CN109609533A - The building of CAR Lentiviral and its application based on humanization CD276 antibody - Google Patents

The building of CAR Lentiviral and its application based on humanization CD276 antibody Download PDF

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CN109609533A
CN109609533A CN201711441809.8A CN201711441809A CN109609533A CN 109609533 A CN109609533 A CN 109609533A CN 201711441809 A CN201711441809 A CN 201711441809A CN 109609533 A CN109609533 A CN 109609533A
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CN109609533B (en
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李峰
张毅
张震
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Saidete Biopharmaceutical Co ltd
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First Affiliated Hospital of Zhengzhou University
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Abstract

The present invention relates to CAR Lentiviral technical fields, it is based especially on the building of CAR Lentiviral and its application of humanization CD276 antibody, by constructing the CAR sequence comprising humanization CD276 specific single-chain antibody (scFv), CD8 signal transduction segment and CD28 or 41BB stimulus signal molecule, it is prepared for CAR (the CD276-28z-CAR or CD276-BBz-CAR) expression plasmid and CAR-T cell of targeting CD276.Present invention employs the anti-CD276scFv of humanization as CAR-T cell recognition area, it is possible to prevente effectively from immunological rejection occurs, facilitates therapeutic effect improvement.By adding stimulus signal (CD28 or 41BB) intracellular, T cell function can be improved.The CAR-T cell of such transgenosis modification has stronger, more longlasting killing ability.

Description

The building of CAR Lentiviral and its application based on humanization CD276 antibody
Technical field
The present invention relates to the technical field of CAR Lentiviral, specific field is based on humanization CD276 antibody The building of CAR Lentiviral.
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 is in hematological system tumor Good therapeutic effect is achieved, but ineffective in solid tumor.
The technical problem underlying that CAR-T cell therapy solid tumor faces includes:
1, whether the expression of target antigen is extensive；2, whether CAR sequence has more strongly immunogenic, leads to immunological rejection； 3, whether CAR can effectively pass to T cell for identification signal.
Summary of the invention
The purpose of the present invention is to provide the CAR Lentiviral building based on humanization CD276 antibody and its answer With to solve the problems, such as CAR-T cell therapy in the prior art.
CD276 is the wide expression in tumour, is preferable CAR-T cell therapy target spot.The present invention passes through building The CAR of humanization CD276 specific single-chain antibody (scFv), CD8 signal transduction segment and CD28 or 41BB stimulus signal molecule Sequence is prepared for CAR (the CD276-28z-CAR or CD276-BBz-CAR) expression plasmid and CAR-T cell of targeting CD276.
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, but many scFv sequences are mouse source, meeting Stronger immunological rejection is caused, causes CAR-T cell therapy invalid.And the present invention has selected high-affinity to identify CD276 Humanization modified scFv sequence (anti-CD276scFv), can either effectively avoid immunological rejection, and being capable of efficient identification Target cell.Intracellular signal area is also the important feature for determining CAR-T cell therapy effect, by the way that costimulatory signal molecule is added (CD28 or 41BB) guarantees that CAR-T cell has stronger killing ability and amplification ability.In addition, the structure in signal transduction area Selection also influences CAR-T cell function.Guarantee there is preferable combination between extracellular cog region and signaling zone using CD8 hinge area, It ensure that the function of CAR-T cell.
To improve CAR-T cell therapy effect, for existing CAR fault of construction, the present invention carries out 3 primary structures Optimization, technical solution are specific as follows:
CAR Lentiviral construction method based on humanization CD276 antibody, comprising the following steps: (1) CAR is tied Structure optimization；(2) CAR sequent synthesis and vector construction；
Wherein, the CAR structure after step (1) optimization is CD276-28z-CAR structure or CD276-BBz-CAR structure； CD276-28z-CAR coding protein sequence is as shown in SEQ ID No:1；CD276-BBz-CAR coding protein sequence such as SEQ ID Shown in No:7.
CAR Lentiviral construction method of the present invention based on humanization CD276 antibody, feature exist It include that people's CD8a molecular signal peptide, humanization CD276 single-chain antibody, people's CD8a molecule are soft in: the CD276-28z-CAR structure Property segment, human CD 28 molecule transmembrane region and intracellular region and people's CD3z molecule intracellular region；
Wherein, people CD8a molecular signal peptide sequence is as shown in SEQ ID No:2；Humanization CD276 single chain antibody sequence is such as Shown in SEQ ID No:3；People's CD8a molecular flexibility fragment sequence is as shown in SEQ ID No:4；Human CD 28 molecule transmembrane region and born of the same parents Interior region sequence is as shown in SEQ ID No:5；People's CD3z molecule region sequence intracellular is as shown in SEQ ID No:6.
CAR Lentiviral construction method of the present invention based on humanization CD276 antibody, feature exist It include that people's CD8a molecular signal peptide, humanization CD276 single-chain antibody, people's CD8a molecule are soft in: the CD276-BBz-CAR structure Property segment and transmembrane region, people's 41BB molecule intracellular region and people's CD3z molecule intracellular region；
Wherein, people CD8a molecular signal peptide sequence is as shown in SEQ ID No:8；Humanization CD276 single-chain antibody such as SEQ Shown in ID No:9；People's CD8a molecular flexibility segment and transmembrane domain are as shown in SEQ ID No:10；People's 41BB molecule intracellular region Sequence is as shown in SEQ ID No:11；People's CD3z molecule region sequence intracellular is as shown in SEQ ID No:12.
CAR Lentiviral construction method of the present invention based on humanization CD276 antibody, feature exist In the step (2) specifically includes: the DNA sequence dna of acquisition is inserted into pCDH- by digestion connection by synthesis CAR coded sequence In EF1-MSC plasmid, slow virus expression plasmid pCDH-EF1-CAR-CD276-28z or pCDH-EF1-CAR-CD276- is constructed BBz。
The CAR based on humanization CD276 antibody that CAR Lentiviral construction method of the present invention obtains is slow Virus expression carrier.
CAR-T cell is prepared using the CAR Lentiviral of the present invention based on humanization CD276 antibody, Its method is the following steps are included: (1) slow virus is packed；(2) T cell purifying and infection；(3) T cell CAR expression efficiency detects； (4) CAR-T cell expansion ex vivo；
Wherein, step (1) is specifically, incasing cells 293T bed board after 24 hours, by pCDH-EF1-CAR-CD276-28z Or pCDH-EF1-CAR-CD276-BBz expression plasmid is mixed with packaging plasmid, carries out 293T cell using calcium phosphate transfection reagent Transfection；After transfection 48 hours, supernatant is collected, prepares to infect for T cell.
The method of the present invention for preparing CAR-T cell, step (2) specifically, human peripheral through density gradient from After the heart, separating peripheral blood mononuclear cells；The CD3 of purifying is obtained using T cell separating kit+T cell, it is thin according still further to 2 The ratio of 1 magnetic bead is added in born of the same parents, and appropriate CD3/CD28 magnetic bead is added and activates 2 days；After 2 days, viral supernatants and polybrene is added It is incubated overnight；After eccentric cleaning T cell 3 times, the RPMI1640 culture of IL-2 containing 1000U and 5% fetal calf serum is added in next day Base expands T cell.
The method of the present invention for preparing CAR-T cell, step (3) utilize stream specifically, after T cell infection 3 days Formula cell art detects the expression of T cell surface C AR.
The method of the present invention for preparing CAR-T cell, step (4) is specifically, count 1 × 106A T cell carries out Infection, and after infection 3,7,10 and 14 days when counted, detect CAR-T cell proliferative conditions.
Compared with prior art, the beneficial effects of the present invention are: antigen presentation intensity and CAR-T cell therapy effect are close It is related.The present invention using tumour extensively highly expressed CD276 as target spot carry out CAR-T cell therapy, as the result is shown the antigen be compared with Good cancer target.It, can be in addition, present invention employs the anti-CD276scFv of humanization as CAR-T cell recognition area Immunological rejection effectively is avoided, facilitates therapeutic effect improvement.It, can by adding stimulus signal (CD28 or 41BB) intracellular Improve T cell function.The CAR-T cell of such transgenosis modification has stronger, more longlasting killing ability, is expected to improve tumour Therapeutic effect.
Detailed description of the invention
Fig. 1 is CD276-28z-CAR structural schematic diagram；
Fig. 2 is CD276-BBz-CAR structural schematic diagram；
Fig. 3 is that Flow cytometry T cell expresses CAR expression；
Fig. 4 is CAR-T cell proliferative conditions；
Fig. 5 illustrates that CAR-T cell being capable of specific killing CD276 positive cell；
Fig. 6 illustrates more preferable using CD276 as the tumor-killing effect of the CAR-T cell of target spot；
Fig. 7 illustrates that CAR-T cell can control tumour growth.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other Embodiment shall fall within the protection scope of the present invention.
1. having been carried out to 3 primary structures excellent to improve CAR-T cell therapy effect for existing CAR fault of construction Change.Specifically include CD276-28z-CAR structure and CD276-BBz-CAR structure.
(1) CD276-28z-CAR structure
As shown in Figure 1, CAR basic structure includes: people's CD8a molecular signal peptide (Leading signal), humanization CD276 single-chain antibody (scFv), people's CD8a molecular flexibility segment (CD8Hinge), human CD 28 molecule transmembrane region and intracellular region (CD28), people CD3z molecule intracellular region (CD3z).
CD276-28z-CAR coding protein sequence is as follows: (shown in SEQ ID No:1)
MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSD SSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKP GSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*
Wherein,
People's CD8a molecular signal peptide (Leading signal) sequence: (shown in SEQ ID No:2)
MALPVTALLLPLALLLHAARPGS
Humanization CD276 single-chain antibody (Humanized scFv) sequence: (shown in SEQ ID No:3)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFT ISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQ SPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQYNNYPFTFGQGTKLEIK
People CD8a molecular flexibility segment (CD8Hinge) sequence: (shown in SEQ ID No:4)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD
Human CD 28 molecule transmembrane region and intracellular region (CD28) sequence: (shown in SEQ ID No:5)
FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
People CD3z molecule intracellular region (CD3z) sequence: (shown in SEQ ID No:6)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
(2) CD276-BBz-CAR structure
As shown in Fig. 2, CAR basic structure includes: people's CD8a molecular signal peptide (Leading signal), humanization CD276 single-chain antibody (scFv), people's CD8a molecular flexibility segment and transmembrane region (CD8Hinge TM), people's 41BB molecule intracellular region (41BB), people's CD3z molecule intracellular region (CD3z).
CD276-BBz-CAR coding protein sequence is as follows: (shown in SEQ ID No:7)
MALPVTALLLPLALLLHAARPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSD SSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKP GSGEGSTKGDIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR GLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG KGHDGLYQGLSTATKDTYDALHMQALPPR*
Wherein,
People CD8a molecular signal peptide (Leadingsignal) sequence: (shown in SEQ ID No:8)
MALPVTALLLPLALLLHAARPGS
Humanization CD276 single-chain antibody (Humanized scFv) sequence: (shown in SEQ ID No:9)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFT ISRDNAKNSLYLQMNSLRDEDTAVYYCARGRENIYYGSRLDYWGQGTTVTVSSGSTSGSGKPGSGEGSTKGDIQLTQ SPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDF ATYYCQQYNNYPFTFGQGTKLEIK
People's CD8a molecular flexibility segment and transmembrane region (CD8Hinge TM) sequence: (shown in SEQ ID No:10)
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT
People 41BB molecule intracellular region (41BB) sequence: (shown in SEQ ID No:11)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
People CD3z molecule intracellular region (CD3z) sequence: (shown in SEQ ID No:12)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
2.CAR sequent synthesis and vector construction
CAR coded sequence is synthesized by Shanghai Sangon Biotech Company, and the DNA sequence dna of acquisition is inserted into pCDH- by digestion connection In EF1-MSC plasmid, slow virus expression plasmid pCDH-EF1-CAR-CD276-28z or pCDH-EF1-CAR-CD276- is constructed BBz。
3. slow virus is packed
Incasing cells 293T bed board is after 24 hours, by pCDH-EF1-CAR-CD276-28z or pCDH-EF1-CAR- (psPAX2 is mixed CD276-BBz expression plasmid with pMD2.G), and it is thin to carry out 293T using calcium phosphate transfection reagent with packaging plasmid Dysuria with lower abdominal colic dye.After transfection 48 hours, supernatant is collected, prepares to infect for T cell.
4.T cell purification 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 2 cells, 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, the RPMI1640 culture medium that IL-2 containing 1000U and 5% fetal calf serum is added expands T cell.
5.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 it shows Show that CAR the positive expression rate reaches~60%-70% (such as Fig. 3), it was demonstrated that the building of CAR expression plasmid and virus are packed successfully.
6.CAR-T cell expansion ex vivo
Count 1 × 106A T cell is infected, and after infection 3,7,10 and 14 days when counted, detect CAR-T Cell proliferative conditions.As shown in figure 4, CAR-T cell can high efficiently multiplying (about 50-60 times is expanded in 14 days), wherein CAR- The proliferative capacity of CD276-BBz cell is more preferable.
7.CAR-T Cell killing efficacy
After T cell infects 14 days, count T cell and target cell, and using cell dye (eFluor670) to target cell into Line flag.Then according to effect target ratio (effector cell: target cell, E:T) 1:1, the ratio of 1:5,1:20, by T cell, (effect is thin Born of the same parents) with CD276 high expression target cell (Calu-3, KYSE70 and SW620), CD276 low expression target cell (A549 and H322) or CD276 negative targets (95D) are incubated for 6 hours altogether.After being incubated for altogether, cell is collected by centrifugation, utilizes apoptosis staining kit Cell is marked, then flow cytometry target cell apoptosis situation (such as Fig. 5).The results show that CAR-T cell is to expression The tumour cell of CD276 has very strong killing ability, and the influence very little to CD276 negative cells illustrates the CAR-T cell With very strong specific killing ability.
The special CAR-T cell of 8.CD276, which has, relatively kills by force ability
In order to further verify the killing ability of the special CAR-T cell of CD276, we utilize cell activity real-time detection system System observes killing of the CAR-T cell to target cell A549 (not only expressed CD276 but also expressed Her2).CAR-T cell (CAR- CD276-28z, CAR-CD276-BBz or CAR-Her2-28z) it is added separately in target cell according to the ratio of E:T=1:10, Then the activity of target cell has been carried out being observed continuously (such as Fig. 6) in 5 days.The results show that with the special CAR-T cell phase of Her2 Than the tumor-killing effect that CD276 targets CAR-T cell is more preferable, illustrates that can be improved CAR-T cell as target spot using CD276 controls Therapeutic effect.
9. CAR-T cell function is verified in zoopery
Immunodeficient mouse inoculates 5 × 106A Calu-3 cell injected 5 × 10 through tail vein after 10 days respectively6It is a CAR-T cell or T cell are treated.At treatment 0,3,7,14 days, using small animal living body imaging device, it is big to observe tumour It is small.As shown in fig. 7,7 days (A) after CAR-T cell infusion, gross tumor volume is obviously reduced, and with the extension for the treatment of time, The gross tumor volume of CAR-T cell therapy group is smaller and smaller (B), and CAR-T cell is prompted to have good tumor-killing ability.
From the above experimental results, we know that antigen presentation intensity is closely related with 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.In addition, present invention employs the anti-CD276scFv of humanization as CAR-T cell recognition area, it is possible to prevente effectively from immune row Reprimand occurs, and facilitates therapeutic effect improvement.By adding stimulus signal (CD28 or 41BB) intracellular, T cell function can be improved. The CAR-T cell of such transgenosis modification has stronger, more longlasting killing ability, is expected to improve oncotherapy effect.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding And modification, the scope of the present invention is defined by the appended.
Sequence table
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<120>building of CAR Lentiviral and its application based on humanization CD276 antibody
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Gly Phe Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro
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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> 2
<211> 23
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
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> 3
<211> 247
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
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> 4
<211> 45
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
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> 5
<211> 68
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
20 25 30
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
35 40 45
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
50 55 60
Ala Tyr Arg Ser
65
<210> 6
<211> 113
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
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> 7
<211> 491
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 7
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 Ile Tyr Ile Trp Ala
305 310 315 320
Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
325 330 335
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn
420 425 430
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
450 455 460
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
465 470 475 480
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 8
<211> 23
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 8
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> 9
<211> 247
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 9
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> 10
<211> 66
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
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
65
<210> 11
<211> 42
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 11
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> 12
<211> 113
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 12
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

Claims (9)

1. the CAR Lentiviral construction method based on humanization CD276 antibody, which comprises the following steps: (1) CAR structure optimization；(2) CAR sequent synthesis and vector construction；
Wherein, the CAR structure after step (1) optimization is CD276-28z-CAR structure or CD276-BBz-CAR structure； CD276-28z-CAR coding protein sequence is as shown in SEQ ID No:1；CD276-BBz-CAR coding protein sequence such as SEQ ID Shown in No:7.
2. the CAR Lentiviral construction method according to claim 1 based on humanization CD276 antibody, special Sign is: the CD276-28z-CAR structure includes people's CD8a molecular signal peptide, humanization CD276 single-chain antibody, people CD8a points Sub- soft segment, human CD 28 molecule transmembrane region and intracellular region and people's CD3z molecule intracellular region；
Wherein, people CD8a molecular signal peptide sequence is as shown in SEQ ID No:2；Humanization CD276 single chain antibody sequence such as SEQ Shown in ID No:3；People's CD8a molecular flexibility fragment sequence is as shown in SEQ ID No:4；Human CD 28 molecule transmembrane region and intracellular region Sequence is as shown in SEQ ID No:5；People's CD3z molecule region sequence intracellular is as shown in SEQ ID No:6.
3. the CAR Lentiviral construction method according to claim 1 based on humanization CD276 antibody, special Sign is: the CD276-BBz-CAR structure includes people's CD8a molecular signal peptide, humanization CD276 single-chain antibody, people CD8a points Sub- soft segment and transmembrane region, people's 41BB molecule intracellular region and people's CD3z molecule intracellular region；
Wherein, people CD8a molecular signal peptide sequence is as shown in SEQ ID No:8；Humanization CD276 single-chain antibody such as SEQ ID Shown in No:9；People's CD8a molecular flexibility segment and transmembrane domain are as shown in SEQ ID No:10；People's 41BB molecule intracellular region sequence Column are as shown in SEQ ID No:11；People's CD3z molecule region sequence intracellular is as shown in SEQ ID No:12.
4. the CAR Lentiviral building side according to claim 1 to 3 based on humanization CD276 antibody Method, which is characterized in that the step (2) specifically includes: synthesis CAR coded sequence connects the DNA sequence dna of acquisition by digestion It is inserted into pCDH-EF1-MSC plasmid, constructs slow virus expression plasmid
PCDH-EF1-CAR-CD276-28z or pCDH-EF1-CAR-CD276-BBz.
5. CAR Lentiviral construction method according to claim 4 obtain based on humanization CD276 antibody CAR Lentiviral.
6. the method for preparing CAR-T cell using CAR Lentiviral described in claim 5, which is characterized in that including Following steps: (1) slow virus is packed；(2) T cell purifying and infection；(3) T cell CAR expression efficiency detects；(4) CAR-T is thin Born of the same parents' amplification in vitro；
Wherein, step (1) is specifically, incasing cells 293T bed board after 24 hours, by pCDH-EF1-CAR-CD276-28z or PCDH-EF1-CAR-CD276-BBz expression plasmid is mixed with packaging plasmid, is carried out 293T cell using calcium phosphate transfection reagent and is turned Dye；After transfection 48 hours, supernatant is collected, prepares to infect for T cell.
7. the method according to claim 6 for preparing CAR-T cell, it is characterised in that: step (2) is specifically, people periphery Blood is after density gradient centrifugation, separating peripheral blood mononuclear cells；The CD3 of purifying is obtained using T cell separating kit+T is thin The ratio of 1 magnetic bead is added according still further to 2 cells by born of the same parents, and appropriate CD3/CD28 magnetic bead is added and activates 2 days；After 2 days, virus is added Supernatant and polybrene are incubated overnight；Next day after eccentric cleaning T cell 3 times, is added and contains 1000 U IL-2 and 5% tire ox blood Clear RPMI1640 culture medium expands T cell.
8. the method according to claim 7 for preparing CAR-T cell, it is characterised in that: step (3) is specifically, T cell sense Dye is detected after 3 days using expression of the flow cytometry to T cell surface C AR.
9. the method according to claim 8 for preparing CAR-T cell, it is characterised in that: step (4) specifically, count 1 × 106A T cell is infected, and after infection 3,7,10 and 14 days when counted, detect CAR-T cell proliferative conditions.
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CN114181972A (en) * 2021-11-23 2022-03-15 上海本导基因技术有限公司 Lentiviral vectors suitable for gene therapy of refractory angiogenic eye diseases
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CN111518773A (en) * 2020-05-09 2020-08-11 山东兴瑞生物科技有限公司 CAR-T cell for resisting novel coronavirus S protein, preparation method and application thereof
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CN112816685A (en) * 2020-12-30 2021-05-18 苏州堪赛尔医学检验有限公司 Immunohistochemical immunoenzyme labeling method and kit for immunoenzyme labeling method
CN114437218A (en) * 2021-01-12 2022-05-06 北京门罗生物科技有限公司 Chimeric antigen receptor targeting CD276 and immune cell comprising same
CN114437218B (en) * 2021-01-12 2022-09-30 北京门罗生物科技有限公司 Chimeric antigen receptor targeting CD276 and immune cell comprising same
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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
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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
CN114763381A (en) * 2021-01-13 2022-07-19 博生吉医药科技(苏州)有限公司 B7-H3 chimeric antigen receptor modified T cell and application thereof
WO2023272924A1 (en) * 2021-06-30 2023-01-05 徐州医科大学 Novel fully human antibody for human b7h3, chimeric antigen receptor and uses thereof
CN114181972A (en) * 2021-11-23 2022-03-15 上海本导基因技术有限公司 Lentiviral vectors suitable for gene therapy of refractory angiogenic eye diseases

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