 365

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

370 375 380

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

385 390 395 400

Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln

405 410 415

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

420 425 430

Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp

435 440 445

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

450 455 460

Leu His Met Gln Ala Leu Pro Pro Arg

465 470

<210> 4

<211> 1419

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gacattgtga tgacacaatc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 60

atgagctgca aatccagtca gagtctgctc aacagtagaa cccggaagaa ctacttggct 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 180

gaatctgggg tccctgatcg cttcataggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctt 300

ccgttcacgt tcggtgctgg gaccaagctg gagctgaaag gtggcggtgg ctcgggcggt 360

ggtgggtcgg gtggcggcgg atctgaagtg aagcttctcg agtctggagg tggcctggtg 420

cagcctggag gatccctgaa actctcctgt gcagcctcag gattcgattt tagtacatgc 480

tggatgaatt gggtccggca ggctccaggg aaaggactag aatggattgg agaaattaat 540

ccagatagta gtaggataaa ttatatgcca tctctaaagg aaaaattcat catctccaga 600

gacaacgcca aaaatacact gtacctgcaa atgagcaaag tgagatctga agacacagcc 660

ctttattact gtgcaagggg aggaactttc tatgctatgg actactgggg tcaaggaacc 720

tcagtcaccg tctcctcaac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 780

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 840

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 900

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaggaa atatagatca 960

aacaaaggag aaagtcctgt ggagcctgca gagccttgtc gttacagctg ccccagggag 1020

gaggagggca gcaccatccc catccaggag gattaccgaa aaccggagcc tgcctgctcc 1080

ccccgcgtga aattcagccg cagcgcagat gctccagcct acaagcaggg gcagaaccag 1140

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1200

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1260

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1320

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1380

acctatgacg ctcttcacat gcaggccctg ccgcctcgg 1419

<210> 5

<211> 1479

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

gacattgtga tgacacaatc tccatcctcc ctggctgtgt cagcaggaga gaaggtcact 120

atgagctgca aatccagtca gagtctgctc aacagtagaa cccggaagaa ctacttggct 180

tggtaccagc agaaaccagg gcagtctcct aaactgctga tctactgggc atccactagg 240

gaatctgggg tccctgatcg cttcataggc agtggatctg ggacagattt cactctcacc 300

atcagcagtg tgcaggctga agacctggca gtttattact gcaagcaatc ttataatctt 360

ccgttcacgt tcggtgctgg gaccaagctg gagctgaaag gtggcggtgg ctcgggcggt 420

ggtgggtcgg gtggcggcgg atctgaagtg aagcttctcg agtctggagg tggcctggtg 480

cagcctggag gatccctgaa actctcctgt gcagcctcag gattcgattt tagtacatgc 540

tggatgaatt gggtccggca ggctccaggg aaaggactag aatggattgg agaaattaat 600

ccagatagta gtaggataaa ttatatgcca tctctaaagg aaaaattcat catctccaga 660

gacaacgcca aaaatacact gtacctgcaa atgagcaaag tgagatctga agacacagcc 720

ctttattact gtgcaagggg aggaactttc tatgctatgg actactgggg tcaaggaacc 780

tcagtcaccg tctcctcaac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 840

atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 900

gtgcacacga gggggctgga cttcgcctgt gatatctaca tctgggcgcc cttggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaggaa atatagatca 1020

aacaaaggag aaagtcctgt ggagcctgca gagccttgtc gttacagctg ccccagggag 1080

gaggagggca gcaccatccc catccaggag gattaccgaa aaccggagcc tgcctgctcc 1140

ccccgcgtga aattcagccg cagcgcagat gctccagcct acaagcaggg gcagaaccag 1200

ctctacaacg aactcaatct tggtcggaga gaggagtacg acgtgctgga caagcggaga 1260

ggacgggacc cagaaatggg cgggaagccg cgcagaaaga atccccaaga gggcctgtac 1320

aacgagctcc aaaaggataa gatggcagaa gcctatagcg agattggtat gaaaggggaa 1380

cgcagaagag gcaaaggcca cgacggactg taccagggac tcagcaccgc caccaaggac 1440

acctatgacg ctcttcacat gcaggccctg ccgcctcgg 1479

<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> 46

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

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

20 25 30

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

35 40 45

<210> 11

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

aggaaatata gatcaaacaa aggagaaagt cctgtggagc ctgcagagcc ttgtcgttac 60

agctgcccca gggaggagga gggcagcacc atccccatcc aggaggatta ccgaaaaccg 120

gagcctgcct gctccccc 138

<210> 12

<211> 112

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

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> 13

<211> 336

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

cgcgtgaaat tcagccgcag cgcagatgct ccagcctaca agcaggggca gaaccagctc 60

tacaacgaac tcaatcttgg tcggagagag gagtacgacg tgctggacaa gcggagagga 120

cgggacccag aaatgggcgg gaagccgcgc agaaagaatc cccaagaggg cctgtacaac 180

gagctccaaa aggataagat ggcagaagcc tatagcgaga ttggtatgaa aggggaacgc 240

agaagaggca aaggccacga cggactgtac cagggactca gcaccgccac caaggacacc 300

tatgacgctc ttcacatgca ggccctgccg cctcgg 336

<210> 14

<211> 20

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

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> 15

<211> 60

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60

Claims

1. a kind of single-chain antibody for targeting DR5, which is characterized in that the single-chain antibody of the targeting DR5 includes such as SEQ ID NO:1 Shown in amino acid sequence.

2. the single-chain antibody of targeting DR5 as described in claim 1, which is characterized in that the volume of the single-chain antibody of the targeting DR5 Code gene includes the nucleotide sequence as shown in SEQ ID NO:2.

3. a kind of Chimeric antigen receptor T cell for targeting DR5, which is characterized in that the Chimeric antigen receptor CAR- including targeting DR5 DR5, the CAR-DR5 include from aminoterminal to c-terminus the sequentially connected targeting single-chain antibody of DR5, extracellular hinge area, across The amino acid sequence in film area and intracellular signal area, wherein the single-chain antibody of the targeting DR5 includes as shown in SEQ ID NO:1 Amino acid sequence.

4. the Chimeric antigen receptor T cell of targeting DR5 as claimed in claim 3, which is characterized in that the extracellular hinge area packet CD8 α hinge area is included, the transmembrane region includes CD8 transmembrane region, and the intracellular signal area includes sequentially connecting from aminoterminal to c-terminus The CD27 signaling zone and CD3 ζ signaling zone connect.

5. the Chimeric antigen receptor T cell of targeting DR5 as claimed in claim 4, which is characterized in that the ammonia of the CAR-DR5 Base acid sequence includes the amino acid sequence as shown in SEQ ID NO:3.

6. the Chimeric antigen receptor T cell of targeting DR5 as described in claim 3 or 4, which is characterized in that the CAR-DR5's Encoding gene includes the nucleotide sequence as shown in SEQ ID NO:4.

7. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes as described in claim any one of 3-6 Targeting DR5 Chimeric antigen receptor T cell CAR-DR5 encoding gene.

8. 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.

9. a kind of preparation method for the Chimeric antigen receptor T cell for targeting DR5 characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-DR5 of targeting DR5 is provided, including sequentially connected from 5 ' ends to 3 ' ends The encoding gene of signal peptide, the encoding gene of single-chain antibody for targeting DR5, the encoding gene of extracellular hinge area, transmembrane region volume The encoding gene of code gene and intracellular signal area, wherein the single-chain antibody of the targeting DR5 is Humanized single chain antibody, described The encoding gene for targeting the single-chain antibody of DR5 includes the nucleotide sequence as shown in SEQ ID NO:2；

(2) encoding gene of the CAR-DR5 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-DR5 recombinant plasmid；

(3) it by the pWPXLD-CAR-DR5 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, is recombinated Slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes；

(5) separate and obtain the Chimeric antigen receptor T cell of targeting DR5.

10. a kind of target the single-chain antibody of DR5, as described in claim any one of 3-6 as claim 1-2 is described in any item Or preparation method as claimed in claim 9 targeting DR5 obtained Chimeric antigen receptor T cell or such as claim 7 institute The recombinant viral vector or host cell as claimed in claim 8 stated are in preparation prevention, the medicine of diagnosing and treating malignant tumour Application in object.