CN109929039A - Chimeric antigen receptor, Lentiviral and its application based on CD276 antibody - Google Patents
Chimeric antigen receptor, Lentiviral and its application based on CD276 antibody Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, more particularly to a kind of Chimeric antigen receptor based on CD276 antibody, Lentiviral and its application, the Chimeric antigen receptor is CD276-28z-CAR Chimeric antigen receptor, CD276-BBz-CAR or CD276-28BBz-CAR Chimeric antigen receptor, the amino acid sequence of the CD276-28z-CAR Chimeric antigen receptor is as shown in SEQ ID NO.1, the amino acid sequence of the CD276-BBz-CAR Chimeric antigen receptor is as shown in SEQ ID NO.2, the amino acid sequence of the CD276-28BBz-CAR Chimeric antigen receptor is as shown in SEQ ID NO.3.The Chimeric antigen receptor that the present invention constructs uses the stronger scFv sequence construct of affinity, and resulting CAR-T cell being capable of effective killing tumor cell.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of Chimeric antigen receptor based on CD276 antibody, slow virus
Expression vector and its application.
Background technique
Chimeric antigen receptor (CAR) is a kind of artificial synthesized structure.Using transgenic technology, CAR sequence is transduceed into T
In cell, the CAR-T cell for identifying special target spot, killing tumor cell can be generated, CAR-T cell display goes out good anti-swollen
Tumor activity.
CAR is mainly made of 3 parts: extracellular cog region, signal transduction area and intracellular signal area.Wherein, extracellular cog region
Determine the specificity of CAR-T cell killing, often by scFv Sequence composition, the specificity and compatibility of scFv sequence are determined
The safety and validity of CAR-T cell therapy.The stronger special scFv of selective affinity helps to improve CAR-T cell therapy
Effect.Costimulatory signal molecule (CD28 and/or 41BB) intracellular is most important to maintenance CAR-T cell killing activity.
CD276 is the membranous antigen of the wide expression in tumour, is preferable CAR-T cell therapy target spot.But needle at present
It is less to the Therapy study of the target spot, it does not know especially and takes which kind of scFv that can obtain optimum therapeuticing effect.
Summary of the invention
Invention broadly provides a kind of Chimeric antigen receptor based on CD276 antibody, Lentiviral and its answer
With the Chimeric antigen receptor of building uses the stronger scFv sequence construct of affinity, and resulting CAR-T cell can be effective
Killing tumor cell.Its technical solution is as follows:
A kind of Chimeric antigen receptor based on CD276 antibody, the Chimeric antigen receptor are CD276-28z-CAR inosculating antibody
Original receptor, CD276-BBz-CAR or CD276-28BBz-CAR Chimeric antigen receptor, the CD276-28z-CAR chimeric antigen by
The amino acid sequence of body is as shown in SEQ ID NO.1, and the amino acid sequence of the CD276-BBz-CAR Chimeric antigen receptor is such as
Shown in SEQ ID NO.2, the amino acid sequence of the CD276-28BBz-CAR Chimeric antigen receptor is as shown in SEQ ID NO.3.
Preferably, the CD276-28z-CAR Chimeric antigen receptor by people's CD8a molecular signal peptide, CD276 single-chain antibody,
People's CD8a molecular flexibility segment, human CD 28 molecule transmembrane region and intracellular region, people's CD3z molecule intracellular region are in series;
Wherein, for the amino acid sequence of the people CD8a molecular signal peptide as shown in SEQ ID NO.4, the CD276 is single-stranded
The amino acid sequence of antibody is as shown in SEQ ID NO.5, the amino acid sequence of human CD 28 molecule transmembrane region and intracellular region such as SEQ
Shown in ID NO.6, the amino acid sequence of people's CD3z molecule intracellular region is as shown in SEQ ID NO.7.
Preferably, the CD276-BBz-CAR Chimeric antigen receptor by people's CD8a molecular signal peptide, CD276 single-chain antibody,
People's CD8a molecular flexibility segment and transmembrane region, people's 41BB molecule intracellular region, people's CD3z molecule intracellular region are in series;
Wherein, the amino acid sequence of people CD8a molecular signal peptide is as shown in SEQ ID NO.4, the ammonia of CD276 single-chain antibody
Base acid sequence is as shown in SEQ ID NO.5, the amino acid sequence of transmembrane region and people's 41BB molecule intracellular region such as SEQ ID NO.8 institute
Show, the amino acid sequence of people's CD3z molecule intracellular region is as shown in SEQ ID NO.7.
Preferably, the CD276-28BBz-CAR Chimeric antigen receptor is single-stranded anti-by people's CD8a molecular signal peptide, CD276
Body, people's CD8a molecular flexibility segment are connected structure with transmembrane region, people's CD28 and 41BB molecule intracellular region, people's CD3z molecule intracellular region
At;
Wherein, the amino acid sequence of people CD8a molecular signal peptide is as shown in SEQ ID NO.4, the ammonia of CD276 single-chain antibody
Base acid sequence is as shown in SEQ ID NO.5, the amino acid sequence of transmembrane region and CD28 and 41BB intracellular region such as SEQ ID NO.9 institute
Show, the amino acid sequence of people's CD3z molecule intracellular region is as shown in SEQ ID NO.7.
A kind of Lentiviral, the carrier are loaded with above-mentioned Chimeric antigen receptor.
Preferably, the Chimeric antigen receptor is to express in pCDH-EF1-MSC plasmid, forms pCDH-EF1-CAR-
CD276-28z, pCDH-EF1-CAR-CD276-BBz or pCDH-EF1-CAR-CD276-28BBz plasmid.
A kind of slow virus, the slow virus are to be total to above-mentioned Lentiviral and packaging plasmid psPAX2 and pMD2.G
Transfected target cells obtain.
A kind of construction method of CAR-T cell, comprising:
(1) CAR sequent synthesis: building CD276-28z-CAR Chimeric antigen receptor, CD276-BBz-CAR or CD276-
28BBz-CAR Chimeric antigen receptor;
(2) expression vector establishment: Chimeric antigen receptor is expressed in pCDH-EF1-MSC plasmid, forms pCDH-EF1-
CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz or pCDH-EF1-CAR-CD276-28BBz plasmid;
(3) slow virus is packed: the plasmid of step (2) being mixed with packaging plasmid, packaging forms slow virus;
(4) T cell purifying and infection: purified T cell obtains CAR-T cell with slow-virus transfection T cell;
(5) T cell CAR expression efficiency detects: detection T cell surface C AR expression;
(6) CAR-T cell expansion ex vivo: CAR-T cell is expanded.
Above-mentioned Chimeric antigen receptor can be applied in the preparation for identifying the CAR-T cell of special target spot CD276.
Using the above scheme, the invention has the following advantages that
The factors such as antigen presentation intensity and identification affinity of antibody affect CAR-T cell therapy effect.The present invention is with swollen
Highly expressed CD276 is that target spot carries out CAR-T cell therapy to tumor extensively, and the antigen is preferable oncotherapy target as the result is shown
Point.Meanwhile present invention employs affinity enhancing anti-CD276scFv as CAR-T cell recognition area, can be more efficiently
Corresponding antigens positive tumor cell is captured, it is effect improved to facilitate immune cell therapy.By adding stimulus signal (CD28 intracellular
Or 41BB), T cell function can be improved.The CAR-T cell of such transgenosis modification has stronger, more longlasting killing ability,
It is expected to improve oncotherapy effect.
Detailed description of the invention
Fig. 1 is CD276-28z-CAR structural schematic diagram in embodiment 1;
Fig. 2 is CD276-BBz-CAR structural schematic diagram;
Fig. 3 is CD276-28BBz-CAR structural schematic diagram;
Fig. 4 is that Flow cytometry T cell expresses CAR expression figure;
Fig. 5 is CAR-T cell proliferative conditions figure;
Fig. 6 is the expression figure of CD276;
Fig. 7 is CAR-T cell-specific killing tumor cell result figure;
Fig. 8 is that CAR-T cell secretion of cytokines can try hard to;
Fig. 9 is CD276-28z-CAR structural schematic diagram in comparative example 1;
Figure 10 is that the structure in embodiment 1 and comparative example 1 transmits T cell activation signal condition comparison diagram;
Figure 11 is CAR-T cells against tumor cells killing activity comparison diagram in embodiment 1 and comparative example 1.
Specific embodiment
Experimental method in following embodiment is conventional method unless otherwise required, related experiment reagent and material
Material is conventional biochemical reagent and material unless otherwise required.
Embodiment 1
One, CAR structure
1.CD276-28z-CAR structure
As shown in Figure 1, CAR basic structure includes: people's CD8a molecular signal peptide (Leadingsignal), CD276 single-stranded anti-
Body (scFv), people's CD8a molecular flexibility segment (CD8Hinge), human CD 28 molecule transmembrane region and intracellular region (CD28), people CD3z points
Sub- intracellular region (CD3z).The amino acid sequence of the CD276-28z-CAR structure is as shown in SEQ ID NO.1.
Wherein, the amino acid sequence of signal peptide (SP) sequence are as follows:
MALPVTALLLPLALLLHAARPGS(SEQ ID NO.4)。
Enhanced CD276 single-chain antibody (CD276-scFv) sequence of affinity:
QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDGSTQYNEKFKGKA
TLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATL
SVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYY
CQNGHSFPLTFGQGTKLELKR(SEQ ID NO.5)。
Soft segment and transmembrane region (TM) and CD28 intracellular region (CD28) sequence:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFI
IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO.6)。
People CD3z molecule intracellular region (CD3z) sequence:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM
AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.7)。
2.CD276-BBz-CAR structure
As shown in Fig. 2, it is by people's CD8a molecular signal peptide (Leadingsignal), CD276 single-chain antibody (scFv), people
CD8a molecular flexibility segment and transmembrane region (CD8HingeTM), people's 41BB molecule intracellular region (41BB), people's CD3z molecule intracellular region
(CD3z) in series, the amino acid sequence of CD276-BBz-CAR structure is as shown in SEQ ID NO.2.
Wherein, signal peptide (Leadingsignal) sequence:
MALPVTALLLPLALLLHAARPGS(SEQ ID NO.4)。
Enhanced CD276 single-chain antibody (scFv) sequence of affinity:
QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDGSTQYNEKFKGKA
TLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATL
SVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYY
CQNGHSFPLTFGQGTKLELKR(SEQ ID NO.5)。
Soft segment and transmembrane region (TM) and 41BB intracellular region (41BB) sequence:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKR
GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.8)。
People CD3z molecule intracellular region (CD3z) sequence:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM
AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.7)。
3.CD276-28BBz-CAR structure
As shown in figure 3, CAR basic structure includes: people's CD8a molecular signal peptide (Leadingsignal), CD276 single-stranded anti-
Body (scFv), people's CD8a molecular flexibility segment and transmembrane region (CD8HingeTM), people's CD28 and 41BB molecule intracellular region (CD28-
41BB), people CD3z molecule intracellular region (CD3z).The amino acid sequence of CD276-28BBz-CAR structure such as SEQ ID NO.3 institute
Show.
Wherein,
Signal peptide (Leadingsignal) sequence:
MALPVTALLLPLALLLHAARPGS(SEQ ID NO.4)。
Enhanced CD276 single-chain antibody (scFv) sequence of affinity:
QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDGSTQYNEKFKGKA
TLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATL
SVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYY
CQNGHSFPLTFGQGTKLELKR(SEQ ID NO.5)。
Soft segment and transmembrane region (TM) and CD28 and 41BB intracellular region (CD28-41BB) sequence:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFI
IFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR
FPEEEEGGCEL(SEQ ID NO.9)。
People CD3z molecule intracellular region (CD3z) sequence:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKM
AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.7)。
Two, CAR sequent synthesis and vector construction
CAR coded sequence is synthesized by Shanghai Sangon Biotech Company, obtains DNA sequence dna (the SEQ ID of CD276-28z-CAR structure
NO.12), the DNA sequence dna (SEQ ID NO.13) of CD276-BBz-CAR structure or the DNA sequence dna of CD276-28BBz-CAR structure
(SEQ ID NO.14) is inserted into pCDH-EF1-MSC plasmid by digestion connection, constructs slow virus expression plasmid pCDH-
EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz or pCDH-EF1-CAR-CD276-28BBz.
Three, slow virus is packed
Incasing cells 293T bed board is after 24 hours, by pCDH-EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-
(psPAX2 is mixed BBz or pCDH-EF1-CAR-CD276-28BBz expression plasmid with pMD2.G), utilizes phosphoric acid with packaging plasmid
Calcium transfection reagent carries out 293T cell transfecting.After transfection 48 hours, supernatant is collected, prepares to infect for T cell.
Four, T cell purifying and infection
Human peripheral is after density gradient centrifugation, separating peripheral blood mononuclear cells.T using German Mei Tian Ni company is thin
Born of the same parents' separating kit obtains the CD3 of purifying+The ratio of 1 magnetic bead is added according still further to 1 cell, appropriate CD3/ is added for T cell
CD28 magnetic bead activates 2 days.After 2 days, viral supernatants are added and polybrene (6 μ g/mL) is incubated overnight.Next day, eccentric cleaning T
After cell 3 times, it is added and expands T cell containing the RPMI-1640 culture medium of 200UIL-2 and 5% fetal calf serum.
Five, T cell CAR expression efficiency
After T cell infects 3 days, detected using expression of the flow cytometry to T cell surface C AR.As a result such as
Shown in Fig. 4.Fig. 4 shows CAR the positive expression rate to of about 80%, it was demonstrated that the building of CAR expression plasmid and virus are packed successfully.
Six, CAR-T cell expansion ex vivo
Count 1 × 106A T cell is infected, and every 2 days counting cells after infection, detects CAR-T cell Proliferation feelings
Condition.As shown in figure 5, CAR-T cell can high efficiently multiplying (about 10 times are expanded in 14 days), compared with normal T-cell (UTD), 3
The no significant difference of proliferation of kind CAR-T cell.
Seven, target antigen detects
In order to verify CAR-T Cell killing efficacy, we have detected the CD276 expression on tumour cell first.Such as
Shown in Fig. 6, high expression CD276 on tumour cell TE-1 and KYSE150, and utilize the cell of gene Knockout preparation
(KYSE150-CD276KO) CD276 is not expressed then.
Eight, CAR-T cell can kill CD276 positive tumor cell
After T cell infects 14 days, T cell and target cell are counted, then according to effect target ratio (effector cell: target cell, E:T)
The ratio of 1:1,1:5,1:10, by T cell (effector cell) and CD276 positive target cell (TE-1 and KYSE150) or CD276 yin
Property target cell (KYSE150-CD276KO) altogether be incubated for 16 hours.After being incubated for altogether, small animal imaging network analysis CAR- is utilized
Killing activity (Fig. 7) of the T cell to tumour cell.The results show that CAR-T cell can Efficient killing effect CD276 positive cell, and
Influence very little to CD276 negative cells illustrates the killing activity that there is CAR-T cell antigen to rely on.
Nine, the research of CAR-T cell secretion of cytokines
In order to further verify the killing ability of the special CAR-T cell of CD276, we analyze Antigen-activated CAR-T
The ability of cell secretion of cytokines.By T cell (UTD, CD276-28z, CD276-BBz or CD276-28BBz) according to E:T=
After the ratio of 1:1 and target cell are incubated for 24 hours, interferon (IFN) and proleulzin (IL-2) in ELISA detection supernatant are utilized
Concentration.As shown in figure 8, CAR-T cell contacted with antigen positive tumour cell after can a large amount of secrete cytokines, and and antigen
Negative cells are not apparent from up-regulation cellular elements surface after being incubated for, illustrate that CAR-T cell could effectively live after specific antigen stimulation
Change.
Comparative example 1
Construct huCD276-CAR structure, the structure the difference from embodiment 1 is that, using humanization CD276 single-chain antibody
For target spot, remaining fragment sequence is identical, and huCD276-CAR structural schematic diagram is as shown in Figure 9.The ammonia of huCD276-CAR structure
Base acid sequence is as shown in SEQ ID NO.10, the wherein amino acid sequence of humanization CD276 single-chain antibody such as SEQ ID NO.11
It is shown.
Interpretation of result
The people that will be constructed in the high-affinity CD276-28z-CAR (CD276-CAR) and comparative example 1 that are constructed in embodiment 1
Source (huCD276-CAR) CAR transduces in Jurkat-NFAT_luc2 cell, then incubates altogether with antigen positive or negative cells
It educates 24 hours.CAR signal transduction intensity can be determined by fluorescence intensity.CD276-28z-CAR can be more as the result is shown
It transmits well T cell activation signal (Figure 10), 1 structure of embodiment is prompted stronger can must to activate T cell.
The people that will be constructed in the high-affinity CD276-28z-CAR (CD276-CAR) and comparative example 1 that are constructed in embodiment 1
After source (huCD276) antibody CAR-T cell is incubated for 24 hours with target cell respectively, the activity of target cell is detected.The results show that
High-affinity CAR-T cell CD276-28z-CAR has better tumor cytotoxicity active (Figure 11), prompts 1 knot of embodiment
Structure can effectively improve the anti-tumor activity of CAR-T cell.
It will be apparent to those skilled in the art that can make various other according to the above description of the technical scheme and ideas
Corresponding change and deformation, and all these changes and deformation all should belong to the protection scope of the claims in the present invention
Within.
Sequence table
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<120>Chimeric antigen receptor, Lentiviral and its application based on CD276 antibody
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Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
485 490 495
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
500 505 510
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
515 520 525
Leu Pro Pro Arg
530
<210> 4
<211> 23
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser
20
<210> 5
<211> 241
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Asp Ile Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Trp Ile Phe Pro Gly Asp Gly Ser Thr Gln Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Arg Gln Thr Thr Ala Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
130 135 140
Ser Val Ser Pro Gly Glu Arg Val Thr Leu Ser Cys Arg Ala Ser Gln
145 150 155 160
Ser Ile Ser Asp Tyr Leu Tyr Trp Tyr Gln Gln Lys Ser His Glu Ser
165 170 175
Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro
180 185 190
Ala Arg Phe Ser Gly Ser Gly Ser Gly Ser Glu Phe Thr Leu Thr Ile
195 200 205
Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly
210 215 220
His Ser Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys
225 230 235 240
Arg
<210> 6
<211> 113
<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 Phe Trp Val
35 40 45
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
50 55 60
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
65 70 75 80
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
85 90 95
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
100 105 110
Ser
<210> 7
<211> 113
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 7
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 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 Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110
Arg
<210> 8
<211> 108
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
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 Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
65 70 75 80
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
85 90 95
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
100 105
<210> 9
<211> 155
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 9
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 Phe Trp Val
35 40 45
Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr
50 55 60
Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu
65 70 75 80
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
85 90 95
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
100 105 110
Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
115 120 125
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
130 135 140
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
145 150 155
<210> 10
<211> 496
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
20 25 30
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
35 40 45
Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro
50 55 60
Gly Lys Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser Ala
65 70 75 80
Ile Tyr Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Arg Glu Asn Ile Tyr Tyr
115 120 125
Gly Ser Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser
130 135 140
Ser Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser
145 150 155 160
Thr Lys Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala
165 170 175
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val
180 185 190
Asp Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
195 200 205
Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg
210 215 220
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
225 230 235 240
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn
245 250 255
Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr
260 265 270
Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln
275 280 285
Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala
290 295 300
Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu Val
305 310 315 320
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
325 330 335
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser
340 345 350
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
355 360 365
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg
370 375 380
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
385 390 395 400
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
405 410 415
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
420 425 430
Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
435 440 445
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
450 455 460
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
465 470 475 480
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490 495
<210> 11
<211> 247
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 11
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser Arg Leu Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Ser Thr Ser Gly Ser
115 120 125
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Ile Gln Leu
130 135 140
Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr
145 150 155 160
Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala Trp Tyr
165 170 175
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
180 185 190
Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
195 200 205
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala
210 215 220
Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe Gly Gln
225 230 235 240
Gly Thr Lys Leu Glu Ile Lys
245
<210> 12
<211> 1470
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
atggccctgc ccgtgaccgc cctgctgctg ccactggccc tgctgctgca tgccgctaga 60
cctggcagcc aggtgcagct ggtgcagtca ggagctgaag tggtgaaacc tggcgcctct 120
gtgaagctga gttgtaagac atctggctat acattcacta attatgatat taattgggtg 180
agacagagac ctggacaggg actggagtgg attggctgga tctttccagg agacggctct 240
acacagtata atgaaaagtt caagggcaaa gctacactga caaccgacac cagcaccagc 300
accgcctaca tggagctgtc cagcctgagg tccgaggata ccgccgtgta cttctgcgct 360
aggcagacca ccgccacctg gttcgcctac tggggccagg gcaccctggt gaccgtgagc 420
agcggcggcg gcggaagcgg cggcggcggc agcggcggcg ggggctccga gatcgtgatg 480
acccagtccc ccgccaccct gagcgtgagc cctggcgaga gggtgaccct gagctgcaga 540
gcatctcaga gcatctccga ctacctgtac tggtaccagc agaagagcca cgaaagcccc 600
agactgctga tcaagtacgc cagccagagc atcagcggca tccctgccag gttctccggc 660
agcggctctg gcagcgagtt caccctgacc attaatagcg tggaaccaga agatgttgga 720
gtgtattatt gtcagaatgg acactctttt ccactgacat ttggacaggg cacaaaactg 780
gagctgaaaa gaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatttt tgggtgctgg tggtggttgg tggagtcctg 960
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc cagagtgaag 1140
ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1200
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260
gagatggggg gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320
cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380
ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440
gcccttcaca tgcaggccct gccccctcgc 1470
<210> 13
<211> 1455
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
atggccctgc ccgtgaccgc cctgctgctg ccactggccc tgctgctgca tgccgctaga 60
cctggcagcc aggtgcagct ggtgcagtca ggagctgaag tggtgaaacc tggcgcctct 120
gtgaagctga gttgtaagac atctggctat acattcacta attatgatat taattgggtg 180
agacagagac ctggacaggg actggagtgg attggctgga tctttccagg agacggctct 240
acacagtata atgaaaagtt caagggcaaa gctacactga caaccgacac cagcaccagc 300
accgcctaca tggagctgtc cagcctgagg tccgaggata ccgccgtgta cttctgcgct 360
aggcagacca ccgccacctg gttcgcctac tggggccagg gcaccctggt gaccgtgagc 420
agcggcggcg gcggaagcgg cggcggcggc agcggcggcg ggggctccga gatcgtgatg 480
acccagtccc ccgccaccct gagcgtgagc cctggcgaga gggtgaccct gagctgcaga 540
gcatctcaga gcatctccga ctacctgtac tggtaccagc agaagagcca cgaaagcccc 600
agactgctga tcaagtacgc cagccagagc atcagcggca tccctgccag gttctccggc 660
agcggctctg gcagcgagtt caccctgacc attaatagcg tggaaccaga agatgttgga 720
gtgtattatt gtcagaatgg acactctttt ccactgacat ttggacaggg cacaaaactg 780
gagctgaaaa gaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 960
ggggtccttc tcctgtcact ggttatcacc aaacggggca gaaagaaact cctgtatata 1020
ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 1080
cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca 1140
gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1200
agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1260
ccgcagagaa ggaagaaccc tcaggaaggc ctgtacaatg aactgcagaa agataagatg 1320
gcggaggcct acagtgagat tgggatgaaa ggcgagcgcc ggaggggcaa ggggcacgat 1380
ggcctttacc agggtctcag tacagccacc aaggacacct acgacgccct tcacatgcag 1440
gccctgcccc ctcgc 1455
<210> 14
<211> 1596
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
atggccctgc ccgtgaccgc cctgctgctg ccactggccc tgctgctgca tgccgctaga 60
cctggcagcc aggtgcagct ggtgcagtca ggagctgaag tggtgaaacc tggcgcctct 120
gtgaagctga gttgtaagac atctggctat acattcacta attatgatat taattgggtg 180
agacagagac ctggacaggg actggagtgg attggctgga tctttccagg agacggctct 240
acacagtata atgaaaagtt caagggcaaa gctacactga caaccgacac cagcaccagc 300
accgcctaca tggagctgtc cagcctgagg tccgaggata ccgccgtgta cttctgcgct 360
aggcagacca ccgccacctg gttcgcctac tggggccagg gcaccctggt gaccgtgagc 420
agcggcggcg gcggaagcgg cggcggcggc agcggcggcg ggggctccga gatcgtgatg 480
acccagtccc ccgccaccct gagcgtgagc cctggcgaga gggtgaccct gagctgcaga 540
gcatctcaga gcatctccga ctacctgtac tggtaccagc agaagagcca cgaaagcccc 600
agactgctga tcaagtacgc cagccagagc atcagcggca tccctgccag gttctccggc 660
agcggctctg gcagcgagtt caccctgacc attaatagcg tggaaccaga agatgttgga 720
gtgtattatt gtcagaatgg acactctttt ccactgacat ttggacaggg cacaaaactg 780
gagctgaaaa gaaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 840
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 900
acgagggggc tggacttcgc ctgtgatttt tgggtgctgg tggtggttgg tggagtcctg 960
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 1020
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 1080
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc 1140
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 1200
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 1260
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 1320
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 1380
gaccctgaga tggggggaaa gccgcagaga aggaagaacc ctcaggaagg cctgtacaat 1440
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1500
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1560
tacgacgccc ttcacatgca ggccctgccc cctcgc 1596
Claims (9)
1. a kind of Chimeric antigen receptor based on CD276 antibody, it is characterised in that: the Chimeric antigen receptor is CD276-28z-
CAR Chimeric antigen receptor, CD276-BBz-CAR or CD276-28BBz-CAR Chimeric antigen receptor, the CD276-28z-CAR
The amino acid sequence of Chimeric antigen receptor is as shown in SEQ ID NO.1, the amino of the CD276-BBz-CAR Chimeric antigen receptor
Acid sequence is as shown in SEQ ID NO.2, the amino acid sequence of the CD276-28BBz-CAR Chimeric antigen receptor such as SEQ ID
Shown in NO.3.
2. Chimeric antigen receptor according to claim 1, it is characterised in that: the CD276-28z-CAR chimeric antigen by
Body by people's CD8a molecular signal peptide, CD276 single-chain antibody, people's CD8a molecular flexibility segment, human CD 28 molecule transmembrane region with it is intracellular
Area, people's CD3z molecule intracellular region are in series;
Wherein, the amino acid sequence of the people CD8a molecular signal peptide is as shown in SEQ ID NO.4, the CD276 single-chain antibody
Amino acid sequence as shown in SEQ ID NO.5, the amino acid sequence of human CD 28 molecule transmembrane region and intracellular region such as SEQ ID
Shown in NO.6, the amino acid sequence of people's CD3z molecule intracellular region is as shown in SEQ ID NO.7.
3. Chimeric antigen receptor according to claim 1, it is characterised in that: the CD276-BBz-CAR chimeric antigen by
Body is intracellular by people's CD8a molecular signal peptide, CD276 single-chain antibody, people's CD8a molecular flexibility segment and transmembrane region, people's 41BB molecule
Area, people's CD3z molecule intracellular region are in series;
Wherein, the amino acid sequence of people CD8a molecular signal peptide is as shown in SEQ ID NO.4, the amino acid of CD276 single-chain antibody
Sequence as shown in SEQ ID NO.5, the amino acid sequence of transmembrane region and people's 41BB molecule intracellular region as shown in SEQ ID NO.8,
The amino acid sequence of people's CD3z molecule intracellular region is as shown in SEQ ID NO.7.
4. Chimeric antigen receptor according to claim 1, it is characterised in that: the CD276-28BBz-CAR chimeric antigen
Receptor is by people's CD8a molecular signal peptide, CD276 single-chain antibody, people's CD8a molecular flexibility segment and transmembrane region, people CD28 and 41BB
Molecule intracellular region, people's CD3z molecule intracellular region are in series;
Wherein, the amino acid sequence of people CD8a molecular signal peptide is as shown in SEQ ID NO.4, the amino acid of CD276 single-chain antibody
Sequence as shown in SEQ ID NO.5, the amino acid sequence of transmembrane region and CD28 and 41BB intracellular region as shown in SEQ ID NO.9,
The amino acid sequence of people's CD3z molecule intracellular region is as shown in SEQ ID NO.7.
5. a kind of Lentiviral, it is characterised in that: the carrier is loaded with Chimeric antigen receptor described in claim 1.
6. Lentiviral according to claim 5, it is characterised in that: the Chimeric antigen receptor is in pCDH-
It is expressed in EF1-MSC plasmid, forms pCDH-EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz or pCDH-
EF1-CAR-CD276-28BBz plasmid.
7. a kind of slow virus, it is characterised in that: the slow virus is by the Lentiviral and packet in claim 5 or 6
Dress plasmid psPAX2 and pMD2.G cotransfection target cell obtains.
8. a kind of construction method of CAR-T cell, it is characterised in that: include:
(1) CAR sequent synthesis: building CD276-28z-CAR Chimeric antigen receptor, CD276-BBz-CAR or CD276-28BBz-
CAR Chimeric antigen receptor;
(2) expression vector establishment: Chimeric antigen receptor is expressed in pCDH-EF1-MSC plasmid, forms pCDH-EF1-CAR-
CD276-28z, pCDH-EF1-CAR-CD276-BBz or pCDH-EF1-CAR-CD276-28BBz plasmid;
(3) slow virus is packed: the plasmid of step (2) being mixed with packaging plasmid, packaging forms slow virus;
(4) T cell purifying and infection: purified T cell obtains CAR-T cell with slow-virus transfection T cell;
(5) T cell CAR expression efficiency detects: detection T cell surface C AR expression;
(6) CAR-T cell expansion ex vivo: CAR-T cell is expanded.
9. the described in any item Chimeric antigen receptors of claim 1-4 are in the CAR-T cell that preparation identifies special target spot CD276
Application.
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CN114763388A (en) * | 2021-01-13 | 2022-07-19 | 博生吉医药科技(苏州)有限公司 | B7-H3-targeted CAR-T cells and application thereof in treatment of acute myeloid leukemia |
CN114763382A (en) * | 2021-01-13 | 2022-07-19 | 博生吉医药科技(苏州)有限公司 | Monoclonal antibody targeting human CD276 and application thereof |
WO2023051663A1 (en) * | 2021-09-30 | 2023-04-06 | 百奥泰生物制药股份有限公司 | Anti-b7-h3 antibody and application thereof |
WO2023060922A1 (en) * | 2021-10-11 | 2023-04-20 | 上海恒润达生生物科技股份有限公司 | Antibody specifically binding to cd276 or antigen-binding fragment thereof, preparation method therefor, and application thereof |
CN116143943A (en) * | 2022-10-31 | 2023-05-23 | 济南泰和医药科技有限公司 | Targeting BAFFR chimeric antigen receptor, CAR-T cell and application |
CN116239699A (en) * | 2022-11-10 | 2023-06-09 | 汕头普罗凯融生物医药科技有限公司 | Chimeric antigen receptor targeting CD276 and application thereof |
WO2024037627A1 (en) * | 2022-08-19 | 2024-02-22 | 盛禾(中国)生物制药有限公司 | Bispecific antibody and use thereof |
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