CN109837247A - A kind of T cell receptor gene modification T cell and its preparation method and application for the targeting TIPE3 that TCR is knocked out - Google Patents
A kind of T cell receptor gene modification T cell and its preparation method and application for the targeting TIPE3 that TCR is knocked out Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of the T cell receptor gene modification T cell of the TIPE3 of TCR targeting knocked out, it include: by knocking out the tcr gene in T lymphocyte, and the T cell receptor of targeting TIPE3 is prepared, obtain the T cell receptor gene modification T cell of the targeting TIPE3 of TCR knockout.The mispairing of the α chain and β interchain of endogenous and exogenous TCR in T cell can be effectively prevented in the T cell receptor gene modification T cell for the targeting TIPE3 that the TCR is knocked out, autoimmune response is avoided, the performance of efficient and specific killer T IPE3 positive cancer cell is kept.The present invention also provides the applications of the T cell receptor gene modification T cell of the TCR targeting TIPE3 knocked out.
Description
Technical field
The present invention relates to field of medical biotechnology, in particular to the T cell receptor gene for the targeting TIPE3 that a kind of TCR is knocked out
Modify T cell 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.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.The TCR and T cell for not having the T cell receptor of targeting TIPE3 also at present while TCR-T technology being avoided to transduce
Between the subunit of endogenous TCR mispairing and research.
Summary of the invention
In view of this, the present invention provides the T cell receptor gene modification T cell of TCR targeting TIPE3 knocked out a kind of,
The T cell receptor gene modification T cell for the targeting TIPE3 that the TCR is knocked out has knocked out tcr gene, is beneficial to prevent in T cell
Endogenous and exogenous TCR α chain and β interchain mispairing, keep the performance of efficient and specific killing cancer cell, and
And not will cause damage to normal cell, avoid autoimmune response.
In a first aspect, the present invention provides the T cell receptor gene modification T cells of TCR targeting TIPE3 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-TIPE3 of targeting TIPE3 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-TIPE3 is inserted into pLenti carrier, obtains pLenti-TCR-TIPE3 weight
Group plasmid；
(4) the pLenti-TCR-TIPE3 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 TCR knockout
To the T cell receptor gene modification T cell of TIPE3.
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 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 IRES.
Wherein, the encoding gene of the TCR-TIPE3 be inserted into pLenti carrier MluI 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 coding of the TCR-TIPE3
When gene is inserted into pLenti carrier, the encoding gene of the TCR-TIPE3 5 ' end can be added initiation codon (such as ATG) with
MluI restriction enzyme site is connected in pLenti carrier, and I enzyme of EcoR in terminator codon (such as TAA) and pLenti carrier can be added in 3 ' ends
Enzyme site is connected.
Optionally, the packaging pLenti-TCR-TIPE3 recombinant plasmid, obtaining recombinant slow virus includes:
By the pLenti-TCR-TIPE3 recombinant plasmid and envelope plasmid and packaging plasmid co-transfecting host cells, obtain
To 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.
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, by the corresponding base of sgRNA of the targeting tcr gene
Because sequence is inserted into pcDNA3.1 carrier, pcDNA3.1-TCR-sgRNA recombinant plasmid is 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,
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.
The preparation of the T cell receptor gene modification T cell for the targeting TIPE3 that the TCR that first aspect present invention provides is knocked out
Method by knocking out the tcr gene in T lymphocyte, and prepares the T cell receptor of targeting TIPE3, obtains the target of TCR knockout
To the T cell receptor gene modification T cell of TIPE3.The T for the targeting TIPE3 that the TCR of preparation method preparation of the present invention is knocked out
The α chain of endogenous and exogenous TCR in T cell and the mistake of β interchain can be effectively prevented in kdr transfected cell modification T cell
Match, avoid autoimmune response, keeps the performance of efficient and specific killer T IPE3 positive cancer cell.
Second aspect is knocked out the present invention provides the TCR being prepared using preparation method as described in relation to the first aspect
Target the T cell receptor gene modification T cell of TIPE3, the T cell receptor gene modification T for the targeting TIPE3 that the TCR is knocked out
Cell is free of tcr gene, and the T cell receptor gene modification T cell for the targeting TIPE3 that the TCR is knocked out includes targeting TIPE3
T cell receptor TCR-TIPE3, the T cell receptor TCR-TIPE3 of the targeting TIPE3 includes α chain and β chain, and the α chain includes
The amino acid sequence as shown in SEQ ID NO:1, the β chain include the amino acid sequence as shown in SEQ ID NO:2.
Wherein, the α chain and β chain that the TCR-TIPE3 includes can form stable heterodimer structure, to TIPE3
Albumen has very strong specificity, will not target normal cell or tissue, increase its safety and reduce undershooting-effect
Risk.
Wherein, TCR-TIPE3 of the present invention can be with specific recognition and in conjunction with TIPE3 albumen, and has optimal target
Point 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.
The T cell receptor gene modification T cell for the targeting TIPE3 that TCR of the present invention is knocked out with efficient identification and can kill
Wound includes the cancer cell that the expression such as chromoma, cancer of the esophagus, oophoroma have TIPE3.
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 TIPE3 that a kind of TCR described in face is knocked out is disliked in preparation prevention, diagnosing and treating
Application in the drug of property tumour.
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 cell for the targeting TIPE3 that a kind of TCR that method is prepared or as described in second aspect is knocked out.
Beneficial effects of the present invention:
The T cell receptor gene modification T cell for the targeting TIPE3 that TCR provided by the invention is knocked out can prevent in T cell
Endogenous and exogenous TCR α chain and β interchain mispairing, avoid autoimmune response, keep efficient and specific
The performance of killing tumor cell, and not will cause damage to normal cell.The T cell for the targeting TIPE3 that the TCR is knocked out by
Body gene modification T cell can promote T cell in the amplification of patient's body with the targeting TIPE3 of specificity, can be efficiently and special
Anisotropic killer T IPE3 positive cancer cell, 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 sun of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR provided in an embodiment of the present invention is knocked out
Property rate；(a) is negative control group in Fig. 2, and the T that (b) is the targeting TIPE3 that TCR provided in an embodiment of the present invention is knocked out in Fig. 2 is thin
The experimental group of born of the same parents' acceptor gene modification T cell.
Fig. 3 is the body of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR provided in an embodiment of the present invention is knocked out
Outer tumor cytotoxicity effect picture.
Fig. 4 is the T cell receptor gene modification T cell treatment for the targeting TIPE3 that TCR provided in an embodiment of the present invention is knocked out
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 TIPE3 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 mMESSAGE T7 kit carries out external
Transcription obtains Cas9 mRNA；
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 73% in the preparation for the CD3 positive t lymphocytes that TCR is knocked out.
(2) 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: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 encoding gene of the T cell receptor TCR-TIPE3 of targeting TIPE3 is obtained.
(3) pLenti-TCR-TIPE3 recombinant plasmid is constructed
The encoding gene for the T cell receptor TCR-TIPE3 for targeting TIPE3 is inserted into the MluI and EcoR of pLenti carrier
Between I restriction enzyme site, and after pLenti carrier EF1 α, using EF1 α as promoter.The encoding gene sequence of the TCR-TIPE3
When column are inserted into pLenti carrier, initiation codon (such as ATG) can be added in 5 ' ends of the coding gene sequence of the TCR-TIPE3
It is connected with MluI restriction enzyme site in pLenti carrier, the I digestion position EcoR in terminator codon and pLenti carrier can be added in 3 ' ends
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, construct pLenti-TCR- as shown in Figure 1
TIPE3 recombinant plasmid.
(4) recombinant slow virus 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 lentiviral disease
Poison.
(5) preparation of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR is knocked out
The CD3 positive t lymphocytes for taking above-mentioned TCR to knock out are added corresponding with the CD3 positive t lymphocytes that TCR is knocked out
The above-mentioned recombinant slow virus of virus titer is cultivated.The 3rd day of culture carries out cell count and changes liquid, adjust cell concentration
It is 1 × 106A/mL is inoculated with, culture；Cell state is observed in the 5th day of culture, if cell density increases, diluting cells
Concentration is 1 × 106A/mL detects cell activity, continues to cultivate.Amplification cultivation collected cell, and obtained TCR and strike by the 9-11 days
The T cell receptor gene modification T cell of the targeting TIPE3 removed.
In order to assess the T cell receptor gene for the targeting TIPE3 that the TCR of above method preparation described in the invention is knocked out
T cell effect is modified, following effect example is carried out.
Effect example one: the T cell receptor gene modification T for the targeting TIPE3 that TCR prepared by the assessment present invention is knocked out
The positive rate of cell
The T cell receptor gene modification T cell (experiment of the targeting TIPE3 of TCR knockout will be prepared by the method for the present invention
Group) and the T lymphocyte (negative control group) without preparation, using its positive rate of flow cytomery, as a result such as Fig. 2 institute
Show, wherein (a) is negative control group in Fig. 2, i.e., without the T cell of preparation, (b) is experimental group in Fig. 2, and the as present invention is made
TCR knock out targeting TIPE3 T cell receptor gene modification T cell.(a) can be obtained compared with (b) in Fig. 2, the present invention
The positive rate of the T cell receptor gene modification T cell for the targeting TIPE3 that prepared TCR is knocked out is 57.8%.
Effect example two: the Vitro Tumor of the T cell receptor gene modification T cell for the targeting TIPE3 that assessment TCR is knocked out
Cell killing situation
By the T cell receptor gene modification T cell (experiment of the targeting TIPE3 knocked out by TCR made from the method for the present invention
Group) it is compared with the Vitro Tumor fragmentation effect of the T lymphocyte (negative control group) without preparation, it is specific: in vitro
By effector cell (TCR knock out targeting TIPE3 T cell receptor gene modification T cell or without the T lymphocyte of preparation) with
Target cell (HCT116 cell) is 1:10,1:3,1:1,3:1 and 10:1 ratio in quantity ratio, at 37 DEG C, 5%CO2Under be total to
Culture after incubation 15-18 hours, collects cell, carries out streaming dyeing, cell killing situation detected, as a result such as Fig. 3 institute
Show.As can be seen from Figure 3, the T cell receptor gene for the targeting TIPE3 that the TCR by method of the present invention preparation is knocked out
The tumor-killing power of T cell is modified 15% or more, even up to 55%, significantly larger than negative control group, this explanation is through this
The T cell receptor gene modification T cell for the targeting TIPE3 that the TCR of inventive method preparation is knocked out has strong tumor-killing ability.
Effect example three: in the Mice Body of the T cell receptor gene modification T cell for the targeting TIPE3 that assessment TCR is knocked out
Tumor cytotoxicity situation
By the T cell receptor gene modification T cell (experiment of the targeting TIPE3 knocked out by the TCR of the method for the present invention preparation
Group), the T lymphocyte (negative control group) without preparation and physiological saline (blank control group), in mouse tumor model,
To every mouse tail vein injection 1 × 106A HCT116 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 the T cell receptor gene modification T cell for the targeting TIPE3 that the TCR by this method preparation is knocked out makes
It obtains mouse survival rate after culture 80 days and is also higher than 40%, considerably beyond negative control group and blank control group.The result of Fig. 4
Show that the T cell receptor gene modification T cell for the targeting TIPE3 that the TCR prepared by this method is knocked out can be protected preferably
Mouse is from because dead caused by 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
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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> 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> 480
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
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> 5
<211> 873
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
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> 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 (10)
1. a kind of preparation method of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR is knocked out, which is characterized in that packet
It includes:
(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-TIPE3 of targeting TIPE3 is provided, including sequentially connected from 5 ' ends to 3 ' ends
The encoding gene of the encoding gene of α chain, the encoding gene of 2A peptide and β chain, wherein the encoding gene of the α chain includes encoding such as
The encoding gene of the nucleotide sequence of amino acid sequence shown in SEQ ID NO:1, the β chain includes coding such as SEQ ID NO:
The nucleotide sequence of amino acid sequence shown in 2, the encoding gene of the 2A peptide include coding ammonia as shown in SEQ ID NO:3
The nucleotide sequence of base acid sequence；
(3) encoding gene of the TCR-TIPE3 is inserted into pLenti carrier, obtains pLenti-TCR-TIPE3 recombination matter
Grain；
(4) the pLenti-TCR-TIPE3 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 targeting of TCR knockout
The T cell receptor gene modification T cell of TIPE3.
2. the preparation method of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR as described in claim 1 is knocked out,
It 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 method of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR as described in claim 1 is knocked out,
It 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 method of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR as described in claim 1 is knocked out,
It 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 method of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR as described in claim 1 is knocked out,
It 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 knocks out the tcr gene of the CD3 positive t lymphocytes.
6. the preparation method of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR as claimed in claim 5 is knocked out,
It is characterized in that, the corresponding gene order of sgRNA of the targeting tcr gene includes the nucleotide as shown in SEQ ID NO:7
Sequence.
7. the preparation method of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR as claimed in claim 5 is knocked out,
It 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 method of the T cell receptor gene modification T cell for the targeting TIPE3 that TCR as described in claim 1 is knocked out,
It is characterized in that, the packaging pLenti-TCR-TIPE3 recombinant plasmid, obtaining recombinant slow virus includes:
By the pLenti-TCR-TIPE3 recombinant plasmid and envelope plasmid and packaging plasmid co-transfecting host cells, institute is obtained
State recombinant slow virus.
9. the T cell receptor base for the targeting TIPE3 that the TCR that the method according to claim 1 is prepared is knocked out
Because modifying T cell, which is characterized in that the T cell receptor gene modification T cell for the targeting TIPE3 that the TCR is knocked out is free of TCR
Gene, the T cell receptor gene modification T cell for the targeting TIPE3 that the TCR is knocked out include the T cell receptor for targeting TIPE3
TCR-TIPE3, the T cell receptor TCR-TIPE3 of the targeting TIPE3 include α chain and β chain, and the α chain includes such as SEQ ID
Amino acid sequence shown in NO:1, the β chain include the amino acid sequence as shown in SEQ ID 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 TIPE3 T cell receptor gene modification T cell preparation prevention, diagnosing and treating malignant tumour drug in answering
With.
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Application publication date: 20190604 |