CN109837248A

CN109837248A - A kind of T cell receptor gene modification T cell and its preparation method and application for the targeting MAGE-A3 that TCR is knocked out

Info

Publication number: CN109837248A
Application number: CN201711197371.3A
Authority: CN
Inventors: 张长风; 吴咏东
Original assignee: Shenzhen Benta Biological Technology Co Ltd
Current assignee: Shenzhen Benta Biological Technology Co Ltd
Priority date: 2017-11-25
Filing date: 2017-11-25
Publication date: 2019-06-04

Abstract

The present invention provides a kind of preparation methods of the T cell receptor gene modification T cell of TCR targeting MAGE-A3 knocked out, it include: by knocking out the tcr gene in T lymphocyte, and the T cell receptor of targeting MAGE-A3 is prepared, obtain the T cell receptor gene modification T cell of the targeting MAGE-A3 of TCR knockout.The mispairing of the α chain and β interchain of endogenous present in T cell and exogenous TCR can be effectively prevented in the T cell receptor gene modification T cell for the targeting MAGE-A3 that the TCR of preparation method preparation of the present invention is knocked out, autoimmune response is avoided, the performance of efficient and specific killing MAGE-A3 positive tumor cell is kept.The present invention also provides the applications of the T cell receptor gene modification T cell of the TCR targeting MAGE-A3 knocked out.

Description

A kind of T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR is knocked out and its Preparation method and application

Technical field

The present invention relates to field of medical biotechnology, in particular to the T cell receptor base for the targeting MAGE-A3 that a kind of TCR is knocked out Because of modification T cell 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.Human melanoma antigen A3 (melanoma antigen-A3, MAGE-A3) is black A member in Su Liu related antigen family.MAGE-A3 is always related to tumour, is expressed in pernicious swollen based on melanoma more It, can be in high expression, such as lung cancer, breast cancer, cervical carcinoma, gastric cancer, knot-rectum in the tumour of various organization types in tumor tissue Cancer, hepatocellular carcinoma etc., but be not present in normal cell.Therefore, therefore MAGE-A3 is tumor-specific immunity treatment and diagnosis One of ideal target antigen.

In recent years, immune cell therapy has become to be had in treating malignant tumor after operation, radiotherapy, chemotherapy A kind for the treatment of method of huge prospect.T cell receptor (T cell receptor, TCR) gene modification T cell technology (TCR-T) For one of current adoptive newest immunocyte technology of cell adoptive therapy technology, itself exempt from because it can be activated in vivo Epidemic disease system, routinely targets neoplastic cells are killed, be finally reached remove malignant cell purpose and by extensive Concern and research.However some researches show that, the subunit of the TCR and the endogenous TCR of T cell that are transduceed by TCR-T technology (such as α chain and β chain) between there are mispairing, cause the TCR of the mispairing generated targets identification Disability or identification autoantigen or Major histocompatibility complex (major histocompatibility complex, MHC) and cause autoimmune response. Meanwhile correct getting high special between the tumour antigen, tcr gene of high-affinity and the subunit of TCR matches still face Face huge challenge.There are not the relevant preparation of T cell receptor and research of targeting MAGE-A3 also at present.

Summary of the invention

In view of this, the present invention provides the T cell receptor gene modification T cells of TCR targeting MAGE-A3 knocked out a kind of The T cell receptor gene modification T cell of (abbreviation TCR-T-MAGE-A3-TCRKO), the targeting MAG E-A3 that the TCR is knocked out are struck In addition to tcr gene, it is beneficial to prevent the mispairing of the α chain and β interchain of endogenous present in T cell and exogenous TCR, is kept The performance of efficient and specific killing tumor cell, and not will cause damage to normal cell, it is anti-to avoid autoimmunity It answers.

In a first aspect, the present invention provides the T cell receptor gene modification T cells of TCR targeting MAGE-A3 knocked out a kind of Preparation method, comprising:

(1) CD3 positive t lymphocytes are provided, the tcr gene of the CD3 positive t lymphocytes is knocked out, obtain TCR knockout CD3 positive t lymphocytes；

(2) encoding gene of the T cell receptor TCR-MAGE-A3 of targeting MAGE-A3 is provided, including suitable from 5 ' ends to 3 ' ends The encoding gene of the encoding gene of the α chain of secondary connection, the encoding gene of 2A peptide and β chain, wherein the encoding gene packet of the α chain The nucleotide sequence of coding amino acid sequence as shown in SEQ ID NO:1 is included, the encoding gene of the β chain includes encoding such as The nucleotide sequence of amino acid sequence shown in SEQ ID NO:2, the encoding gene of the 2A peptide include coding such as SEQ ID The nucleotide sequence of amino acid sequence shown in NO:3；

(3) encoding gene of the TCR-MAGE-A3 is inserted into pLenti carrier, obtains pLent i-TCR- MAGE-A3 recombinant plasmid；

(4) the pLenti-TCR-MAGE-A3 recombinant plasmid is packed, recombinant slow virus is obtained；

(5) recombinant slow virus is transfected into the CD3 positive t lymphocytes that the TCR is knocked out, obtains the target of TC R knockout To the T cell receptor gene modification T cell of MAGE-A3.

Optionally, in step (1), 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.

Optionally, the tcr gene of the tcr gene for knocking out CD3 positive t lymphocytes uses electrotransfection and Crispr/ Cas9 technology knocks out the tcr gene of the tcr gene of the CD3 positive t lymphocytes.

Further, the tcr gene of the tcr gene for knocking out CD3 positive t lymphocytes, comprising the following steps:

The encoding gene of the Cas9 is inserted into pcDNA3.1 carrier, pcDNA3.1-cas9 recombinant plasmid is obtained, with The pcDNA3.1-cas9 recombinant plasmid is that template is transcribed in vitro to obtain Cas9mRNA；

The corresponding gene order of sgRNA of targeting tcr gene is provided；The sgRN A of the targeting tcr gene is corresponding Gene order is inserted into pcDNA3.1 carrier, obtains pcDNA3.1-TCR-sgRNA recombinant plasmid, with the pcDNA3.1- TCR-sgRNA recombinant plasmid is that template is transcribed in vitro to obtain sgRNA；

The sgRNA of the Cas9mRNA and the targeting tcr gene are mixed with the CD3 positive t lymphocytes, It is placed in electroporation and carries out electricity turn, complete the knockout of the tcr gene of the CD3 positive t lymphocytes.

Optionally, the corresponding gene order of sgRNA of the targeting tcr gene includes the core as shown in SEQ ID NO:7 Nucleotide sequence.

Optionally, the mass ratio of the sgRNA of the Cas9mRNA and the tcr gene is 1:1-1:5.

Further alternative, the mass ratio of the sgRNA of the Cas9mRNA and the tcr gene is 1:3.

Optionally, the coding gene sequence of the α chain includes the nucleotide sequence as shown in SEQ ID NO:4.

Optionally, the encoding gene of the α chain should consider degeneracy base, the i.e. amino acid as shown in SEQ ID NO:1 The encoding gene of sequence includes the nucleotide sequence as shown in SEQ ID NO:4, and protection scope should also be protected 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:1。

Optionally, the coding gene sequence of the β chain includes the nucleotide sequence as shown in SEQ ID NO:5.

Optionally, the encoding gene of the β chain should consider degeneracy base, the i.e. amino acid as shown in SEQ ID NO:2 The encoding gene of sequence includes the nucleotide sequence as shown in SEQ ID NO:5, and protection scope should also be protected and SEQ ID NO:5 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ ID NO:2。

Optionally, the coding gene sequence of the 2A peptide includes the nucleotide sequence as shown in SEQ ID NO:6.

Optionally, the coding gene sequence of the 2A peptide 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: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:3。

Wherein, above-mentioned " being sequentially connected with from 5 ' ends to 3 ' ends " specifically: 3 ' ends of the coding gene sequence of the α chain and 2A 5 ' ends of the coding gene sequence of peptide are connected, and 3 ' ends of the coding gene sequence of the 2A peptide are connected with 5 ' ends of the β chain.

Wherein, the 2A peptide be can " self splicing " short and small peptide chain, the 2A peptide can be sheared in protein translation. The 2A peptide has the advantage that (1) 2A peptide sequence is short, can effectively realize coexpression of the linker because between；(2) it is located at The gene in the downstream 2A can equally obtain very high expression.Since the expression efficiency of gene after traditional IRES sequence is significant Lower than the expression of gene before IRES sequence, so 2A peptide of the present invention is more advantageous compared with IR ES.

Wherein, the encoding gene of the TCR-MAGE-A3 is inserted into I restriction enzyme site of BamH I and Ec oR in pLenti carrier Between, and be located at after the extension factor 1 α (EF1 α) of pLenti carrier, using EF1 α as promoter.The TCR-MAGE-A3's When encoding gene is inserted into pLenti carrier, initiation codon can be added (such as in 5 ' ends of the encoding gene of the TCR-MAGE-A3 ATG) it is connected with Ba mH1 restriction enzyme site in pLenti carrier, terminator codon (such as TAA) and pLenti carrier can be added in 3 ' ends Middle EcoR1 restriction enzyme site is connected.

Optionally, the packaging pLenti-TCR-MAGE-A3 recombinant plasmid, obtaining recombinant slow virus includes:

By the pLenti-TCR-MAGE-A3 recombinant plasmid and envelope plasmid and packaging plasmid co-transfecting host cells, Obtain the recombinant slow virus.

Optionally, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2.

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.

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.

Further alternative, the host cell is HEK293T cell.

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.

The system of the T cell receptor gene modification T cell for the targeting MAGE-A3 that the TCR that first aspect present invention provides is knocked out Preparation Method by knocking out the tcr gene in T lymphocyte, and prepares the T cell receptor of targeting MAGE-A3, obtains TCR knockout Targeting MAGE-A3 T cell receptor gene modification T cell.The targeting that the TCR of preparation method preparation of the present invention is knocked out The T cell receptor gene modification T cell of MAGE-A3 can be effectively prevented endogenous present in T cell and exogenous TCR's The mispairing of α chain and β interchain avoids autoimmune response, keeps efficient and specific killing MAGE-A3 positive tumor cell Performance.

Second aspect is knocked out the present invention provides the TCR being prepared using preparation method as described in relation to the first aspect The T cell receptor gene modification T cell of MAGE-A3 is targeted, the T cell receptor gene for the targeting MAGE-A3 that the TCR is knocked out is repaired Adoring T cell does not include tcr gene, and the T cell receptor gene modification T cell for the targeting MAGE-A3 that the TCR is knocked out includes targeting The T cell receptor TCR-MAGE-A3 of MAGE-A3, the T cell receptor TCR-MAGE-A3 of the targeting MAGE-A3 includes α chain and β Chain, the α chain include the amino acid sequence as shown in SEQ ID NO:1, and the β chain includes the ammonia as shown in SEQ ID NO:2 Base acid sequence.

Wherein, α chain and β chain included by the T cell receptor (TCR-MAGE-A3) of the targeting MAGE-A3 can be formed Stable heterodimer structure has very strong specificity to MAGE-A3 albumen, will not target normal cell or tissue, increases Add its safety and reduces the risk of undershooting-effect.

Wherein, TCR-MAGE-A3 of the present invention can be with specific recognition and in conjunction with MAGE-A3 albumen, and has best Target spot affinity.The target spot affinity refer to the TCR of genetic modification for target spot (such as in the present invention, TCR-MAGE-A3 To MAGE-A3 albumen) bond strength.Influence of the target spot affinity to TC R is huge, if the bond strength of target spot affinity It is too low, then can not inducing T cell to the specific killing of cancer cell；, whereas if bond strength is excessively high, then specificity, T are lost Cell can kill other low expressions TCR target spot other than tumour cell normal cell (once for example, these low expressions TCR target spot is distributed in the vitals such as heart, brain, can cause serious even lethal side effect).

The T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR of the present invention is knocked out can be with efficient identification simultaneously Killing includes the cancer cell that the expression such as lung cancer, breast cancer, gastric cancer, the knot-carcinoma of the rectum, hepatocellular carcinoma have MAGE-A3, is particularly suitable for The malignant cell of Humanmachine tumour or tissue.

The third aspect, a kind of be prepared the present invention provides preparation method as described in relation to the first aspect or such as second party The T cell receptor gene modification T cell for the targeting MAGE-A3 that a kind of TCR described in face is knocked out is in preparation prevention, diagnosing and treating Application in the drug of malignant tumour.Specifically, suitable for expressing prevention, the diagnosing and treating of the solid tumor of MAGE-A3, such as Lung cancer, breast cancer, cervical carcinoma, gastric cancer, the knot-carcinoma of the rectum, hepatocellular carcinoma etc., the malignant tumour for being particularly suitable for Humanmachine tumour is thin Born of the same parents or tissue.

The application specifically: provide a kind of kit, the kit includes preparation side as described in relation to the first aspect The T cell receptor gene modification T for the targeting MAGE-A3 that a kind of TCR that method is prepared or as described in second aspect is knocked out is thin Born of the same parents.

Beneficial effects of the present invention:

The T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR provided by the invention is knocked out can prevent T cell Present in endogenous and exogenous TCR α chain and β interchain mispairing, avoid autoimmune response, keep efficiently and special The performance of the killing tumor cell of property, and not will cause damage to normal cell.The T for the targeting MAGE-A3 that the TCR is knocked out Kdr transfected cell, which modifies T cell, to promote T cell in the amplification of patient's body with the targeting MAGE-A3 of specificity, can Efficient and specific killing MAGE-A3 positive tumor cell, while MAGE-A3 wide expression in tumour cell, and general Express very faint in logical cell, therefore the T cell receptor gene modification T cell of targeting MAGE-A3 that the TCR is knocked out has very Extensive tumor-killing spectrum.

Detailed description of the invention

Fig. 1 is the plasmid map of pLenti-TCR-MAGE-A3 recombinant plasmid provided in an embodiment of the present invention.

Fig. 2 is the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR provided in an embodiment of the present invention is knocked out Positive rate；(A) is negative control group in Fig. 2, and (B) is the targeting MAGE-A3 that TCR provided in an embodiment of the present invention is knocked out in Fig. 2 T cell receptor gene modification T cell experimental group.

Fig. 3 is the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR provided in an embodiment of the present invention is knocked out Tumor cell in vitro fragmentation effect figure.

Fig. 4 is that the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR provided in an embodiment of the present invention is knocked out is controlled Treat the effect picture of mice with tumor.

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 of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR is knocked out, including following step It is rapid:

(1) preparation for the CD3 positive t lymphocytes that TCR is knocked out

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) tcr gene is knocked out

The encoding gene of the Cas9 is inserted into pcDNA3.1 carrier, pcDNA3.1-cas9 recombinant plasmid is obtained, with The pcDNA3.1-cas9 recombinant plasmid is template, utilizes mMESSAGET7 kit carries out external Transcription obtains Cas9mRNA；

The corresponding gene order of sgRNA of targeting tcr gene is provided, sequence is as shown in SEQ ID NO:7；By the target It is inserted into pcDNA3.1 carrier to the corresponding gene order of sgRNA of tcr gene, obtains pcDNA3.1-TCR-sgRNA recombination Plasmid is transcribed in vitro to obtain sgRNA as template using the pcDNA3.1-TCR-sgRNA recombinant plasmid；

The sgRNA sequence and CD3 positive T lymph obtained above of the Cas9mRNA and the targeting tcr gene is thin Born of the same parents mix, and are placed in electroporation and carry out electricity turn, knock out the tcr gene of the CD3 positive t lymphocytes；After electricity is turned T cell cultivated, obtain TCR knockout CD3 positive t lymphocytes.

The TCR expression quantity of the preparation for the CD3 positive t lymphocytes that above-mentioned TCR is knocked out is measured using flow cytometer, is calculated Knockout rate, as a result, it has been found that the knockout rate of tcr gene is up to 72% in the preparation for the CD3 positive t lymphocytes that TCR is knocked out.

(2) gene order of preparation targeting MAGE-A3T cell receptor TCR-MAGE-A3

The coding gene sequence of α chain, 2A peptide and β chain is prepared respectively, and the coding gene sequence of the α chain includes such as SEQ ID Nucleotide sequence shown in NO:4, the coding gene sequence of the β chain include the nucleotide sequence as shown in SEQ ID NO:5, The coding gene sequence of the 2A peptide includes the nucleotide sequence as shown in SEQ ID NO:6.

The coding gene sequence of above-mentioned α chain, 2A peptide and β chain is successively connected to from 5 ' ends to 3 ' ends by the method for PCR Together, the coding gene sequence of the T cell receptor TCR-MAGE-A3 of targeting MAGE-A3 is obtained.

(3) pLenti-TCR-MAGE-A3 recombinant plasmid is constructed

By the coding gene sequence of TCR-MAGE-A3 be inserted into pLenti carrier BamH1 and EcoR1 restriction enzyme site it Between, and after pLenti carrier EF1 α, using EF1 α as promoter.The coding gene sequence of the TCR-MAGE-A3 is inserted into When pLenti carrier, the coding gene sequence of the TCR-MAGE-A3 5 ' end added with initiation codon (such as ATG) with BamH1 restriction enzyme site is connected in pLenti carrier, and 3 ' ends are also added with EcoR1 digestion position in terminator codon and pLenti carrier Point is connected.Then it is transferred to competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.It is produced by PCR Object detected through gel electrophoresis and sequencing identification meet target fragment size and sequence, successfully construct pLenti-TCR- as shown in Figure 1 MAGE-A3 recombinant plasmid.

(4) recombined lentivirus vector constructs

PLenti-TCR-MAGE-A3 recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three's cotransfection are entered Cultured HEK293T cell.Supernatant of the 48h harvest containing virus, through 0.45 μm of membrane filtration, in -80 DEG C of ultra low temperature freezers It saves；Supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration, after merging with the viral supernatants of 48h harvest together It is added in ultracentrifugation pipe, is put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, when centrifugation Between be 2h, centrifuging temperature control at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, is added Virus saves liquid, and gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant glimmering after being centrifuged 5min Light method measures titre, and 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 lentiviral disease Poisonous carrier.

(5) preparation of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR is knocked out

The CD3 positive t lymphocytes for taking the above-mentioned TCR obtained by gene knockout to knock out, are added the CD3 knocked out with TCR The recombinant slow virus of the corresponding virus titer of positive t lymphocytes is cultivated.The 3rd day of culture carries out cell count With change liquid, adjustment cell concentration is 1 × 10⁶A/mL is inoculated with, culture；Cell state is observed, if cell in the 5th day of culture Density increases, then diluting cells concentration is 1 × 10⁶A/mL detects cell activity, continues to cultivate.Amplification cultivation is to 9-11 It, collects cell, obtains the preparation of the T cell receptor gene modification T cell of the targeting MAGE-A3 of TCR knockout.

In order to assess the T cell receptor base for the targeting MAGE-A3 that the TCR of above method preparation described in the invention is knocked out Because modifying T cell effect, following effect example is carried out.

Effect example one: the T cell receptor gene for the targeting MAGE-A3 that TCR prepared by the assessment present invention is knocked out is repaired Adorn the positive rate of T cell

The T cell receptor gene modification T cell TCR- of the targeting MAGE-A3 of TCR knockout will be prepared by the method for the present invention T-MAGE-A3-TCRKO (experimental group) and the T lymphocyte (negative control group) without preparation, using flow cytomery its Positive rate, as a result as shown in Fig. 2, wherein (A) is negative control group in Fig. 2, i.e., without the T cell of preparation, (B) is real in Fig. 2 Test group, the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR as produced by the present invention is knocked out.By (A) in Fig. 2 It can be obtained compared with (B), the positive rate of TCR-T-MAGE-A3-TCRKO prepared by the present invention is 38.5%.

Effect example two: the external of T cell receptor gene modification T cell for the targeting MAGE-A3 that assessment TCR is knocked out is swollen Cytotoxic effect situation

The T cell receptor gene modification T cell of the targeting MAGE-A3 knocked out by TCR made from the method for the present invention is (real Test group) it is compared with the Vitro Tumor fragmentation effect of the T lymphocyte (negative control group) without preparation, it is specific: in body It is outer by effector cell (TCR-T-MAGE-A3-TCRKO or without the T lymphocyte of preparation) with target cell (Hela cell) by number Amount is than being 1:10,1:3,1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Under co-cultured, 15-18 after incubation Hour, cell is collected, streaming dyeing is carried out, detects cell killing situation, as a result as shown in Figure 3.As can be seen from Figure 3, it adds TCR-T-MAGE-A3-TCRKO ratio it is higher, they are stronger to the lethality of tumour cell.By side of the present invention The tumor-killing power of the TCR-T-MAGE-A3-TCRKO of method preparation is in 20% or more, even up to 60%, significantly larger than feminine gender Control group, the T cell receptor gene modification T cell tool for the targeting MAGE-A3 that the TCR that this explanation is prepared through the method for the present invention is knocked out There is strong tumor-killing ability.

Effect example three: the Mice Body of the T cell receptor gene modification T cell for the targeting MAGE-A3 that assessment TCR is knocked out Inner tumour cell kills situation

The T cell receptor gene modification T cell of the targeting MAGE-A3 knocked out by the TCR of the method for the present invention preparation is (real Test group), the T lymphocyte (negative control group) without preparation and physiological saline (blank control group), in mouse tumor model In, give every mouse tail vein injection 1 × 10⁶A Hela cell (n=9), draws the survivorship curve of mouse, as a result such as Fig. 4 institute Show.From fig. 4, it can be seen that making mouse survival when cultivating 70 days by TCR-T-MAGE-A3-TCRKO prepared by this method Rate is also higher than 50%, and the survival rate when cultivating 80 days and the longer time is close to 25%, considerably beyond negative control group and blank Control group.Fig. 4's the result shows that, by this method preparation TCR knock out targeting MAGE-A3 T cell receptor gene modification T Cell is dead caused by capable of preferably protecting mice against because of tumour.

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

<110>Shenzhen Bin De Bioisystech Co., Ltd

<120>the T cell receptor gene modification T cell and its preparation method and application for the targeting MAGE-A3 that a kind of TCR is knocked out

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 117

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Ala Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Leu Ile Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu His Ser Ala

50 55 60

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

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Pro Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Ala Arg Asp Tyr Arg Ser Met Asp Tyr Trp Gly Ala Gly Thr

100 105 110

Ile Val Thr Val Asn

115

<210> 2

<211> 108

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Asp Val Val Met Thr Gln Thr Pro Ser Ser Met Tyr Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr

20 25 30

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

35 40 45

Tyr Arg Ala Asn Gly Leu Val Ala Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr

65 70 75 80

Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Phe Asp Asp Phe Pro Tyr

85 90 95

Thr Phe Gly Glu Gly Thr Lys Leu Glu Ile Asn Leu

100 105

<210> 3

<211> 21

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 4

<211> 351

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gaggtgaagc tggtggagtc tggaggaggc ttggtacagc ctgggggttc tctgagactc 60

tcctgtgcaa cttctgggtt cacgttctct gattactaca tggcctgggt ccgccagcct 120

ccaggaaagg cacttgagtg gttgggttta attagaaaca aaggtaatgg ttacacaaca 180

gagcacagtg catctgtgag gggtcggttc accatctcca gagataattc ccaaagcatc 240

ctctatcttc aaatgaacac cctgagacct gaggacagtg ccacttatta ctgtgcaaga 300

gattaccggt ctatggacta ctggggcgca ggaactatag tcacagtcaa t 351

<210> 5

<211> 324

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gatgttgtga tgacccagac tccatcttcc atgtatgcat ctctaggaga gagagtcact 60

atcacttgca aggcgagtca ggacattaat agctatttaa gctggttcca gcagaaacca 120

gggaaatctc ctaagaccct gatctatcgt gcaaacggat tggtggctgg ggtcccatca 180

aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240

gaagatatgg gaatttatta ttgtctacag tttgatgact ttccgtacac gttcggtgag 300

gggacaaaac tggaaataaa tctc 324

<210> 6

<211> 63

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc 60

cca 63

<210> 7

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

tcaacctttg gggatgggac tac 23

Claims

1. a kind of preparation method of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR is knocked out, which is characterized in that Include:

(2) encoding gene of the T cell receptor TCR-MAGE-A3 of targeting MAGE-A3 is provided, including is sequentially connected from 5 ' ends to 3 ' ends The encoding gene of the encoding gene of the α chain connect, the encoding gene of 2A peptide and β chain, wherein the encoding gene of the α chain includes compiling The nucleotide sequence of code amino acid sequence as shown in SEQ ID NO:1, the encoding gene of the β chain include coding such as SEQ ID The nucleotide sequence of amino acid sequence shown in NO:2, the encoding gene of the 2A peptide include coding as shown in SEQ ID NO:3 Amino acid sequence nucleotide sequence；

(3) encoding gene of the TCR-MAGE-A3 is inserted into pLenti carrier, obtains pLenti-TCR-MAGE-A3 weight Group plasmid；

(5) recombinant slow virus is transfected into the CD3 positive t lymphocytes that the TCR is knocked out, obtains the targeting of TCR knockout The T cell receptor gene modification T cell of MAGE-A3.

2. the preparation side of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR as described in claim 1 is knocked out Method, which is characterized in that the encoding gene of the α chain includes the nucleotide sequence as shown in SEQ ID NO:4.

3. the preparation side of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR as described in claim 1 is knocked out Method, which is characterized in that the encoding gene of the β chain includes the nucleotide sequence as shown in SEQ ID NO:5.

4. the preparation side of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR as described in claim 1 is knocked out Method, which is characterized in that the encoding gene of the 2A peptide includes the nucleotide sequence as shown in SEQ ID NO:6.

5. the preparation side of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR as described in claim 1 is knocked out Method, which is characterized in that the tcr gene for knocking out the CD3 positive t lymphocytes, comprising:

The encoding gene of the Cas9 is inserted into pcDNA3.1 carrier, pcDNA3.1-cas9 recombinant plasmid is obtained, with described PcDNA3.1-cas9 recombinant plasmid is that template is transcribed in vitro to obtain Cas9mRNA；

The corresponding gene order of sgRNA of targeting tcr gene is provided；By the corresponding gene sequence of sgRNA of the targeting tcr gene Column are inserted into pcDNA3.1 carrier, pcDNA3.1-TCR-sgRNA recombinant plasmid are obtained, with the pcDNA3.1-TCR- SgRNA recombinant plasmid is that template is transcribed in vitro to obtain sgRNA；

The sgRNA of the Cas9mRNA and the targeting tcr gene are mixed with the CD3 positive t lymphocytes, juxtaposition Electricity is carried out in electroporation to turn, and completes the knockout of the tcr gene of the CD3 positive t lymphocytes.

6. the preparation side of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR as claimed in claim 5 is knocked out Method, which is characterized in that the corresponding gene order of sgRNA of the targeting tcr gene includes the nucleosides as shown in SEQ ID NO:7 Acid sequence.

7. the preparation side of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR as claimed in claim 5 is knocked out Method, which is characterized in that the mass ratio of the sgRNA of the Cas9mRNA and the tcr gene is 1:1-1:5.

8. the preparation side of the T cell receptor gene modification T cell for the targeting MAGE-A3 that TCR as described in claim 1 is knocked out Method, which is characterized in that the packaging pLenti-TCR-MAGE-A3 recombinant plasmid, obtaining recombinant slow virus includes:

9. the T cell receptor for the targeting MAGE-A3 that the TCR that the method according to claim 1 is prepared is knocked out Gene modification T cell, which is characterized in that the T cell receptor gene modification T cell for the targeting MAGE-A3 that the TCR is knocked out is not wrapped Tcr gene is included, the T cell receptor gene modification T cell for the targeting MAGE-A3 that the TCR is knocked out includes the T for targeting MAGE-A3 Cell receptor TCR-MAGE-A3, the T cell receptor TCR-MAGE-A3 of the targeting MAGE-A3 includes α chain and β chain, the α chain Including the amino acid sequence as shown in SEQ ID NO:1, the β chain includes the amino acid sequence as shown in SEQID NO:2.

10. one kind is as made from the described in any item preparation methods of claim 1-8 or TCR as claimed in claim 9 is knocked out Targeting MAGE-A3 T cell receptor gene modification T cell preparation prevention, diagnosing and treating malignant tumour drug in Using.