CN109836491A - It is a kind of to target the T cell receptor of TIPE3, 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 TIPE3, the T cell receptor of the targeting TIPE3 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 TIPE3.The T cell receptor gene modification T cell of the targeting TIPE3 efficiently and specifically killer T IPE3 positive cancer cell can 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 TIPE3.

Description

It is a kind of target the T cell receptor of TIPE3, T cell receptor gene modification T cell and its 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 cell for targeting TIPE3 And its preparation method and application.

Background technique

8 sample 3 (TNFAIP8L3, TIPE3) of tumor necrosis factor α inducible protein is TNFAIP3 family member, is used as rouge Class second messenger transmits albumen to promote canceration, strengthens son as feedback and is regulating and controlling canceration and phosphatide access.TIPE3mRNA It can be detected in many tissues, but TIPE3 albumen is expressed seldom in the normal tissue, and have in cancerous issue very bright Aobvious expression lifting.Therefore, TIPE3 is the tumour-specific target spot being highly suitable in cellular immunotherapy.

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-affinity special to tumour antigen Tcr gene and TCR double-strand between it is correct pairing be still TCR-T technology facing challenges.In addition, not there is targeting also at present The relevant preparation of the T cell receptor of TIPE3 and research.

Summary of the invention

In view of this, the present invention provides a kind of T cell receptors for targeting TIPE3, and including the targeting TIPE3's The T cell receptor of T cell receptor gene modification T cell, the targeting TIPE3 can have high parent with the TIPE3 of specific recognition And property.It is described targeting TIPE3 T cell receptor gene modification T cell can be efficient and specific killer T IPE3 positive carcinoma it is thin Born of the same parents have lasting vigor and lethality, and not will cause damage to normal cell.The present invention also provides a kind of targetings The preparation method and application of the T cell receptor gene modification T cell of TIPE3.

In a first aspect, the present invention provides it is a kind of target TIPE3 T cell receptor, it is described targeting TIPE3 T cell by Body 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 ID Amino acid sequence shown in NO:2.

Wherein, α chain and β chain included by the T cell receptor (TCR-TIPE3) of the targeting TIPE3 can be formed heterologous Dimeric structure has very strong specificity to TIPE3 albumen.

Wherein, TCR-TIPE3 of the present invention can be with specific recognition and in conjunction with TIPE3 albumen, and has optimal Target spot affinity.The target spot affinity refer to the TCR of genetic modification for target spot (such as in the present invention, TCR-TIPE3 pairs TIPE3 albumen) bond strength.Influence of the target spot affinity to TCR is huge, if the bond strength of target spot affinity 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 cell are lost The normal cell of other low expressions TCR target spot other than tumour cell can be killed.

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 TIPE3, the T cell receptor of the targeting TIPE3 TIPE3 albumen is specifically bound, promotes identification of the immunocyte with the T cell receptor to tumour cell, to tumour Cell has stronger affinity.The solid tumor that the T cell receptor of the targeting TIPE3 can especially be targeted to expression TIPE3 is thin Born of the same parents, 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 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 the T is thin Born of the same parents' receptor complex can be on the α chain or β chain of α chain, the compound molecule of β chain and CD3 or the tcr complex also With other protein moleculars etc..

The third aspect, the present invention provides a kind of T cell receptor gene modification T cells for targeting TIPE3, including the present invention The T cell receptor of TIPE3 is targeted described in first aspect.

The T cell receptor gene modification T cell of targeting TIPE3 of the present invention can be with specific recognition TIPE3 positive carcinoma Cell, and it is high with the compatibility of TIPE3 positive cancer cell, with efficient identification and the cancer cell expressed and have TIPE3 can be killed.

The T cell receptor gene modification T cell for the targeting TIPE3 that third aspect present invention provides, including targeting The T cell receptor TCR-TIPE3 of TIPE3, this receptor for T cell targeted expression TIPE3 in specific manner cancer cell, After TCR-TIPE3 is in conjunction with TIPE3, T cell can be promoted in the amplification of patient's body, and efficient and specific killing cancer Cell, 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 TIPE3 is targeted described in aspect.

Optionally, the coding gene sequence of the T cell receptor of the targeting TIPE3 includes being sequentially connected with from 5 ' ends to 3 ' ends α chain, 2A peptide and β chain gene order, the coding gene sequence of the α chain includes the nucleotide as shown in SEQ ID NO:3 Sequence, the coding gene sequence of the β chain include the nucleotide sequence as shown in SEQ ID NO:4, the coding base of the 2A peptide Because sequence 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-TIPE3 encoding gene segment entered can by genetic recombination be inserted into host genome, obtain targeting TIPE3 T cell by Body gene modification T cell, to make it continue, the T cell receptor TCR-TIPE3 of steadily expression targeting TIPE3.

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 it is a kind of target TIPE3 T cell receptor gene modification T cell preparation method, The following steps are included:

(1) gene order of the T cell receptor TCR-TIPE3 of targeting TIPE3 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 SEQID NO:4, the coding base of the 2A peptide Because sequence includes the nucleotide sequence as shown in SEQ ID NO:5；

(2) gene order of the TCR-TIPE3 is inserted into pLenti carrier, obtains pLenti-TCR-TIPE3 weight Group plasmid；

(3) it by the pLenti-TCR-TIPE3 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains To recombinant slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, obtains the T cell receptor gene of targeting TIPE3 Modify 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.

In the present invention the 2A peptide be can " self splicing " short and small peptide chain, the 2A peptide can pass through in protein translation Ribosomal skip is broken from itself last 2 amino acid C-terminal.The 2A peptide has the advantage that (1) 2A peptide sequence is short, energy Enough effectively realize coexpression of the linker because between；(2) gene positioned at the downstream 2A can equally obtain very high expression water It is flat.Since the expression efficiency of gene after traditional IRES sequence is substantially less than the expression of gene before IRES sequence, so and IRES It compares, 2A peptide of the present invention is more advantageous.

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-TIPE3 is inserted into pLenti carrier between I restriction enzyme site of MluI and EcoR, And be located at after the extension factor 1 α (EF1 α) of carrier, using EF1 α as promoter.The gene order of the TCR-TIPE3 is inserted into When pLenti carrier, initiation codon (such as ATG) can also be added for 5 ' ends of the gene order of the TCR-TIPE3 and pLenti is carried MluI restriction enzyme site is connected in body, and EcoR1 restriction enzyme site in terminator codon (such as TAA) and pLenti carrier can be also added in 3 ' ends It 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 receptors of targeting TIPE3 as described in relation to the first aspect a kind of, such as third Described in aspect or the T cell receptor gene modification T cell of targeting TIPE3 made from the preparation method as described in terms of the 6th, Recombinant viral vector as described in fourth aspect or the host cell as described in terms of the 5th is disliked in preparation prevention, diagnosing and treating Application in property tumour medicine.

Wherein, application of the present invention is suitable for the prevention of each quasi-cancer cell of high expression TIPE3, diagnosing and treating drug In application.

The application specifically: provide a kind of kit, the kit includes targeting TIPE3 described in first aspect T cell receptor, as described in second aspect tcr complex, as described in the third aspect targeting TIPE3 T cell by One of body gene modification T cell, the recombinant viral vector as described in fourth aspect, host cell as described in terms of the 5th Or it is a variety of.

Beneficial effects of the present invention:

A kind of T cell receptor targeting TIPE3 provided by the invention can be with the TIPE3 of specific recognition, compatibility and spy It is anisotropic high, the targeting TIPE3 that the T cell receptor gene modification T cell for targeting TIPE3 can be efficient and specific, especially table Up to the positive cancer cell of TIPE3, lasting activity and lethality are generated, and not will cause damage to normal cell.

Detailed description of the invention

Fig. 1 is the plasmid map of pLenti-TCR-TIPE3 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 TIPE3 provided in an embodiment of the present invention；Fig. 2 In (a) be negative control group, the T cell receptor gene modification T that (b) is targeting TIPE3 provided in an embodiment of the present invention in Fig. 2 is thin The experimental group of born of the same parents.

Fig. 3 is that the Vitro Tumor of the T cell receptor gene modification T cell of targeting TIPE3 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 of targeting TIPE3 provided in an embodiment of the present invention treats mice with tumor Effect picture.

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 TIPE3, comprising the following steps:

(1) gene order of the T cell receptor TCR-TIPE3 of preparation targeting TIPE3

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-TIPE3 of targeting TIPE3 is obtained.

(2) pLenti-TCR-TIPE3 recombinant plasmid is constructed

The coding gene sequence of TCR-TIPE3 is inserted between MluI the and EcoR1 restriction enzyme site of pLenti carrier, and After carrier EF1 α, using EF1 α as promoter.When the coding gene sequence of the TCR-TIPE3 is inserted into pLenti carrier, MluI enzyme in initiation codon (such as ATG) and pLenti carrier can be added in 5 ' ends of the coding gene sequence of the TCR-TIPE3 Enzyme site is connected, and 3 ' ends can be added terminator codon (such as TAA) and be connected with EcoR1 restriction enzyme site in pLenti carrier.Then turn Enter competent escherichia coli cell DH5 α, carries out positive colony PCR identification and sequencing identification.It is examined by PCR product gel electrophoresis It surveys and sequencing identification meets target fragment size and sequence, successfully construct pLenti-TCR-TIPE3 recombination matter as shown in Figure 1 Grain.

(3) recombined lentivirus vector constructs

PLenti-TCR-TIPE3 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 add together after merging with the viral supernatants of 48h harvest Enter in the centrifuge tube that exceeds the speed limit, be put into Beckman ultracentrifuge one by one, setting parameter of noncentricity is 25000rpm, centrifugation time For 2h, centrifuging temperature is controlled at 4 DEG C；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and disease is added Poison saves liquid, and gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence after being centrifuged 5min 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 slow virus Carrier.

(4) preparation of the T cell receptor gene modification T cell of TIPE3 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, and the T cell receptor gene modification T for obtaining targeting TIPE3 is thin Born of the same parents, 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 assess the T cell receptor gene modification T cell for targeting TIPE3 of above method preparation described in the invention Effect carries out following effect example.

Effect example one: the sun of the T cell receptor gene modification T cell of targeting TIPE3 prepared by the assessment present invention Property rate

It will be by the T cell receptor gene modification T cell (experimental group) of the method for the present invention preparation targeting TIPE3 and without system Standby T lymphocyte (negative control group), using its positive rate of flow cytomery, as a result as shown in Fig. 2, wherein in Fig. 2 It (a) is negative control group, i.e., without the T cell of preparation, (b) is experimental group, targeting TIPE3 as produced by the present invention in Fig. 2 T cell receptor gene modification T cell.(a) can be obtained compared with (b) in Fig. 2, the T of targeting TIPE3 prepared by the present invention The positive rate that kdr transfected cell modifies T cell is 37.1%.

Effect example two: the tumor cell in vitro killing of the T cell receptor gene modification T cell of assessment targeting TIPE3 Situation

Will by the T cell receptor gene modification T cell (experimental group) of targeting TIPE3 made from the method for the present invention with without The Vitro Tumor fragmentation effect of the T lymphocyte (negative control group) of preparation is compared, specifically: in vitro by effector cell (the T cell receptor gene modification T cell of targeting TIPE3 or the T lymphocyte without preparation) is pressed with target cell (Hela cell) Quantity ratio is 1:10,1:3,1:1,3:1 and 10:1 ratio, at 37 DEG C, 5%CO₂Under co-cultured, 15- after incubation 18 hours, cell is collected, carries out streaming dyeing, detects cell killing situation, as a result as shown in Figure 3.As can be seen from Figure 3, it passes through Cross the tumor-killing power of the TCR-T-TIPE3 cell of method of the present invention preparation 25% or more, even up to 65%, Significantly larger than negative control group.The result of Fig. 3 illustrates that the T cell receptor gene of the targeting TIPE3 through the method for the present invention preparation is repaired Adoring T cell has very strong tumor-killing ability.

Effect example three: the mouse interior tumor cell of the T cell receptor gene modification T cell of assessment targeting TIPE3 Kill situation

Will by the method for the present invention preparation targeting TIPE3 T cell receptor gene modification T cell (experimental group), without The T lymphocyte (negative control group) and physiological saline (blank control group) of preparation, in mouse tumor model, to every small Tail vein injection 1 × 10⁶A Hela cell (n=9), draws the survivorship curve of mouse, as a result as shown in Figure 4.It can be with from Fig. 4 Find out that the T cell receptor gene modification T cell of the targeting TIPE3 by this method preparation makes mouse when cultivating 85 days, Survival rate is up to 40%, considerably beyond negative control group and blank control group.Fig. 4's the result shows that, by this method preparation The T cell receptor gene modification T cell of targeting TIPE3 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>a kind of T cell receptor for targeting TIPE3, T cell receptor gene modification T cell and its preparation method and application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 160

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly His

20 25 30

Tyr Met His Trp Val Lys Gln Ser His Gly Gln Ser Leu Glu Trp Ile

35 40 45

Gly Arg Val Asn Pro Asn Asn Gly Gly Thr Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Pro Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Gly Ala Tyr Tyr Arg Ser Asp Gly Asn Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro

115 120 125

Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp Thr Thr Gly Ser

130 135 140

Ser Val Thr Leu Gly Cys Leu Val Lys Gly Trp Ser Trp Leu Phe Pro

145 150 155 160

<210> 2

<211> 291

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Met Gly Lys Pro Arg Gln Asn Pro Ser Thr Leu Val Ser Thr Leu Cys

1 5 10 15

Glu Ala Glu Pro Lys Gly Lys Leu Trp Val Asn Gly Tyr Ala Gly Thr

20 25 30

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

35 40 45

Phe His Leu Gln Arg Gly Lys Gly Leu Ala Ala Pro Leu Ser Ala Leu

50 55 60

Ser Ala Pro Arg Leu Pro Glu Arg Pro Ala Asp Gly Arg Val Ala Val

65 70 75 80

Asp Ala Gln Pro Ala Ala Arg Ser Met Asp Ser Asp Ser Gly Glu Gln

85 90 95

Ser Glu Gly Glu Pro Val Thr Ala Ala Gly Pro Asp Val Phe Ser Ser

100 105 110

Lys Ser Leu Ala Leu Gln Ala Gln Lys Lys Ile Leu Ser Lys Ile Ala

115 120 125

Ser Lys Thr Val Ala Asn Met Leu Ile Asp Asp Thr Ser Ser Glu Ile

130 135 140

Phe Asp Glu Leu Tyr Lys Val Thr Lys Glu His Thr His Asn Lys Lys

145 150 155 160

Glu Ala His Lys Ile Met Lys Asp Leu Ile Lys Val Ala Ile Lys Ile

165 170 175

Gly Ile Leu Tyr Arg Asn Asn Gln Phe Ser Gln Glu Glu Leu Val Ile

180 185 190

Val Glu Lys Phe Arg Lys Lys Leu Asn Gln Thr Ala Met Thr Ile Val

195 200 205

Ser Phe Tyr Glu Val Glu Tyr Thr Phe Asp Arg Asn Val Leu Ser Asn

210 215 220

Leu Leu His Glu Cys Lys Asp Leu Val His Glu Leu Val Gln Arg His

225 230 235 240

Leu Thr Pro Arg Thr His Gly Arg Ile Asn His Val Phe Asn His Phe

245 250 255

Ala Asp Val Glu Phe Leu Ser Thr Leu Tyr Ser Leu Asp Gly Asp Cys

260 265 270

Arg Pro Asn Leu Lys Arg Ile Cys Glu Gly Ile Asn Lys Leu Leu Asp

275 280 285

Glu Lys Val

290

<210> 3

<211> 480

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

gaggtccagc tgcagcagtc tggacctgac ctggtgaagc ctggggcttc agtgaagata 60

tcctgcaagg cttctggtta ctcattcact ggccactaca tgcactgggt gaagcagagc 120

catggacaga gccttgagtg gattggacgt gttaatccta acaatggtgg tactggctac 180

aaccagaagt tcaaggacaa ggccatatta actgtagaca agccatccag cacagcctac 240

atggagctcc gcagcctgac atctgaggac tctgcggtct attactgtgc aagaaacgga 300

gcctactata ggtccgatgg gaactacttt gactactggg gccaaggcac cactctcaca 360

gtctcctcag ccaaaacaac acccccatca gtctatccac tggcccctgg gtgtggagat 420

acaactggtt cctccgtgac tctgggatgc ctggtcaagg gttggtcatg gctgtttcct 480

<210> 4

<211> 873

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

atggggaaac cacggcaaaa cccaagcaca ctggtttcca cactctgtga ggcagagcca 60

aaggggaaac tgtgggtcaa cggatatgca gggacccaag gcacaaggga tgccacgtta 120

caaacaagac tcatcccctt atcttttcat cttcagaggg gaaagggact tgccgccccg 180

ctgtccgccc tgagtgcgcc gcggctgccc gagcgccccg cagacgggcg ggtggccgtg 240

gacgcccagc cagcagcccg cagcatggat tcggattccg gggagcagag cgagggcgag 300

cccgtgaccg ccgcaggtcc tgatgttttt agttcaaaga gtcttgcgct tcaagcccag 360

aagaagattc tgagcaaaat agccagcaaa actgtggcca acatgttgat tgatgacacc 420

agcagcgaga tctttgatga gctctacaaa gtcaccaaag agcacacaca caacaagaag 480

gaagcccaca agatcatgaa agacttaatc aaggtggcga tcaaaatcgg gatcctctac 540

cggaacaacc agtttagcca agaggagctg gttattgtgg agaagttccg gaagaagctg 600

aaccagaccg ccatgaccat tgtcagcttc tatgaggtgg aatacacctt cgataggaac 660

gtgctctcca atctcctgca tgagtgcaag gacctggtgc atgaactggt gcagcggcac 720

ctgacgccca ggacccacgg gcgcatcaac cacgtcttta accactttgc cgatgtggag 780

ttcctctcca ccctctatag tctggatgga gactgtaggc ccaacctcaa gaggatttgt 840

gaaggaatca ataagttgct agatgagaaa gtc 873

<210> 5

<211> 63

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc 60

cca 63

Claims (10)

1. a kind of T cell receptor for targeting TIPE3, which is characterized in that the T cell receptor of the targeting TIPE3 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.

2. the T cell receptor of targeting TIPE3 as described in claim 1, which is characterized in that the coding gene sequence of the α chain Including the nucleotide sequence as shown in SEQ ID NO:3, the coding gene sequence of the β chain includes as shown in SEQ ID NO:4 Nucleotide sequence.

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 TIPE3, which is characterized in that any including such as claim 1-2 The T cell receptor of targeting TIPE3 described in.

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 TIPE3 T cell receptor encoding gene.

6. recombinant viral vector as claimed in claim 5, which is characterized in that the coding of the T cell receptor of the targeting TIPE3 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 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, the coding gene sequence of the 2A peptide include 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 TIPE3, which is characterized in that including following step It is rapid:

(1) gene order of the T cell receptor TCR-TIPE3 of targeting TIPE3 is provided, including sequentially connected from 5 ' ends to 3 ' ends The gene order of α chain, 2A peptide and β chain, the coding gene sequence of the α chain include the nucleotides sequence as shown in SEQ ID NO:3 Column, the coding gene sequence of the β chain include the nucleotide sequence as shown in SEQ ID NO:4, the encoding gene of the 2A peptide Sequence includes the nucleotide sequence as shown in SEQ ID NO:5；

(2) gene order of the TCR-TIPE3 is inserted into pLenti carrier, obtains pLenti-TCR-TIPE3 recombination matter Grain；

(3) by the pLenti-TCR-TIPE3 recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, weight is obtained Group slow virus；

(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, obtains the T cell receptor gene modification T of targeting TIPE3 Cell.

9. the preparation method of the T cell receptor gene modification T cell of targeting TIPE3, 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 TIPE3 of claim 1-2, it is as claimed in claim 4 or The T cell receptor gene modification T cell or such as of TIPE3 is targeted as made from the described in any item preparation methods of claim 8-9 The described in any item recombinant viral vectors of claim 5-6 or host cell as claimed in claim 7 are in preparation prevention, diagnosis With the application in treatment malignant tumor medicine.