CN110526979A - Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of FAP - Google Patents
Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of FAP Download PDFInfo
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Abstract
The present invention provides a kind of single-chain antibody for targeting FAP, the single-chain antibody of the targeting FAP includes the amino acid sequence as shown in SEQ ID NO:1.The present invention also provides a kind of Chimeric antigen receptor T cells of single-chain antibody including the targeting FAP, the Chimeric antigen receptor for wherein targeting FAP can in specific manner on target tumor FAP, T cell is activated to play immunization of cell, the malignant cell of the efficient and specific killing FAP positive, has lasting cell viability and lethality.The present invention also provides a kind of preparation method and application of Chimeric antigen receptor T cell for targeting FAP.
Description
Technical field
The present invention relates to field of medical biotechnology, in particular to a kind of single-chain antibody, Chimeric antigen receptor T for targeting FAP is thin
Born of the same parents and its preparation method and application.
Background technique
Malignant tumour (cancer) has become the arch-criminal for threatening human life, and disease incidence is at ascendant trend.For example, ovary
The current death rate of malignant tumour occupies the first place of gynaecology's genital tract malignant tumour, the malignant tumor of ovary of 60-70% when being diagnosed
Patient has belonged to advanced stage.The unconventionality expression of fibroblast activation protein (Fibroblast Activation Protein, FAP)
May be related with the generation of oophoroma, development, it can promote the proliferation of ovarian cancer cell, invasion and migration, FAP are often expressed in ovum
In nest cancer Stromal fibroblasts.The traditional remedies such as radiotherapy, chemotherapy are bad to the therapeutic effect of oophoroma, and it is easy to recur that there are tumours
The problems such as serious with side reaction.
CAR-T (Chimeric antigen receptor T cell) technology is a kind of novel cell therapy, it is that the T by CAR transformation is thin
Born of the same parents are fed back to human body, activate self immune system, kill to tumour cell, it is considered to be current most effective malignant tumour
One of therapeutic modality, the drawbacks of traditional remedies can be made up.However there is not also the Chimeric antigen receptor T of targeting FAP thin at present
The preparation and research of born of the same parents.Therefore, in conjunction with CAR-T technology, the diseases such as oophoroma are cured with being expected to targeting.
Summary of the invention
In view of this, the present invention provides a kind of single-chain antibodies for targeting FAP, and including the single-stranded of the targeting FAP
The Chimeric antigen receptor T cell of antibody.The Chimeric antigen receptor of the targeting FAP can targeted expression FAP in specific manner it is swollen
Oncocyte, activation T cell play immunization of cell, efficiently and specifically kill FAP positive tumor cell, preferably maintain
The vigor and lethality of Chimeric antigen receptor T cell.The present invention also provides a kind of Chimeric antigen receptor T cells for targeting FAP
Preparation method and related application.
In a first aspect, the present invention provides a kind of single-chain antibody for targeting FAP, the single-chain antibody of the targeting FAP includes
The amino acid sequence as shown in SEQ ID NO:1.
Optionally, the encoding gene of the single-chain antibody of the targeting FAP includes the nucleotides sequence as shown in SEQ ID NO:2
Column.
Optionally, the single-chain antibody encoding gene of the targeting FAP should consider degeneracy base, i.e., such as SEQ ID NO:1
Shown in the encoding gene of amino acid sequence include the nucleotide sequence as shown in SEQ ID NO:2, protection scope should also protect
Shield has the nucleotide sequence of base degeneracy matter with SEQ ID NO:2, and the corresponding amino acid sequence of these nucleotide sequences is still
It is so SEQ ID NO:1.
First aspect present invention provide the targeting FAP single-chain antibody, can specific recognition FAP albumen, and with
It specifically binds, and to tumour cell, the oophoroma Stromal fibroblasts of especially expression FAP have stronger affine
Activity and internalization activity.
Second aspect, the present invention provides a kind of Chimeric antigen receptor T cells for targeting FAP, including the chimeric of targeting FAP
Antigen receptor CAR-FAP, the CAR-FAP include the single-chain antibody of sequentially connected targeting FAP, born of the same parents from aminoterminal to c-terminus
The amino acid sequence of outer hinge area, transmembrane region and intracellular signal area, wherein the single-chain antibody of the targeting FAP includes such as SEQ ID
Amino acid sequence shown in NO:1.
Wherein, described " being sequentially connected with from aminoterminal to c-terminus " specifically: the amino of the single-chain antibody of the targeting FAP
The c-terminus of acid sequence is connected with the aminoterminal of the amino acid sequence of the extracellular hinge area, the amino acid of the extracellular hinge area
The c-terminus of sequence is connected with the aminoterminal of the amino acid sequence of the transmembrane region, the carboxyl of the amino acid sequence of the transmembrane region
End is connected with the aminoterminal of the amino acid sequence in the intracellular signal area.
In the present invention, the extracellular hinge area is used to promote the FAP knot on the single-chain antibody and tumour of the targeting FAP
It closes.Optionally, the extracellular hinge area includes CD8 α hinge area, CD28 hinge area, CD4 hinge area, CD5 hinge area, CD134 hinge
One of sequence, CD137 hinge area, ICOS hinge area or a variety of combinations.
Still optionally further, the extracellular hinge area is CD8 α hinge area.
Optionally, the amino acid sequence of the CD8 α hinge area includes the amino acid sequence as shown in SEQ ID NO:6.
Optionally, the encoding gene of the CD8 α hinge area includes the nucleotide sequence as shown in SEQ ID NO:7.
Optionally, the encoding gene of the CD8 α hinge area should consider degeneracy base, i.e., as shown in SEQ ID NO:6
The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:7, and protection scope should also protect and SEQ
ID NO:7 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ
ID NO:6。
In the present invention, the transmembrane region is used to fix the Chimeric antigen receptor CAR-FAP of the targeting FAP.Optionally, institute
Stating transmembrane region includes one of CD3 transmembrane region, CD4 transmembrane region, CD8 transmembrane region, CD28 transmembrane region or a variety of combinations.
Still optionally further, the transmembrane region is CD8 transmembrane region.
Optionally, the amino acid sequence of the CD8 transmembrane region includes the amino acid sequence as shown in SEQ ID NO:8.
Optionally, the encoding gene of the CD8 transmembrane region includes the nucleotide sequence as shown in SEQ ID NO:9.
Optionally, the encoding gene of the CD8 transmembrane region should consider degeneracy base, i.e., as shown in SEQ ID NO:8
The encoding gene of amino acid sequence includes the nucleotide sequence as shown in SEQ ID NO:9, and protection scope should also protect and SEQ
ID NO:9 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ
ID NO:8。
In the present invention, the intracellular signal area for providing the signal of T cell activation, maintain T cell life span and
Activate T cell proliferation signal access.Optionally, the intracellular signal area includes 4-1BB signaling zone, CD3 ζ signaling zone, ICOS letter
Number area, CD27 signaling zone, OX40 signaling zone, CD28 signaling zone, IL1R1 signaling zone, CD70 signaling zone, TNFRSF19L signaling zone
One of or a variety of combinations.
Optionally, the intracellular signal Qu Weicong aminoterminal is believed to the sequentially connected 4-1BB signaling zone of c-terminus and CD3 ζ
Number area.Correspondingly, the encoding gene in the intracellular signal area includes the volume from 5 ' ends to 3 ' the sequentially connected 4-1BB signaling zones in end
The encoding gene of code gene and CD3 ζ signaling zone.
Wherein, CD3 ζ signaling zone is signal transduction structural domain, and 4-1BB signaling zone is costimulation structural domain, in being total to for they
Under same-action, T cell is activated completely after identifying antigen.Still optionally further, the carboxylic of the amino acid sequence of the transmembrane region
Cardinal extremity is connected with the aminoterminal of the amino acid sequence of the 4-1BB signaling zone, the carboxylic of the amino acid sequence of the 4-1BB signaling zone
Cardinal extremity is connected with the aminoterminal of the amino acid sequence of the CD3 ζ signaling zone.
Optionally, the amino acid sequence of the 4-1BB signaling zone includes the amino acid sequence as shown in SEQ ID NO:10.
Optionally, the encoding gene of the 4-1BB signaling zone includes the nucleotide sequence as shown in SEQ ID NO:11.
Optionally, the encoding gene of the 4-1BB signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:10
Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:11 shown in, protection scope should also protect and
SEQ ID NO:11 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as
SEQ ID NO:10。
Optionally, the amino acid sequence of the CD3 ζ signaling zone includes the amino acid sequence as shown in SEQ ID NO:12.
Optionally, the encoding gene of the CD3 ζ signaling zone includes the nucleotide sequence as shown in SEQ ID NO:13.
Optionally, the encoding gene of the CD3 ζ signaling zone should consider degeneracy base, i.e., as shown in SEQ ID NO:12
Amino acid sequence encoding gene include the nucleotide sequence such as SEQ ID NO:13 shown in, protection scope should also protect and
SEQ ID NO:13 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as
SEQ ID NO:12。
Optionally, the amino acid sequence of the CAR-FAP includes the amino acid sequence as shown in SEQ ID NO:3.
Optionally, the encoding gene of the CAR-FAP includes the nucleotide sequence as shown in SEQ ID NO:4.
Optionally, the encoding gene of the CAR-FAP should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:3
The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:4, and protection scope should also protect and SEQ
ID NO:4 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ
ID NO:3。
The Chimeric antigen receptor T cell (CAR-T cell) for the targeting FAP that second aspect of the present invention provides, including target
To the Chimeric antigen receptor CAR-FAP of FAP, receptor CAR-FAP is for CAR-T cell targeted expression FAP in specific manner
Tumour cell, after CAR-FAP and FAP protein binding, the intracellular signal area of the CAR-T cell is activated, and T cell is promoted to exist
The amplification of patient's body, efficient and specific killing tumor cell, and normal ovarian tissue is hardly caused to damage, from
And thoroughly cure oophoroma.
The third aspect, the present invention provides a kind of recombinant viral vector, the recombinant viral vector includes such as second aspect
The encoding gene of the CAR-FAP of the Chimeric antigen receptor T cell of the targeting FAP.
Optionally, the encoding gene of the CAR-FAP includes the nucleotide sequence as shown in SEQ ID NO:4.
Optionally, the encoding gene of the CAR-FAP includes the nucleotide sequence as shown in SEQ ID NO:5.Such as SEQ
Nucleotide sequence shown in ID NO:5 is compared with the nucleotide sequence as shown in SEQ ID NO:4, more coding bases of link peptide
Cause.The encoding gene of the signal peptide can be expressed with Chimeric antigen receptor CAR-FAP described in guide to cell surface.
Optionally, the viral vectors in the recombinant viral vector includes slow virus carrier, adenovirus vector or reverse transcription
Viral vectors.Still optionally further, the viral vectors is slow virus carrier.
Further optionally, the slow virus carrier include pWPXLD carrier, pLEX-MCS carrier, pSico carrier and
At least one of pCgpV carrier.Specifically, when the slow virus carrier is pWPXLD carrier, gained recombinant viral vector
In, the encoding gene of CAR-FAP is located between I restriction enzyme site of I restriction enzyme site of BamH and EcoR of pWPXLD carrier.
The recombinant viral vector that third aspect present invention provides is a safe and reliable carrier tool, can be efficiently
Shift the encoding gene of the CAR-FAP.The recombinant viral vector, which can be used for preparing, carries the CAR-FAP encoding gene
Virus, and preparation targeting FAP Chimeric antigen receptor T cell, make the T cell continue, play consistently targeting, killing effect
Power.
The recombinant viral vector that third aspect present invention provides, efficiency of infection and transcriptional efficiency with higher,
The encoding gene segment of CAR-FAP therein can be inserted into host genome by genetic recombination, and it is thin to obtain above-mentioned targeting T lymph
Born of the same parents continue it, play consistently targeting, killing effect.
Fourth aspect, the present invention provides a kind of host cell, the host cell includes the weight as described in the third aspect
Group viral vectors.
The host cell that fourth aspect present invention provides is used to provide and carries the recombinant virus as described in second aspect
Body is assembled and is prepared the place for generating corresponding virus, and the something lost of the CAR-FAP is carried by virus prepared by host cell
Communication breath, has strong 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.Still optionally further, described
Host cell is HEK293T cell.
5th aspect, the present invention provides a kind of preparation methods of Chimeric antigen receptor T cell for targeting FAP, comprising:
(1) encoding gene of the Chimeric antigen receptor CAR-FAP of targeting FAP is provided, including is sequentially connected from 5 ' ends to 3 ' ends
The encoding gene of the signal peptide connect, the encoding gene of single-chain antibody, the encoding gene of extracellular hinge area, transmembrane region for targeting FAP
Encoding gene and intracellular signal area encoding gene, wherein it is described targeting FAP single-chain antibody encoding gene include such as
Nucleotide sequence corresponding to amino acid sequence shown in SEQ ID NO:1；
(2) encoding gene of the CAR-FAP is inserted into pWPXLD carrier, obtains pWPXLD-CAR-FAP recombination matter
Grain；
(3) it by the pWPXLD-CAR-FAP recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, obtains
Recombinant slow virus；
(4) by the recombinant slow virus transfect CD3 positive t lymphocytes, through separation obtain targeting FAP chimeric antigen by
Body T cell.
It is above-mentioned " from 5 ' end to 3 ' end be sequentially connected with " specifically: the coding gene sequence of the signal peptide 3 ' end with it is described
Target FAP single-chain antibody encoding gene 5 ' end be connected, it is described targeting FAP single-chain antibody encoding gene 3 ' end with
5 ' ends of the encoding gene of the extracellular hinge area are connected, 3 ' ends of the encoding gene of the extracellular hinge area and the transmembrane region
5 ' ends of encoding gene be connected, the 3 ' ends and the 5 ' of the encoding gene in the intracellular signal area of the encoding gene of the transmembrane region
End is connected.
The signal peptide is for instructing the Chimeric antigen receptor CAR-FAP expression to cell surface, institute in the present invention
Signal peptide is stated to be cut in protein translation maturation by signal peptidase.
Optionally, the amino acid sequence of the signal peptide includes the amino acid sequence as shown in SEQ ID NO:14.
Optionally, the encoding gene of the signal peptide includes the nucleotide sequence as shown in SEQ ID NO:15.
Optionally, the encoding gene of the signal peptide should consider degeneracy base, the i.e. ammonia as shown in SEQ ID NO:14
The encoding gene of base acid sequence includes the nucleotide sequence as shown in SEQ ID NO:15, and protection scope should also protect and SEQ
ID NO:15 has the nucleotide sequence of base degeneracy matter, and the corresponding amino acid sequence of these nucleotide sequences remains as SEQ
ID NO:14。
The extracellular hinge area, transmembrane region, the specific choice in intracellular signal area and corresponding coding gene sequence such as this hair
Described in bright second aspect part, which is not described herein again.
Optionally, the encoding gene of the CAR-FAP includes corresponding to the amino acid sequence as shown in SEQ ID NO:16
Nucleotide sequence.
Still optionally further, the coding gene sequence of CAR-FAP nucleotide sequence as shown in SEQ ID NO:5.
The nucleotide sequence as shown in SEQ ID NO:5 is compared with the nucleotide sequence as shown in SEQ ID NO:4, more connections
The encoding gene of peptide, but when Chimeric antigen receptor CAR-FAP expression is to cell surface, the signal peptide is in protein translation
It is cut in maturation by signal peptidase.Therefore, in the amino acid sequence of the Chimeric antigen receptor CAR-FAP translated into
And not with the amino acid sequence as shown in SEQ ID NO:14.
The encoding gene of the CAR-FAP is inserted into pWPXLD carrier between I restriction enzyme site of BamH I and EcoR, and position
After the EF1 α of pWPXLD carrier, using EF1 α as promoter.The encoding gene of the CAR-FAP is inserted into pWPXLD carrier
When, initiation codon (such as ATG) can be added in 5 ' ends of the encoding gene of the CAR-FAP, with BamH1 digestion in pWPXLD carrier
Site (ggatcc) is connected, and EcoR1 restriction enzyme site in terminator codon (such as TAA) and pWPXLD carrier can be added in 3 ' ends
(gaattc) it is connected, thus makes the encoding gene of the CAR-FAP between BamH1 and EcoR1 restriction enzyme site.
Optionally, the envelope plasmid is PMD2G, and the packaging plasmid is psPAX2, and the host cell is HEK293T
Cell.
The envelope plasmid PMD2G encodes vesicular stomatitis virus glycoprotein capsid, the vesicular stomatitis virus sugar egg
White capsid assists recombinant slow virus to adhere to cell membrane, and keeps the infectivity of recombinant slow virus.
Recombinant slow virus of the present invention can further contain the envelope protein from other viruses.For example, as this
Kind protein, is preferably the virus enveloped protein for carrying out self-infection human cell.To this protein, there is no particular limitation, can example
The amphophilic virus hand epithelium albumen etc. for enumerating retrovirus, can be used for example from mouse leukemia virus (MuMLV)
4070A plants of envelope protein.Alternatively, it is also possible to use the envelope protein from MuMLV 10Al.In addition, as herpetoviridae
Albumen, it can be cited for example that, gB, gD, gH, gp85 albumen of herpes simplex virus, gp350, gp220 albumen of Epstein-Barr virus
Deng.As the albumen of Hepadna Virus section, the S protein etc. of hepatitis B virus can be included.The envelope protein can also be morbilli
It is formed after viral glycoprotein and other single chain antibody fusions.
The packaging of recombinant slow virus is generallyd use transient transfection or is packed using cell line.It may be used as wrapping when transient transfection
Human cell's strain that dress cell uses, for example including 293 cells, 293T cell, 293FT cell, 293LTV cell, 293EBNA
Cell and other clones separated from 293 cells；SW480 cell, u87MG cell, HOS cell, C8166 cell, MT-4 are thin
Born of the same parents, Molt-4 cell, HeLa cell, HT1080 cell, TE671 cell etc..It can also be using the cell strain from monkey, example
Such as, COS1 cell, COS7 cell, CV-1 cell, BMT10 cell etc..Moreover, the calcium phosphate and PEI transfection reagent that generally use,
It is also well used there are also some transfection reagents such as Lipofectamine2000, FuGENE and S93fectin.
The packaging of recombinant slow virus also uses some slow virus package cell lines, such as most common Env glycoprotein of use,
Stable cell lines caused by VSVG albumen or HIV-1gag-pol albumen.
For the sake of security, the slow virus carrier system of large-scale use is all the method using split gene group, i.e., will
The assignment of genes gene mapping of different miscellaneous functions is played in different plasmids.There are four pUC pUCs (encoding gag-pol gene, Rev base at present
Cause, VSVG gene, SIN metastatic gene be located at four different plasmids), three pUC pUCs (eliminate coding Rev gene
Plasmid, gag-pol gene uses the codon of the preferences in people's cell in gag-pol plasmid) and two pUC pUCs are (slowly
Auxiliary gene necessary to viral vectors is packed is located on the same plasmid, these auxiliary genes are single gene orders；Separately
One is then transgenosis plasmid).Also the slow virus packaging system for having more than four pUC pUCs is using.
Optionally, in step (4), the CD3 positive t lymphocytes are to separate to obtain from source of people peripheral blood mononuclear cells
.
Optionally, the source of people peripheral blood mononuclear cells derives from autologous vein blood, autologous bone marrow, Cord blood and placenta
Blood etc..
Still optionally further, the fresh peripheral acquired after cancer patient's operation one month, after chemicotherapy one month
Blood or marrow.
Specifically, the acquisition process of the CD3 positive t lymphocytes is as follows: into peripheral blood mononuclear cells by certain
CD3/CD28 immunomagnetic beads are added in ratio, after being incubated for a period of time, are put into magnet and are screened, it is coated to obtain immunomagnetic beads
CD3 positive t lymphocytes after removing magnetic bead, obtain CD3 positive t lymphocytes.
6th aspect, the present invention provides the single-chain antibodies of targeting FAP as described in relation to the first aspect a kind of, such as second aspect
Being fitted into for FAP is targeted made from the Chimeric antigen receptor T cell of the described targeting FAP or the preparation method as described in the 5th aspect
Antigen receptor T cell, the recombinant viral vector as described in the third aspect or the host cell as described in fourth aspect are examined in preparation
Application in disconnected and/treatment malignant tumour drug.Particularly, it can be used for diagnosing and/or treating the malignant tumour of expression FAP,
Such as oophoroma.
The application specifically: provide a kind of kit, the kit includes targeting FAP described in first aspect
Single-chain antibody, the Chimeric antigen receptor T cell for targeting FAP as described in second aspect, the recombinant virus as described in the third aspect
One of carrier, host cell as described in fourth aspect are a variety of.
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 pWPXLd-CAR-FAP recombinant plasmid provided in an embodiment of the present invention.
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Embodiment one
A kind of preparation method for the Chimeric antigen receptor T cell targeting FAP, comprising the following steps:
(1) gene order of the Chimeric antigen receptor CAR-FAP of preparation targeting FAP
Prepare respectively signal peptide, target the single-chain antibody of FAP, CD8 α hinge area, CD8 transmembrane region, 4-1BB signaling zone and
The encoding gene of CD3 ζ signaling zone, for the encoding gene of the signal peptide as shown in SEQ ID NO:15, the targeting FAP's is single-stranded
The encoding gene of antibody is as shown in SEQ ID NO:2, and the encoding gene of the CD8 α hinge area is as shown in SEQ ID NO:7, institute
The encoding gene of CD8 transmembrane region is stated as shown in SEQ ID NO:9, the encoding gene of the 4-1BB signaling zone such as SEQ ID NO:
Shown in 11, the encoding gene of the CD3 ζ signaling zone is as shown in SEQ ID NO:13.
By the method for PCR by above-mentioned signal peptide, target single-chain antibody, the CD8 α hinge area, CD8 transmembrane region, 4- of FAP
1BB signaling zone is successively connected together from 5 ' ends to 3 ' ends with the encoding gene of CD3 ζ signaling zone, obtains the inosculating antibody of targeting FAP
The encoding gene of original receptor CAR-FAP, the encoding gene of the CAR-FAP is as shown in SEQ ID NO:5.
(2) pWPXLd-CAR-FAP recombinant plasmid is constructed
The encoding gene of CAR-FAP is inserted between BamH1 the and EcoR1 restriction enzyme site of pWPXLD carrier, and
After pWPXLD carrier EF1 α, using EF1 α as promoter.When the encoding gene of the CAR-FAP is inserted into pWPXLD carrier, institute
Initiation codon (such as ATG) and BamH1 restriction enzyme site phase in pWPXLD carrier can be added in the 5 ' ends for stating the encoding gene of CAR-FAP
Even, 3 ' ends can also be added terminator codon (such as TAA) and be connected with EcoR1 restriction enzyme site in pWPXLD carrier.Then it is transferred to big
Enterobacteria competent cell DH5 α carries out positive colony PCR identification and sequencing identification.By PCR product detected through gel electrophoresis and
Sequencing identification meets target fragment size and sequence, successfully constructs pWPXLd-CAR-FAP recombinant plasmid, is as shown in Figure 1
PWPXLd-CAR-FAP recombinant plasmid.
(3) recombinant slow virus constructs
PWPXLd-CAR-FAP recombinant plasmid, packaging plasmid psPAX2, envelope plasmid pMD2G three cotransfection are entered into culture
Good HEK293T cell.48h harvest saves in -80 DEG C of ultra low temperature freezers containing the supernatant of virus through 0.45 μm of membrane filtration；
It is super to merge addition together with the viral supernatants of 48h harvest for supernatant of the 72h aftercrop containing virus, 0.45 μm of membrane filtration
In fast centrifuge tube, be put into Beckman ultracentrifuge one by one, setting parameter of noncentricity be 25000rpm, centrifugation time 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 virus is added and saves
Liquid, gently piping and druming is resuspended repeatedly；Through after completely dissolution, high speed centrifugation 10000rpm takes supernatant fluorescence spectrometry after being centrifuged 5min
Titre, virus is according to 100 μ l, and 2 × 108A/mL packing, is stored in -80 DEG C of ultra low temperature freezers, obtains recombinant slow virus.
(4) preparation of the Chimeric antigen receptor T cell of FAP is targeted
A) separation of PBMC (peripheral blood mononuclear cells)
PBMC is from autologous vein blood, autologous bone marrow, Cord blood and placental blood etc..Preferably derive from cancer patient's hand
The fresh peripheral blood or marrow acquired after art one month, after chemicotherapy one month.
Extract patient blood, sample presentation to blood separating chamber；Peripheral blood mononuclear cells is acquired, is taken after Ficoll centrifuge separation
Intermediate layer cell；After PBS is washed, PBMC is obtained.
B) immunomagnetic beads antigenspecific T lymphocyte
Above-mentioned PBMC is taken, the basal medium for being free of serum is added, is made into cell suspension；Ratio in magnetic bead and cell is
3:1, is added CD3/CD28 immunomagnetic beads, and room temperature incubates 1-2h；The cell for being incubated for magnetic bead is screened using magnet；PBS is washed
It washs, after removing immunomagnetic beads, obtains CD3 positive t lymphocytes.
C) virus transfection method prepares antigenspecific T lymphocyte
The above-mentioned CD3 positive t lymphocytes obtained by magnetic activated cell seperation are taken, are added and CD3 positive cell number phase
The recombinant slow virus for the virus titer answered is cultivated.
The 3rd day of culture carries out cell count and changes liquid, and adjustment cell concentration is 1 × 106A/mL is inoculated with, culture；Training
Cell state is observed in feeding the 5th day, if cell density increases, diluting cells concentration is 1 × 106A/mL detects cell
Activity continues to cultivate.Amplification cultivation collected cell by the 9-11 days, obtained the Chimeric antigen receptor T cell of targeting FAP, and protect
In the presence of in the dedicated cells frozen storing liquid of feedback.
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 Xianjin Technology Academe
<120>single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of FAP are targeted
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 237
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Arg Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Phe Trp Asp Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
130 135 140
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr
145 150 155 160
Thr Phe Thr Glu Tyr Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln
165 170 175
Arg Leu Glu Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn
180 185 190
Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Thr Ser
195 200 205
Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
210 215 220
Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
225 230 235
<210> 2
<211> 711
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gacattgtga tgacccaatc tccagactct ttggctgtgt ctctagggga gagggccacc 60
atcaactgca agtccagtca gagcctttta tattctagaa atcaaaagaa ctacttggcc 120
tggtatcagc agaaaccagg acagccaccc aaactcctca tcttttggga tagcactagg 180
gaatctgggg tacctgatag gttcagtggc agtgggtttg ggacagactt caccctcacc 240
attagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttttagctat 300
ccgctcacgt tcggacaagg gaccaaggtg gaaataaaag gaggcggagg atctggcggc 360
ggaggaagtg gcggaggggg atctggggga ggcggaagcc aggtgcaact agtgcagtcc 420
ggcgccgaag tgaagaaacc cggtgcttcc gtgaaagtca gctgtaaaac tagtagatac 480
accttcactg aatacaccat acactgggtt agacaggccc ctggccaaag gctggagtgg 540
ataggaggta ttaatcctaa caatggtatt cctaactaca accagaagtt caagggccgg 600
gtcaccatca ccgtagacac ctctgccagc accgcctaca tggaactgtc cagcctgcgc 660
tccgaggaca ctgcagtctg gggtcaagga acccttgtca ccgtctcctc a 711
<210> 3
<211> 460
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Arg Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Pro Pro Lys Leu Leu Ile Phe Trp Asp Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
130 135 140
Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Thr Ser Arg Tyr
145 150 155 160
Thr Phe Thr Glu Tyr Thr Ile His Trp Val Arg Gln Ala Pro Gly Gln
165 170 175
Arg Leu Glu Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Ile Pro Asn
180 185 190
Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr Val Asp Thr Ser
195 200 205
Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
210 215 220
Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr
225 230 235 240
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
245 250 255
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
260 265 270
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
275 280 285
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
290 295 300
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
305 310 315 320
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
325 330 335
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
340 345 350
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu
355 360 365
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
370 375 380
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
385 390 395 400
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
405 410 415
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
420 425 430
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
435 440 445
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
450 455 460
<210> 4
<211> 1380
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gacattgtga tgacccaatc tccagactct ttggctgtgt ctctagggga gagggccacc 60
atcaactgca agtccagtca gagcctttta tattctagaa atcaaaagaa ctacttggcc 120
tggtatcagc agaaaccagg acagccaccc aaactcctca tcttttggga tagcactagg 180
gaatctgggg tacctgatag gttcagtggc agtgggtttg ggacagactt caccctcacc 240
attagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttttagctat 300
ccgctcacgt tcggacaagg gaccaaggtg gaaataaaag gaggcggagg atctggcggc 360
ggaggaagtg gcggaggggg atctggggga ggcggaagcc aggtgcaact agtgcagtcc 420
ggcgccgaag tgaagaaacc cggtgcttcc gtgaaagtca gctgtaaaac tagtagatac 480
accttcactg aatacaccat acactgggtt agacaggccc ctggccaaag gctggagtgg 540
ataggaggta ttaatcctaa caatggtatt cctaactaca accagaagtt caagggccgg 600
gtcaccatca ccgtagacac ctctgccagc accgcctaca tggaactgtc cagcctgcgc 660
tccgaggaca ctgcagtctg gggtcaagga acccttgtca ccgtctcctc aaccacgacg 720
ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 780
ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 840
tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 900
gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 960
tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 1020
gaagaagaag gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg 1080
tacaagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1140
gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1200
aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1260
gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1320
ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1380
<210> 5
<211> 1440
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
gacattgtga tgacccaatc tccagactct ttggctgtgt ctctagggga gagggccacc 120
atcaactgca agtccagtca gagcctttta tattctagaa atcaaaagaa ctacttggcc 180
tggtatcagc agaaaccagg acagccaccc aaactcctca tcttttggga tagcactagg 240
gaatctgggg tacctgatag gttcagtggc agtgggtttg ggacagactt caccctcacc 300
attagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttttagctat 360
ccgctcacgt tcggacaagg gaccaaggtg gaaataaaag gaggcggagg atctggcggc 420
ggaggaagtg gcggaggggg atctggggga ggcggaagcc aggtgcaact agtgcagtcc 480
ggcgccgaag tgaagaaacc cggtgcttcc gtgaaagtca gctgtaaaac tagtagatac 540
accttcactg aatacaccat acactgggtt agacaggccc ctggccaaag gctggagtgg 600
ataggaggta ttaatcctaa caatggtatt cctaactaca accagaagtt caagggccgg 660
gtcaccatca ccgtagacac ctctgccagc accgcctaca tggaactgtc cagcctgcgc 720
tccgaggaca ctgcagtctg gggtcaagga acccttgtca ccgtctcctc aaccacgacg 780
ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 840
ccagaggcgt gccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 900
tgtgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 960
gttatcaccc tttactgcaa acggggcaga aagaaactcc tgtatatatt caaacaacca 1020
tttatgagac cagtacaaac tactcaagag gaagatggct gtagctgccg atttccagaa 1080
gaagaagaag gaggatgtga actgagagtg aagttcagca ggagcgcaga cgcccccgcg 1140
tacaagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 1200
gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gagaaggaag 1260
aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1320
gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1380
ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1440
<210> 6
<211> 45
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 7
<211> 135
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 8
<211> 24
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 9
<211> 72
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
accctttact gc 72
<210> 10
<211> 42
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 11
<211> 126
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 12
<211> 112
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 12
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 13
<211> 336
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 14
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro
20
<210> 15
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gccctgcctg tgacagccct gctgctgcct ctggctctgc tgctgcatgc cgctagaccc 60
<210> 16
<211> 480
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 16
Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His
1 5 10 15
Ala Ala Arg Pro Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala
20 25 30
Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser
35 40 45
Leu Leu Tyr Ser Arg Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln
50 55 60
Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Phe Trp Asp Ser Thr Arg
65 70 75 80
Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Phe Gly Thr Asp
85 90 95
Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr
100 105 110
Tyr Cys Gln Gln Tyr Phe Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr
115 120 125
Lys Val Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser
145 150 155 160
Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys
165 170 175
Thr Ser Arg Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp Val Arg Gln
180 185 190
Ala Pro Gly Gln Arg Leu Glu Trp Ile Gly Gly Ile Asn Pro Asn Asn
195 200 205
Gly Ile Pro Asn Tyr Asn Gln Lys Phe Lys Gly Arg Val Thr Ile Thr
210 215 220
Val Asp Thr Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg
225 230 235 240
Ser Glu Asp Thr Ala Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser
245 250 255
Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
260 265 270
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
275 280 285
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
290 295 300
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
305 310 315 320
Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
325 330 335
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
340 345 350
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
355 360 365
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
370 375 380
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
385 390 395 400
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
405 410 415
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
420 425 430
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
435 440 445
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
450 455 460
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
465 470 475 480
Claims (10)
1. a kind of single-chain antibody for targeting FAP, which is characterized in that the single-chain antibody of the targeting FAP includes such as SEQ ID NO:1
Shown in amino acid sequence.
2. the single-chain antibody of targeting FAP as described in claim 1, which is characterized in that the volume of the single-chain antibody of the targeting FAP
Code gene includes the nucleotide sequence as shown in SEQ ID NO:2.
3. a kind of Chimeric antigen receptor T cell for targeting FAP, which is characterized in that the Chimeric antigen receptor CAR- including targeting FAP
FAP, the CAR-FAP include from aminoterminal to c-terminus the sequentially connected targeting single-chain antibody of FAP, extracellular hinge area, across
The amino acid sequence in film area and intracellular signal area, wherein the single-chain antibody of the targeting F AP includes as shown in SEQ ID NO:1
Amino acid sequence.
4. the Chimeric antigen receptor T cell of targeting FAP as claimed in claim 3, which is characterized in that the extracellular hinge area packet
CD8 α hinge area is included, the transmembrane region includes CD8 transmembrane region, and the intracellular signal area includes sequentially connecting from aminoterminal to c-terminus
The 4-1BB signaling zone and CD3 ζ signaling zone connect.
5. the Chimeric antigen receptor T cell of targeting FAP as claimed in claim 4, which is characterized in that the ammonia of the CAR-FAP
Base acid sequence includes the amino acid sequence as shown in SEQ ID NO:3.
6. the Chimeric antigen receptor T cell of targeting FAP as described in claim 3 or 4, which is characterized in that the CAR-FAP's
Encoding gene includes the nucleotide sequence as shown in SEQ ID NO:4.
7. a kind of recombinant viral vector, which is characterized in that the recombinant viral vector includes as described in claim any one of 3-6
Targeting FAP Chimeric antigen receptor T cell CAR-FAP encoding gene.
8. a kind of host cell, which is characterized in that the host cell includes recombinant viral vector as claimed in claim 7.
9. a kind of preparation method for the Chimeric antigen receptor T cell for targeting FAP characterized by comprising
(1) encoding gene of the Chimeric antigen receptor CAR-FAP of targeting FAP is provided, including sequentially connected from 5 ' ends to 3 ' ends
The encoding gene of signal peptide, the encoding gene of single-chain antibody for targeting FAP, the encoding gene of extracellular hinge area, transmembrane region volume
The encoding gene of code gene and intracellular signal area, wherein the encoding gene of the single-chain antibody of the targeting FAP includes such as SEQ ID
Nucleotide sequence corresponding to amino acid sequence shown in NO:1；
(2) encoding gene of the CAR-FAP is inserted into pWPXLD carrier, obtains pWPXL D-CAR-FAP recombinant plasmid；
(3) it by the pWPXLD-CAR-FAP recombinant plasmid and envelope plasmid, packaging plasmid cotransfection host cell, is recombinated
Slow virus；
(4) recombinant slow virus is transfected into CD3 positive t lymphocytes, the Chimeric antigen receptor T of targeting FAP is obtained through separation
Cell.
10. a kind of target the single-chain antibody of FAP, as described in claim any one of 3-6 as claim 1-2 is described in any item
Or preparation method as claimed in claim 9 targeting FAP obtained Chimeric antigen receptor T cell or such as claim 7 institute
The recombinant viral vector or host cell as claimed in claim 8 stated diagnose and/or treat the drug of malignant tumour in preparation
In application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810515287.XA CN110526979A (en) | 2018-05-25 | 2018-05-25 | Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of FAP |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810515287.XA CN110526979A (en) | 2018-05-25 | 2018-05-25 | Target single-chain antibody, the Chimeric antigen receptor T cell and its preparation method and application of FAP |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110526979A true CN110526979A (en) | 2019-12-03 |
Family
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Cited By (3)
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| CN111574634A (en) * | 2019-12-16 | 2020-08-25 | 四川大学华西医院 | Double-target chimeric antigen receptor simultaneously targeting mesothelin and FAP and application thereof |
| CN112870227A (en) * | 2021-03-09 | 2021-06-01 | 深圳市体内生物医药科技有限公司 | Liver cancer treatment medicine and preparation method and application thereof |
| CN113004423A (en) * | 2021-03-09 | 2021-06-22 | 湖南昭泰生物医药有限公司 | CAR-T cell for specifically targeting and activating hepatic stellate cell and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111574634A (en) * | 2019-12-16 | 2020-08-25 | 四川大学华西医院 | Double-target chimeric antigen receptor simultaneously targeting mesothelin and FAP and application thereof |
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| CN112870227A (en) * | 2021-03-09 | 2021-06-01 | 深圳市体内生物医药科技有限公司 | Liver cancer treatment medicine and preparation method and application thereof |
| CN113004423A (en) * | 2021-03-09 | 2021-06-22 | 湖南昭泰生物医药有限公司 | CAR-T cell for specifically targeting and activating hepatic stellate cell and preparation method and application thereof |
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