CN109836498A - It is a kind of to target the single-chain antibody of ROR1, Chimeric antigen receptor T cell and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of single-chain antibody for targeting ROR1, the single-chain antibody of the targeting ROR1 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 ROR1, the Chimeric antigen receptor of the targeting HER2 can be with the targeting ROR1 of specificity, T cell is activated to play immunization of cell, efficient and specific killing ROR1 positive tumor cell, with lasting cell viability and lethality, and damage is not will cause to normal cell.The present invention also provides a kind of preparation method and application of Chimeric antigen receptor T cell for targeting ROR1.

Description

It is a kind of to target the single-chain antibody of ROR1, 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 ROR1 is thin Born of the same parents and its preparation method and application.

Background technique

Malignant tumour is to seriously endanger the disease of human health the most, and about three million peoples of annual China are diagnosed as newly sending out Tumor patient, two million peoples die of cancer.Wherein, the morbidity and mortality of lung cancer increase most fast, are population health and life Threaten one of maximum malignant tumour.Nearest studies have shown that tyrosine protein kinase transmembrane receptor (Receptor Tyrosine kinase like orphan receptor 1, ROR1) be expressed in a variety of B cell malignant tumours, and And then include lung cancer, lymphoma mantle cell (MCL), acute lymphoblastic leukemia (ALL), breast cancer, colon cancer, ROR1 is identified on the surface of many cancers including cancer of pancreas, kidney and oophoroma subgroup.In addition, ROR1 can be dry thin in cancer High expression on born of the same parents such as ovary cancer stem cell, and it is mediated to migrate and invasion.Therefore, ROR1 is a kind of tumour-specific height, and Ideal tumour target antigen with wide spectrum expression in all kinds of tumours.

Chimeric antigen receptor T cell technology (Chimeric Antigen Receptor-Modified T Cells, CAR- T) as one of current newest immunocyte technology, because it can activate self immune system in vivo, routinely target It is killed to tumour cell, is finally reached fully erased malignant cell, is widely paid close attention to and studied.But The current application of CART technology is also limited to blood tumor, does not there is the correlative study of the Chimeric antigen receptor T cell of targeting ROR1 also.

Summary of the invention

To solve the above problems, the present invention provides a kind of single-chain antibodies for targeting ROR1, and including the targeting The Chimeric antigen receptor T cell of the single-chain antibody of ROR1.The single-chain antibody of the targeting ROR1 has very ROR1 antigen protein Strong specificity, can be with efficient identification ROR1 positive cell；The Chimeric antigen receptor T cell of the single-chain antibody of the targeting ROR1 The tumour cell for having ROR1 is expressed in killing that can be efficient and specific, has very high vigor and lethality, and to normal Cell not will cause damage.The present invention also provides a kind of preparation method of Chimeric antigen receptor T cell for targeting ROR1 and answer With.

In a first aspect, the present invention provides a kind of single-chain antibody for targeting ROR1, the single-chain antibody packet of the targeting ROR1 Include the amino acid sequence as shown in SEQ ID NO:1.

Optionally, the encoding gene of the single-chain antibody of the targeting ROR1 includes the nucleotide as shown in SEQ ID NO:2 Sequence.

Optionally, the single-chain antibody encoding gene of the targeting ROR1 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 ROR1 that first aspect present invention provides can occur special with ROR1 antigen protein Property combine, have very high specificity, stability is good, especially for expression ROR1 solid tumor cell have it is stronger affine Activity and internalization activity.

Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells for targeting ROR1, including the embedding of targeting ROR1 Antigen receptor CAR-ROR1 is closed, the CAR-ROR1 includes the single-stranded anti-of the sequentially connected targeting ROR1 from aminoterminal to c-terminus Body, extracellular hinge area, transmembrane region and intracellular signal area amino acid sequence, wherein it is described targeting ROR1 single-chain antibody include such as Amino acid sequence 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 ROR1 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 the ROR1 on the single-chain antibody and tumour of the targeting ROR1 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 N O:6 Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:7 shown in, 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-ROR1 of the targeting ROR1 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.

Optionally, the intracellular signal area includes 4-1BB signaling zone, CD3 ζ signaling zone, ICOS signaling zone, CD27 signal One of area, OX40 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRSF19L signaling zone are a variety of Combination.

Optionally, the intracellular signal area is 4-1BB signaling zone and 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:10.

Optionally, the encoding gene of the 4-1BB signaling zone includes the nucleotide sequence as shown in SEQ ID NO:11.

Optionally, the encoding gene of the 4-1BB 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-ROR1 includes the amino acid sequence as shown in SEQ ID NO:3.

Optionally, the encoding gene of the CAR-ROR1 includes the nucleotide sequence as shown in SEQ ID NO:4.

Optionally, the encoding gene of the CAR-ROR1 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。

The Chimeric antigen receptor T cell of targeting HER2 of the present invention can include that acute lymphoblastic is female with efficient identification and killing The expression such as cell leukemia, breast cancer, colon cancer, cancer of pancreas, kidney and oophoroma have the cancer cell of ROR1, are particularly suitable for The lung carcinoma cell or tissue of the ROR1 positive.

The Chimeric antigen receptor T cell for the targeting ROR1 that second aspect of the present invention provides, including the embedding of targeting ROR1 Close antigen receptor CAR-ROR1, this receptor for T cell targeted expression ROR1 in specific manner tumour cell, in CAR- After ROR1 is in conjunction with ROR1, the intracellular signal area of the T cell is activated, and promotes T cell in the amplification of patient's body, and efficiently And the killing tumor cell of specificity, 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-ROR1 of the Chimeric antigen receptor T cell of the targeting ROR1.

Optionally, the encoding gene of the CAR-ROR1 includes the nucleotide sequence as shown in SEQ ID NO:4.

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 recombinant viral vector that third aspect present invention provides is safe and efficient, can steadily realize CAR-ROR1 Encoding gene import in host cell or replicate, and can be used for targeting the preparation of the Chimeric antigen receptor T cell of HER2, and Realize the expression of height tissue specificity.

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 ROR1, comprising:

(1) encoding gene of the Chimeric antigen receptor CAR-ROR1 of targeting ROR1 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 for targeting ROR1, CD8 α hinge area encoding gene, CD8 The encoding gene of the encoding gene of transmembrane region, the encoding gene of 4-1BB signaling zone and CD3 ζ signaling zone, wherein the targeting The encoding gene of the single-chain antibody of ROR1 includes as shown in SEQ ID NO:2；

(2) encoding gene of the CAR-ROR1 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-ROR1 recombination Plasmid；

(3) it by the pWPXLD-CAR-ROR1 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains To recombined lentivirus vector；

(4) by the recombined lentivirus vector transfect CD3 positive t lymphocytes, obtain targeting ROR1 chimeric antigen by Body T cell.

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 coding gene sequence of the single-chain antibody of ROR1 are connected, the encoding gene sequence of the single-chain antibody of the targeting ROR1 3 ' ends of column are connected with 5 ' ends of the coding gene sequence of the extracellular hinge area, the coding gene sequence of the extracellular hinge area 3 ' ends be connected with the 5 ' of the coding gene sequence of transmembrane region ends, the 3 ' of the coding gene sequence of the transmembrane region are held and institute 5 ' the ends for stating the coding gene sequence in intracellular signal area are connected.

The signal peptide is for instructing the Chimeric antigen receptor CAR-ROR1 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-ROR1 is as shown in SEQ ID NO:5.

The encoding gene of the CAR-ROR1 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-ROR1 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-ROR1 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 single-chain antibodies of targeting ROR1 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 ROR1, 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 controlled in preparation treatment and/or prevention and/or auxiliary Treat the application in the drug of malignant tumour.Specifically, can be used for preventing, diagnosing and treating kinds of tumors, including acute lymphoblastic is female The expression such as cell leukemia and breast cancer, colon cancer, cancer of pancreas, kidney and oophoroma have the cancer cell of ROR1, especially suitable In the lung carcinoma cell or tissue of the ROR1 positive.

The application specifically: provide a kind of kit, the kit includes targeting ROR1 described in first aspect Single-chain antibody, as described in second aspect targeting ROR1 Chimeric antigen receptor T cell, as described in the third aspect recombination disease One of poisonous 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 ROR1 provided by the invention can promote T cell with the targeting ROR1 of specificity In the amplification of patient's body, killing tumor cell that can be efficient and specific, while ROR1 wide expression in tumour cell, And expressed in ordinary cells it is very faint, therefore target ROR1 Chimeric antigen receptor T cell can specificity combination ROR1 Positive tumor cell generates fragmentation effect to ROR1 positive tumor cell, not will cause damage to normal cell.

Detailed description of the invention

Fig. 1 is the plasmid map of pWPXLd-CAR-ROR1 recombinant plasmid provided in an embodiment of the present invention.

Fig. 2 is the positive rate of the Chimeric antigen receptor T cell of targeting ROR1 provided in an embodiment of the present invention；(A) is in Fig. 2 Negative control group, (B) is the experimental group of the Chimeric antigen receptor T cell of targeting ROR1 provided in an embodiment of the present invention in Fig. 2.

Fig. 3 is the tumor cell in vitro killing of the Chimeric antigen receptor T cell of targeting ROR1 provided in an embodiment of the present invention Effect picture.

Fig. 4 is that the Chimeric antigen receptor T cell of targeting ROR1 provided in an embodiment of the present invention treats the effect of mice with tumor Figure.

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 ROR1, comprising the following steps:

(1) gene order of the Chimeric antigen receptor CAR-ROR1 of preparation targeting ROR1

Prepare respectively signal peptide, target the single-chain antibody of ROR1, 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:15, the list of the targeting ROR1 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:7, The encoding gene of the CD8 transmembrane region is as shown in SEQID NO:9, the encoding gene of the 4-1BB signaling zone such as SEQ ID NO: 11, the encoding gene of the CD3 ζ signaling zone such as 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, 4- of ROR1 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 ROR1 The encoding gene of antigen receptor CAR-ROR1, the encoding gene of the CAR-ROR1 is as shown in SEQ ID NO:5.

(2) pWPXLd-CAR-ROR1 recombinant plasmid is constructed

The encoding gene of CAR-ROR1 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-ROR1 is inserted into pWPXLD carrier, institute 5 ' end additions initiation codon (such as ATG) for stating the encoding gene of CAR-ROR1 and BamH1 restriction enzyme site phase in pWPXLD carrier Even, 3 ' ends are also connected added with terminator codon (such as TAA) with EcoR1 restriction enzyme site in pWPXLD carrier.Then it is transferred to large intestine Bacillus competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and survey Sequence identification meets target fragment size and sequence, successfully constructs pWPXLd-CAR-ROR1 recombinant plasmid, is as shown in Figure 1 PWPXLd-CAR-ROR1 recombinant plasmid.

(3) recombinant slow virus constructs

PWPXLd-CAR-ROR1 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 ROR1 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 ROR1, and It is stored in and feeds back in dedicated cells frozen storing liquid.

Effect example

In order to assess the Chimeric antigen receptor T cell effect for targeting ROR1 of above method preparation described in the invention, Carry out following effect example.

Effect example one assesses the positive rate of the Chimeric antigen receptor T cell of targeting ROR1 prepared by the present invention

It will be by the Chimeric antigen receptor T cell (experimental group) of the method for the present invention preparation targeting ROR1 and without the T of preparation Lymphocyte (negative control group), using its positive rate of flow cytomery, as a result as shown in Fig. 2, wherein (A) is in Fig. 2 Negative control group, i.e., without the T cell of preparation, (B) is experimental group in Fig. 2, and targeting ROR1's as produced by the present invention is chimeric Antigen receptor T cell.It can be obtained compared with (B) by (A) in Fig. 2, the Chimeric antigen receptor T of targeting ROR1 prepared by the present invention The positive rate of cell is 48.2%.

The tumor cell in vitro of effect example two, the Chimeric antigen receptor T cell of assessment targeting ROR1 kills situation

Will by the Chimeric antigen receptor T cell (being abbreviated as CAR-T-ROR1) of targeting ROR1 made from the method for the present invention with The Vitro Tumor fragmentation effect of T lymphocyte (negative control group) without preparation is compared, specific: in vitro by effect Cell (CAR-T-ROR1 or the T lymphocyte without preparation) and target cell (H526 cell) quantity ratio are 1:10,1:3,1: 1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Under co-cultured, after incubation 15-18 hours, collect cell, carry out Streaming dyeing, detects cell killing situation, as a result as shown in Figure 3.As can be seen from Figure 3, by method system of the present invention The Chimeric antigen receptor T cell tumor-killing power of standby targeting ROR1 is in 20% or more, even up to 37%, significantly larger than yin Property control group, therefore through the method for the present invention preparation targeting ROR1 Chimeric antigen receptor T cell have strong tumor-killing energy Power.

Effect example three, the mouse interior tumor cell killing feelings of the Chimeric antigen receptor T cell of assessment targeting ROR1 Condition

By the Chimeric antigen receptor T cell (CAR-T-ROR1) of the targeting ROR1 by the method for the present invention preparation, without system Standby T lymphocyte (negative control group) and physiological saline (blank control group) gives every mouse in mouse tumor model Tail vein injection 1 × 10⁶A H526 cell (n=9), obtains the survivorship curve of mouse, as a result as shown in Figure 4.It can from Fig. 4 The Chimeric antigen receptor T cell of the targeting ROR1 by this method preparation is also higher than mouse survival rate when cultivating 35 days out 70%, when cultivating 66 days, survival rate is also higher than 36% or more, considerably beyond negative control group and blank control group.The knot of Fig. 4 Fruit shows that the Chimeric antigen receptor T cell of the targeting ROR1 prepared by this method can preferably be protected mice against because of tumour Caused death.

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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tgtccacctg gacaccatat ctcagaagac ggtagagatt gcatctcctg caaatatgga 60

caggactata gcactcactg gaatgacctc cttttctgct tgcgctgcac caggtgtgat 120

tcaggtgaag tggagctaag tccctgcacc acgaccagaa acacagtgtg tcagtgcgaa 180

gaaggcacct tccgggaaga agattctcct gagatgtgcc ggaagtgccg cacagggtgt 240

cccagaggga tggtcaaggt cggtgattgt acaccctgga gtgacatcga atgtgtccac 300

aaagaagagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 360

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 420

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 480

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 540

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 600

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 660

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 720

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 780

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 840

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 900

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 960

aaccactaca cgcagaagag cctctccctg tctccg 996

<210> 3

<211> 555

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 3

Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg Asp Cys Ile Ser

1 5 10 15

Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe

20 25 30

Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro

35 40 45

Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe

50 55 60

Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys

65 70 75 80

Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp Ile

85 90 95

Glu Cys Val His Lys Glu Glu Pro Lys Ser Cys Asp Lys Thr His Thr

100 105 110

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

115 120 125

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

130 135 140

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

145 150 155 160

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

165 170 175

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

180 185 190

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

195 200 205

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

210 215 220

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

225 230 235 240

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

245 250 255

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

260 265 270

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

275 280 285

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

290 295 300

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

305 310 315 320

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Thr Thr Thr Pro

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

545 550 555

<210> 4

<211> 1665

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

tgtccacctg gacaccatat ctcagaagac ggtagagatt gcatctcctg caaatatgga 60

caggactata gcactcactg gaatgacctc cttttctgct tgcgctgcac caggtgtgat 120

tcaggtgaag tggagctaag tccctgcacc acgaccagaa acacagtgtg tcagtgcgaa 180

gaaggcacct tccgggaaga agattctcct gagatgtgcc ggaagtgccg cacagggtgt 240

cccagaggga tggtcaaggt cggtgattgt acaccctgga gtgacatcga atgtgtccac 300

aaagaagagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 360

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 420

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 480

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 540

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 600

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 660

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 720

tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 780

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 840

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 900

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 960

aaccactaca cgcagaagag cctctccctg tctccgacca ctaccccagc accgaggcca 1020

cccaccccgg ctcctaccat cgcctcccag cctctgtccc tgcgtccgga ggcatgtaga 1080

cccgcagctg gtggggccgt gcatacccgg ggtcttgact tcgcctgcga tatctacatt 1140

tgggcccctc tggctggtac ttgcggggtc ctgctgcttt cactcgtgat cactctttac 1200

tgtaagcgcg gtcggaagaa gctgctgtac atctttaagc aacccttcat gaggcctgtg 1260

cagactactc aagaggagga cggctgttca tgccggttcc cagaggagga ggaaggcggc 1320

tgcgaactgc gcgtgaaatt cagccgcagc gcagatgctc cagcctacaa gcaggggcag 1380

aaccagctct acaacgaact caatcttggt cggagagagg agtacgacgt gctggacaag 1440

cggagaggac gggacccaga aatgggcggg aagccgcgca gaaagaatcc ccaagagggc 1500

ctgtacaacg agctccaaaa ggataagatg gcagaagcct atagcgagat tggtatgaaa 1560

ggggaacgca gaagaggcaa aggccacgac ggactgtacc agggactcag caccgccacc 1620

aaggacacct atgacgctct tcacatgcag gccctgccgc ctcgg 1665

<210> 5

<211> 575

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 5

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

1 5 10 15

Ala Ala Arg Pro Cys Pro Pro Gly His His Ile Ser Glu Asp Gly Arg

20 25 30

Asp Cys Ile Ser Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn

35 40 45

Asp Leu Leu Phe Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val

50 55 60

Glu Leu Ser Pro Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu

65 70 75 80

Glu Gly Thr Phe Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys

85 90 95

Arg Thr Gly Cys Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro

100 105 110

Trp Ser Asp Ile Glu Cys Val His Lys Glu Glu Pro Lys Ser Cys Asp

115 120 125

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

130 135 140

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

145 150 155 160

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

165 170 175

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

180 185 190

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

195 200 205

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

210 215 220

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

225 230 235 240

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

245 250 255

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

<210> 6

<211> 46

<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 Ile

35 40 45

<210> 7

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

accactaccc cagcaccgag gccacccacc ccggctccta ccatcgcctc ccagcctctg 60

tccctgcgtc cggaggcatg tagacccgca gctggtgggg ccgtgcatac ccggggtctt 120

gacttcgcct gcgatatc 138

<210> 8

<211> 26

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly

20 25

<210> 9

<211> 78

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

tacatttggg cccctctggc tggtacttgc ggggtcctgc tgctttcact cgtgatcact 60

ctttactgta agcgcggt 78

<210> 10

<211> 39

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

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

1 5 10 15

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

20 25 30

Glu Glu Gly Gly Cys Glu Leu

35

<210> 11

<211> 117

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

cggaagaagc tgctgtacat ctttaagcaa cccttcatga ggcctgtgca gactactcaa 60

gaggaggacg gctgttcatg ccggttccca gaggaggagg aaggcggctg cgaactg 117

<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

gccctccctg tcaccgccct gctgcttccg ctggctcttc tgctccacgc cgctcggccc 60

Claims (10)

1. a kind of single-chain antibody for targeting ROR1, which is characterized in that the single-chain antibody of the targeting ROR1 includes such as SEQ ID Amino acid sequence shown in NO:1.

2. the single-chain antibody of targeting ROR1 as described in claim 1, which is characterized in that the single-chain antibody of the targeting ROR1 Encoding gene includes the nucleotide sequence as shown in SEQ ID NO:2.

3. a kind of Chimeric antigen receptor T cell for targeting ROR1, which is characterized in that the Chimeric antigen receptor including targeting ROR1 CAR-ROR1, the CAR-ROR1 include the single-chain antibody of sequentially connected targeting ROR1, extracellular hinge from aminoterminal to c-terminus The amino acid sequence of sequence, transmembrane region and intracellular signal area, wherein the single-chain antibody of the targeting ROR1 includes such as SEQ ID Amino acid sequence shown in NO:1.

4. the Chimeric antigen receptor T cell of targeting ROR1 as claimed in claim 3, which is characterized in that the CAR-ROR1's Amino acid sequence includes 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-ROR1 of the Chimeric antigen receptor T cell of the targeting ROR1 stated.

6. recombinant viral vector as claimed in claim 5, which is characterized in that the encoding gene of the CAR-ROR1 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 ROR1 characterized by comprising

(1) encoding gene of the Chimeric antigen receptor CAR-ROR1 of targeting ROR1 is provided, including is sequentially connected with from 5 ' ends to 3 ' ends Signal peptide encoding gene, target ROR1 the encoding gene of single-chain antibody, the encoding gene of CD8 α hinge area, CD8 cross-film The encoding gene of the encoding gene in area, the encoding gene of 4-1BB signaling zone and CD3 ζ signaling zone, wherein the targeting ROR1's The encoding gene of single-chain antibody includes as shown in SEQ ID NO:2；

(2) encoding gene of the CAR-ROR1 is inserted into pWPXLD carrier, obtains pWPXLD-CAR-ROR1 recombination matter Grain；

(3) by the pWPXLD-CAR-ROR1 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, weight is obtained Group slow virus carrier；

(4) recombined lentivirus vector is transfected into CD3 positive t lymphocytes, the Chimeric antigen receptor T for obtaining targeting ROR1 is thin Born of the same parents.

9. the preparation method of the Chimeric antigen receptor T cell of targeting ROR1 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 ROR1, 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 ROR1 is targeted made from preparation method state or as described in claim 8-9 or as right is wanted Ask the described in any item recombinant viral vectors of 5-6 or host cell as claimed in claim 7 or in preparation treatment and/or prevention And/or the application in the drug of adjuvant therapy of malignant tumor.