CN109836490A - It is a kind of to target the T cell receptor of MAGE-A3, T cell receptor gene modification T cell and its preparation method and application - Google Patents

Abstract

The present invention provides a kind of T cell receptors for targeting MAGE-A3, the T cell receptor of the targeting MAGE-A3 includes α chain and β chain, the α chain includes the amino acid sequence as shown in SEQ ID NO:1, and the β chain includes the amino acid sequence as shown in SEQ ID NO:2.The present invention also provides a kind of T cell receptor gene modification T cells for targeting MAGE-A3.The T cell receptor gene modification T cell of the targeting MAGE-A3 can efficiently and specifically kill MAGE-A3 positive cancer cell, have lasting vigor and lethality, and not will cause damage to normal cell.The present invention also provides a kind of preparation method and application of T cell receptor gene modification T cell for targeting MAGE-A3.

Description

It is a kind of target the T cell receptor of MAGE-A3, T cell receptor gene modification T cell and Preparation method and application

Technical field

The present invention relates to field of medical biotechnology, in particular to a kind of T cell receptor, T cell receptor for targeting MAGE-A3 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.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, liver cancer etc., but be not present in normal cell.

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.There is immune cell therapy the huge advantage of high specificity, almost non-toxic side effect to make up The drawbacks of traditional remedies, at home and abroad have been used to clinical treatment malignant tumour.T cell receptor (T cell receptor, TCR) gene modification T cell technology (TCR-T) be the current adoptive newest immunocyte technology of cell adoptive therapy technology it One, because it can activate self immune system in vivo, routinely targets neoplastic cells are killed, and are finally reached removing The purpose of malignant cell and widely paid close attention to and studied.However, getting high special to tumour antigen, Gao Qinhe Property T CR gene and TCR subunit (such as α chain and β chain) between it is correct pairing be still TCR-T technology facing challenges. 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 a kind of T cell receptors for targeting MAGE-A3, and including the targeting MAGE- The T cell receptor gene modification T cell of A3, it is described targeting MAGE-A3 T cell receptor can with the MAGE-A3 of specific recognition, With high-affinity.It is described targeting MAGE-A3 T cell receptor gene modification T cell can be efficient and specific killing MAGE-A3 positive cancer cell has lasting vigor and lethality, and not will cause damage to normal cell.The present invention is also Provide a kind of preparation method and application of T cell receptor gene modification T cell for targeting MAGE-A3.

In a first aspect, the T of the targeting MAGE-A3 is thin the present invention provides a kind of T cell receptor for targeting MAGE-A3 Born of the same parents' receptor includes α chain and β chain, and the α chain includes the amino acid sequence as shown in SEQ ID NO:1, and the β chain includes such as SEQ Amino acid sequence shown in ID NO:2.

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 TCR is huge, if the bond strength mistake of target spot affinity It is low, then can not inducing T cell to the specific killing of cancer cell；, whereas if bond strength is excessively high, then specificity is lost, T is thin Born of the same parents can kill other low expressions TCR target spot other than tumour cell normal cell (once for example, the TCR of these low expressions Target spot is distributed in the vitals such as heart, brain, can cause serious even lethal side effect).

Optionally, the coding gene sequence of the α chain includes the nucleotide sequence as shown in SEQ ID NO:3, the β chain Coding gene sequence include 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:3, and protection scope should also be protected and SEQ ID NO:3 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 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: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:2。

First aspect present invention provides a kind of T cell receptor for targeting MAGE-A3, the T cell of the targeting MAGE-A3 Receptor specifically binds MAGE-A3 albumen, promotes the immunocyte pair of the T cell receptor containing the targeting MAGE-A3 The identification of tumour cell has stronger affinity to tumour cell.The T cell receptor of the targeting MAGE-A3 especially can target To the solid tumor cell of expression MAGE-A3, and hardly target normal cell.

Second aspect, the present invention provides a kind of tcr complex, the tcr complex include α chain and The one or two of β chain, the α chain include the amino acid sequence as shown in SEQ ID NO:1, and the β chain includes such as SEQ ID Amino acid sequence shown in NO:2.

Wherein, the tcr complex may include any one of α chain and β chain, and combine other antibody chains, Such as δ chain or γ chain；The tcr complex can also include the composite construction of α chain, β chain and other molecules, such as institute Stating tcr complex can be the α chain or β chain of α chain, the compound molecule of β chain and CD3 or the tcr complex On also there are other protein moleculars etc..

The third aspect, the present invention provides a kind of T cell receptor gene modification T cells for targeting MAGE-A3, including this hair The T cell receptor of MAGE-A3 is targeted described in bright first aspect.

The T cell receptor gene modification T cell of targeting MAGE-A3 of the present invention can be with specific recognition MAGE-A3 sun Property tumour cell, and with MAGE-A3 positive tumor cell have high-affinity, can with efficient identification and kill include lung cancer, The expression such as breast cancer, gastric cancer, the knot-carcinoma of the rectum, liver cancer have the cancer cell of MAGE-A3, are particularly suitable for the pernicious of Humanmachine tumour Tumour cell or tissue.

The T cell receptor gene modification T cell for the targeting MAGE-A3 that third aspect present invention provides, including targeting The T cell receptor TCR-MAGE-A3 of MAGE-A3, this receptor are swollen for T cell targeted expression MAGE-A3's in specific manner Oncocyte can promote T cell in the amplification of patient's body after TCR-MAGE-A3 is in conjunction with MAGE-A3, and efficiently and The killing tumor cell of specificity, and normal cell is hardly caused to damage.

Fourth aspect, the present invention provides a kind of recombinant viral vector, the recombinant viral vector includes the present invention first The encoding gene of the T cell receptor of MAGE-A3 is targeted described in aspect.

Optionally, the coding gene sequence of the T cell receptor of the targeting MAGE-A3 includes sequentially connecting from 5 ' ends to 3 ' ends The gene order of the α chain, 2A peptide and β chain that connect, the coding gene sequence of the α chain include the nucleosides as shown in SEQ ID NO:3 Acid sequence, the coding gene sequence of the β chain include the nucleotide sequence as shown in SEQ ID NO:4, the coding of the 2A peptide Gene order includes the nucleotide sequence as shown in SEQ ID NO:5.

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 fourth aspect present invention provides has very high efficiency of infection and transcriptional efficiency, inserts The TCR-MAGE-A3 encoding gene segment entered can be inserted into host genome by genetic recombination, and the T for obtaining targeting MAGE-A3 is thin Born of the same parents' acceptor gene modifies T cell, to make it continue, the T cell receptor TCR-MAGE-A3 of steadily expression targeting MAGE-A3.

5th aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in fourth 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.

6th aspect, the present invention provides a kind of preparation sides of T cell receptor gene modification T cell for targeting MAGE-A3 Method, comprising the following steps:

(1) gene order of the T cell receptor TCR-MAGE-A3 of targeting MAGE-A3 is provided, including suitable from 5 ' ends to 3 ' ends The gene order of the α chain of secondary connection, 2A peptide and β chain, the coding gene sequence of the α chain include as shown in SEQ ID NO:3 Nucleotide sequence, the coding gene sequence of the β chain include the nucleotide sequence as shown in SEQ ID NO:4, the 2A peptide Coding gene sequence includes the nucleotide sequence as shown in SEQ ID NO:5；

(2) gene order of the TCR-MAGE-A3 is inserted into pLenti carrier, obtains pLenti-TCR-MAGE- A3 recombinant plasmid；

(3) by the pLenti-TCR-MAGE-A3 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, Obtain recombinant slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, obtains the T cell receptor base of targeting MAGE-A3 Because modifying T cell.

Above-mentioned " being sequentially connected with from 5 ' ends to 3 ' ends " specifically: 3 ' ends and the 2A peptide of the coding gene sequence of the α chain 5 ' ends of coding gene sequence 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 IRES.

Optionally, the amino acid sequence of the 2A peptide includes the amino acid sequence as shown in SEQ ID NO:6.

Optionally, the encoding gene of the 2A peptide includes the nucleotide sequence as shown in SEQ ID NO:5.

Optionally, the coding gene sequence of the 2A peptide should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:6 The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:5, and protection scope should also protect 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:6。

For the coding gene sequence of the α chain and β chain, reference can be made to the description of second aspect of the present invention part, here not It repeats again.

The coding gene sequence of the TCR-MAGE-A3 be inserted into pLenti carrier BamH I and I restriction enzyme site of EcoR it Between, and be located at after the extension factor 1 α (EF1 α) of pLenti carrier, using EF1 α as promoter.The base of the TCR-MAGE-A3 When being inserted into pLenti carrier because of sequence, also initiation codon can be added (such as in 5 ' ends of the gene order of the TCR-MAGE-A3 ATG) it is connected with BamH1 restriction enzyme site in pLenti carrier, EcoR1 in terminator codon and pLenti carrier can be also added in 3 ' ends Restriction enzyme site is connected.

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 can assist recombinant slow virus to adhere to cell membrane, and keep 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..It is further alternative, from cancer patient perform the operation one month after, the fresh peripheral blood that acquires after chemicotherapy one month 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.

7th aspect, the present invention provides the T cell receptor of targeting MAGE-A3 as described in relation to the first aspect a kind of, such as the The T cell receptor gene modification T that MAGE-A3 is targeted described in three aspects or made from the preparation method as described in terms of the 6th is thin Born of the same parents, the recombinant viral vector as described in fourth aspect or the host cell as described in terms of the 5th in preparation prevention, diagnose and control Treat the application in malignant tumor medicine.

Wherein, application of the present invention is suitable for the prevention of all kinds of tumour cells of high expression MAGE-A3, diagnoses and control It treats, including lung cancer, breast cancer, cervical carcinoma, gastric cancer, the knot-carcinoma of the rectum, liver cancer etc., especially suitable for the pernicious swollen of Humanmachine tumour Oncocyte or tissue.

The application specifically: provide a kind of kit, the kit includes targeting MAGE- described in first aspect The T cell receptor of A3, tcr complex, the T for targeting MAGE-A3 as described in the third aspect are thin as described in second aspect Born of the same parents' acceptor gene modifies T cell, the recombinant viral vector as described in fourth aspect, in the host cell as described in terms of the 5th It is one or more.

Advantages of the present invention will be illustrated partially in the following description, and a part is apparent according to specification , or can implementation through the embodiment of the present invention and know.

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 positive rate of the T cell receptor gene modification T cell of targeting MAGE-A3 provided in an embodiment of the present invention；Figure (A) is negative control group in 2, and (B) is the T cell receptor gene modification of targeting MAGE-A3 provided in an embodiment of the present invention in Fig. 2 The experimental group of T cell.

Fig. 3 is that the T cell receptor gene modification T cell Vitro Tumor of targeting MAGE-A3 provided in an embodiment of the present invention is thin Born of the same parents' fragmentation effect figure.

Fig. 4 is that the T cell receptor gene modification T cell treatment tumour of targeting MAGE-A3 provided in an embodiment of the present invention is small The effect picture of mouse.

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 T cell receptor gene modification T cell targeting MAGE-A3, comprising the following steps:

(1) gene order of the T cell receptor TCR-MAGE-A3 of preparation targeting 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:3, the coding gene sequence of the β chain include the nucleotide sequence as shown in SEQ ID NO:4, The coding gene sequence of the 2A peptide includes the nucleotide sequence as shown in SEQ ID NO:5.

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.

(2) 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.

(3) 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.

(4) preparation of the T cell receptor gene modification T cell of MAGE-A3 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；To screening Cell out removes magnetic bead, and PBS washing 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 recombined lentivirus vector 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 T cell receptor gene modification T of targeting MAGE-A3 Cell, and be stored in and feed back in dedicated cells frozen storing liquid, it is used so that patient feeds back.

Effect example

In order to which the T cell receptor gene modification T for assessing the targeting MAGE-A3 of above method preparation described in the invention is thin Born of the same parents' effect carries out following effect example.

Effect example one assesses the T cell receptor gene modification T cell of targeting MAGE-A3 prepared by the present invention Positive rate

By by the method for the present invention preparation targeting MAGE-A3 T cell receptor gene modification T cell (experimental group) with without The T lymphocyte (negative control group) of preparation, using its positive rate of flow cytomery, as a result as shown in Fig. 2, wherein Fig. 2 In (A) be negative control group, i.e., without the T cell of preparation, (B) is experimental group, targeting as produced by the present invention in Fig. 2 The T cell receptor gene modification T cell of MAGE-A3.It can be obtained compared with (B) by (A) in Fig. 2, targeting prepared by the present invention The positive rate of the T cell receptor gene modification T cell of MAGE-A3 is 50.6%.

The tumor cell in vitro of effect example two, the T cell receptor gene modification T cell of assessment targeting MAGE-A3 kills Condition of the injury condition

(TCR- will be abbreviated as by the T cell receptor gene modification T cell of targeting MAGE-A3 made from the method for the present invention T-MAGE-A3 it) is compared with the Vitro Tumor fragmentation effect of the T lymphocyte (negative control group) without preparation, specifically: Effector cell (TCR-T-MAGE-A3 or without the T lymphocyte of preparation) and target cell (Hela cell) are pressed into quantity in vitro Than for 1:10,1:3,1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Under co-cultured, 15-18 after incubation is small When, 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, by this The tumor-killing power of the TCR-T-MAGE-A3 cell of the invention method preparation is 20% or more, even up to 60%, far Much higher than negative control group.The result of Fig. 3 illustrates that the T cell receptor gene of the targeting MAGE-A3 through the method for the present invention preparation is repaired Adoring T cell has very strong tumor-killing ability.

The mouse interior tumor of effect example three, the T cell receptor gene modification T cell of assessment targeting MAGE-A3 is thin Born of the same parents kill situation

By the T cell receptor gene modification T cell (TCR-T-MAGE- of the targeting MAGE-A3 by the method for the present invention preparation A3), the T lymphocyte without preparation (negative control group) and physiological saline (blank control group), in mouse tumor model, To every mouse tail vein injection 1 × 10⁶A Hela cell (n=9), draws the survivorship curve of mouse, as a result as shown in Figure 4. The T cell receptor gene modification T cell of the targeting MAGE-A3 by this method preparation is training mouse as can be seen from Figure 4 It supports 50 days or more, survival rate is close to 80% or more, and survival rate when cultivating 80 days is close to 40%, considerably beyond negative control group And blank control group.Fig. 4's the result shows that, by this method preparation targeting MAGE-A3 T cell receptor gene modification T it is thin Born of the same parents are 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.

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Claims (10)

1. a kind of T cell receptor for targeting MAGE-A3, which is characterized in that the T cell receptor of the targeting MAGE-A3 includes α chain With β chain, the α chain includes the amino acid sequence as shown in SEQ ID NO:1, and the β chain includes as shown in SEQ ID NO:2 Amino acid sequence.

2. the T cell receptor of targeting MAGE-A3 as described in claim 1, which is characterized in that the encoding gene sequence of the α chain Column include the nucleotide sequence as shown in SEQ ID NO:3, and the coding gene sequence of the β chain includes such as SEQ ID NO:4 institute The nucleotide sequence shown.

3. a kind of tcr complex, which is characterized in that the tcr complex include α chain and β chain one kind or Two kinds, the α chain includes the amino acid sequence as shown in SEQ ID NO:1, and the β chain includes as shown in SEQ ID NO:2 Amino acid sequence.

4. a kind of T cell receptor gene modification T cell for targeting MAGE-A3, which is characterized in that appoint including such as claim 1-2 The T cell receptor of MAGE-A3 is targeted described in one.

5. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes as described in claim any one of 1-2 Targeting MAGE-A3 T cell receptor encoding gene.

6. recombinant viral vector as claimed in claim 5, which is characterized in that the volume of the T cell receptor of the targeting MAGE-A3 Code gene order includes from the gene order of the sequentially connected α chain in 5 ' ends to 3 ' ends, 2A peptide and β chain, the encoding gene of the α chain Sequence includes the nucleotide sequence as shown in SEQ ID NO:3, and the coding gene sequence of the β chain includes such as SEQ ID NO:4 Shown in nucleotide sequence, the coding gene sequence of the 2A peptide includes the nucleotide sequence as shown in SEQ ID NO:5.

7. a kind of host cell, which is characterized in that the host cell includes such as the described in any item recombination diseases of claim 5-6 Poisonous carrier.

8. a kind of preparation method for the T cell receptor gene modification T cell for targeting MAGE-A3, which is characterized in that including following step It is rapid:

(1) gene order 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 gene order of the α chain, 2A peptide and β chain that connect, the coding gene sequence of the α chain include the nucleosides as shown in SEQ ID NO:3 Acid sequence, the coding gene sequence of the β chain include the nucleotide sequence as shown in SEQ ID NO:4, the coding of the 2A peptide Gene order includes the nucleotide sequence as shown in SEQ ID NO:5；

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

(3) it by the pLenti-TCR-MAGE-A3 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, the T cell receptor gene for obtaining targeting MAGE-A3 is repaired Adorn T cell.

9. the preparation method of the T cell receptor gene modification T cell of targeting MAGE-A3, feature exist as claimed in claim 8 In the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T cell.

10. a kind of T cell receptor such as the described in any item targeting MAGE-A3 of claim 1-2, as claimed in claim 4 Or the T cell receptor gene modification T cell of targeting MAGE-A3 as made from claim 8-9 described in any item preparation methods, Or as the described in any item recombinant viral vectors of claim 5-6 or host cell as claimed in claim 7 preparation prevention, Application in diagnosing and treating malignant tumor medicine.