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 PDF

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CN109929039A
CN109929039A CN201910243041.6A CN201910243041A CN109929039A CN 109929039 A CN109929039 A CN 109929039A CN 201910243041 A CN201910243041 A CN 201910243041A CN 109929039 A CN109929039 A CN 109929039A
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李峰
张毅
盛誉乔
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First Affiliated Hospital of Zhengzhou University
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First Affiliated Hospital of Zhengzhou University
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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

Chimeric antigen receptor based on CD276 antibody, lentiviral expression vector and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a chimeric antigen receptor based on a CD276 antibody, a lentivirus expression vector and application thereof.
Background
Chimeric Antigen Receptors (CARs) are an artificially synthesized structure. By utilizing a transgenic technology, the CAR sequence is transferred into the T cell, the CAR-T cell which can recognize a specific target spot and kill tumor cells can be generated, and the CAR-T cell shows good anti-tumor activity.
CAR is mainly composed of 3 parts: an extracellular recognition region, a signal transduction region, and an intracellular signaling region. Among these, the extracellular recognition region determines the specificity of CAR-T cell killing, often consisting of scFv sequences, the specificity and affinity of which determine the safety and efficacy of CAR-T cell therapy. The selection of a specific scFv with stronger affinity helps to improve the therapeutic effect of CAR-T cells. Intracellular costimulatory signaling molecules (CD28 and/or 41BB) are critical to maintain CAR-T cell killing activity.
CD276 is a membrane antigen widely expressed in tumors and is a good target for CAR-T cell therapy. However, few therapeutic studies are currently aimed at this target, and in particular it is unclear which scFv can be used to achieve the best therapeutic effect.
Disclosure of Invention
The invention mainly provides a chimeric antigen receptor based on a CD276 antibody, a lentivirus expression vector and application thereof, wherein the constructed chimeric antigen receptor is constructed by adopting an scFv sequence with stronger affinity, and the generated CAR-T cell can effectively kill tumor cells. The technical scheme is as follows:
a chimeric antigen receptor based on a CD276 antibody, wherein the chimeric antigen receptor is a CD276-28z-CAR chimeric antigen receptor, a CD276-BBz-CAR or a CD276-28BBz-CAR chimeric antigen receptor, the amino acid sequence of the CD276-28z-CAR chimeric antigen receptor is shown in SEQ ID NO.1, the amino acid sequence of the CD276-BBz-CAR chimeric antigen receptor is shown in SEQ ID NO.2, and the amino acid sequence of the CD276-28BBz-CAR chimeric antigen receptor is shown in SEQ ID NO. 3.
Preferably, the CD276-28z-CAR chimeric antigen receptor is composed of a human CD8a molecule signal peptide, a CD276 single-chain antibody, a human CD8a molecule flexible fragment, a human CD28 molecule transmembrane region, an intracellular region and a human CD3z molecule intracellular region in tandem;
wherein, the amino acid sequence of the signal peptide of the human CD8a molecule is shown as SEQ ID NO.4, the amino acid sequence of the CD276 single-chain antibody is shown as SEQ ID NO.5, the amino acid sequences of the transmembrane region and the intracellular region of the human CD28 molecule are shown as SEQ ID NO.6, and the amino acid sequence of the intracellular region of the human CD3z molecule is shown as SEQ ID NO. 7.
Preferably, the CD276-BBz-CAR chimeric antigen receptor is composed of a human CD8a molecule signal peptide, a CD276 single-chain antibody, a human CD8a molecule flexible fragment and a transmembrane region, a human 41BB molecule intracellular region and a human CD3z molecule intracellular region which are connected in series;
wherein, the amino acid sequence of the signal peptide of the human CD8a molecule is shown as SEQ ID NO.4, the amino acid sequence of the single-chain antibody of the CD276 is shown as SEQ ID NO.5, the amino acid sequences of the transmembrane region and the intracellular region of the human 41BB molecule are shown as SEQ ID NO.8, and the amino acid sequence of the intracellular region of the human CD3z molecule is shown as SEQ ID NO. 7.
Preferably, the CD276-28BBz-CAR chimeric antigen receptor is composed of a human CD8a molecule signal peptide, a CD276 single-chain antibody, a human CD8a molecule flexible fragment, a transmembrane region, human CD28 and 41BB molecule intracellular regions and a human CD3z molecule intracellular region in tandem;
wherein, the amino acid sequence of the signal peptide of the human CD8a molecule is shown as SEQ ID NO.4, the amino acid sequence of the single-chain antibody of the CD276 is shown as SEQ ID NO.5, the amino acid sequences of the transmembrane region and the intracellular regions of the CD28 and 41BB are shown as SEQ ID NO.9, and the amino acid sequence of the intracellular region of the human CD3z molecule is shown as SEQ ID NO. 7.
A lentiviral expression vector carrying the chimeric antigen receptor described above.
Preferably, the chimeric antigen receptor is expressed in a pCDH-EF1-MSC plasmid, forming a pCDH-EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz, or pCDH-EF1-CAR-CD276-28BBz plasmid.
A lentivirus, wherein the lentivirus is obtained by co-transfecting a target cell with the lentivirus expression vector and packaging plasmids psPAX2 and pMD2. G.
A method of constructing a CAR-T cell, comprising:
(1) CAR sequence synthesis: constructing a CD276-28z-CAR chimeric antigen receptor, a CD276-BBz-CAR or a CD276-28BBz-CAR chimeric antigen receptor;
(2) construction of an expression vector: expressing the chimeric antigen receptor in a pCDH-EF1-MSC plasmid to form a pCDH-EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz, or pCDH-EF1-CAR-CD276-28BBz plasmid;
(3) and (3) slow virus packaging: mixing the plasmid in the step (2) with a packaging plasmid, and packaging to form a lentivirus;
(4) t cell purification and infection: purifying the T cells, and transfecting the T cells with lentiviruses to obtain CAR-T cells;
(5) t cell CAR expression efficiency assay: detecting the expression of the CAR on the surface of the T cell;
(6) CAR-T cell expansion in vitro: CAR-T cells were expanded.
The chimeric antigen receptor can be applied to the preparation of CAR-T cells for recognizing a specific target CD 276.
By adopting the scheme, the invention has the following advantages:
factors such as antigen expression intensity and recognition antibody affinity influence the therapeutic effect of CAR-T cells. The invention carries out CAR-T cell therapy by taking CD276 with wide and high expression of tumors as a target spot, and the result shows that the antigen is a better tumor therapy target spot. Meanwhile, the invention adopts the anti-CD 276scFv with enhanced affinity as the CAR-T cell recognition region, can more effectively capture corresponding antigen positive tumor cells, and is beneficial to improving the treatment effect of immune cells. T cell function can be improved by the addition of intracellular stimulatory signals (CD28 or 41 BB). The transgenic modified CAR-T cell has stronger and more durable killing capability and is expected to improve the tumor treatment effect.
Drawings
FIG. 1 is a schematic structural diagram of CD276-28z-CAR from example 1;
FIG. 2 is a schematic structural diagram of CD 276-BBz-CAR;
FIG. 3 is a schematic structural diagram of CD276-28 BBz-CAR;
FIG. 4 is a graph of flow cytometry to detect expression of CAR by T cells;
FIG. 5 is a graph of CAR-T cell proliferation;
FIG. 6 is a graph showing the expression of CD 276;
FIG. 7 is a graph showing the results of CAR-T cells specifically killing tumor cells;
FIG. 8 is a CAR-T cell secretion cytokine map;
FIG. 9 is a schematic structural view of CD276-28z-CAR of comparative example 1;
FIG. 10 is a graph comparing the transmission of T cell activation signals by the constructs of example 1 and comparative example 1;
FIG. 11 is a graph comparing the killing activity of CAR-T cells on tumor cells in example 1 and comparative example 1.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental reagents and materials involved are conventional biochemical reagents and materials unless otherwise specified.
Example 1
A, CAR structure
CD276-28z-CAR structure
As shown in fig. 1, the CAR infrastructure includes: human CD8a molecular signal peptide (LeadingSignal), CD276 single-chain antibody (scFv), human CD8a molecular flexible fragment (CD8Hinge), human CD28 molecular transmembrane region and intracellular region (CD28), and human CD3z molecular intracellular region (CD3 z). The amino acid sequence of the CD276-28z-CAR structure is shown as SEQ ID NO. 1.
Wherein the amino acid sequence of the Signal Peptide (SP) sequence is:
MALPVTALLLPLALLLHAARPGS(SEQ ID NO.4)。
affinity-enhanced CD276 single chain antibody (CD276-scFv) sequence:
QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGHSFPLTFGQGTKLELKR(SEQ ID NO.5)。
flexible fragments with Transmembrane (TM) and CD28 intracellular (CD28) sequences:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO.6)。
sequence of intracellular region (CD3z) of human CD3z molecule:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.7)。
CD276-BBz-CAR structures
As shown in FIG. 2, the polypeptide is composed of a human CD8a molecule signal peptide (LeadingSignal), a CD276 single-chain antibody (scFv), a human CD8a molecule flexible fragment, a transmembrane region (CD8HingeTM), a human 41BB molecule intracellular region (41BB), and a human CD3z molecule intracellular region (CD3z) which are connected in series, and the amino acid sequence of the CD276-BBz-CAR structure is shown as SEQ ID NO. 2.
Wherein, the signal peptide (leader) sequence:
MALPVTALLLPLALLLHAARPGS(SEQ ID NO.4)。
affinity-enhanced CD276 single chain antibody (scFv) sequence:
QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGHSFPLTFGQGTKLELKR(SEQ ID NO.5)。
flexible fragments with Transmembrane (TM) and 41BB intracellular (41BB) regions sequences:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.8)。
sequence of intracellular region (CD3z) of human CD3z molecule:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.7)。
CD276-28BBz-CAR structures
As shown in fig. 3, the CAR infrastructure includes: human CD8a molecular signal peptide (LeadingSignal), CD276 single chain antibody (scFv), human CD8a molecular flexible fragment and transmembrane region (CD8HingeTM), human CD28 and 41BB molecular intracellular region (CD28-41BB), and human CD3z molecular intracellular region (CD3 z). The amino acid sequence of the CD276-28BBz-CAR structure is shown in SEQ ID NO. 3.
Wherein,
signal peptide (Leadingsignal) sequence:
MALPVTALLLPLALLLHAARPGS(SEQ ID NO.4)。
affinity-enhanced CD276 single chain antibody (scFv) sequence:
QVQLVQSGAEVVKPGASVKLSCKTSGYTFTNYDINWVRQRPGQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSTSTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPATLSVSPGERVTLSCRASQSISDYLYWYQQKSHESPRLLIKYASQSISGIPARFSGSGSGSEFTLTINSVEPEDVGVYYCQNGHSFPLTFGQGTKLELKR(SEQ ID NO.5)。
flexible fragments with Transmembrane (TM) and CD28 and 41BB intracellular domain (CD28-41BB) sequences:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.9)。
sequence of intracellular region (CD3z) of human CD3z molecule:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.7)。
II, CAR sequence synthesis and vector construction
The CAR coding sequence is synthesized by Shanghai chemical company, the DNA sequence of a CD276-28z-CAR structure (SEQ ID NO.12), the DNA sequence of a CD276-BBz-CAR structure (SEQ ID NO.13) or the DNA sequence of a CD276-28BBz-CAR structure (SEQ ID NO.14) is obtained, and the obtained product is inserted into a pCDH-EF1-MSC plasmid through enzyme digestion connection to construct a lentivirus expression plasmid pCDH-EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz or pCDH-EF1-CAR-CD276-28 BBz.
Three, slow virus package
24 hours after the packaging cells 293T were plated, the pCDH-EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz or pCDH-EF1-CAR-CD276-28BBz expression plasmids were mixed with packaging plasmids (psPAX2 and pMD2.G) and 293T cells were transfected using calcium phosphate transfection reagents. At 48 hours post transfection, supernatants were collected and prepared for T cell infection.
Fourth, T cell purification and infection
After the human peripheral blood is centrifuged by density gradient, the peripheral blood mononuclear cells are separated. Purified CD3 was obtained using T cell isolation kit from German America, whirlpool+And adding an appropriate amount of CD3/CD28 magnetic beads into the T cells according to the proportion that 1 cell is added into 1 magnetic bead for activation for 2 days. After 2 days, the viral supernatant was added and incubated with polybrene (6. mu.g/mL) overnight. The next day, after the T cells were washed by centrifugation 3 times, the T cells were amplified by adding RPMI-1640 medium containing 200UIL-2 and 5% fetal bovine serum.
Fifth, T cell CAR expression efficiency
After 3 days of T cell infection, expression of CAR on the T cell surface was examined by flow cytometry. The results are shown in FIG. 4. Figure 4 shows that CAR expression positive rate reaches about 80%, demonstrating that CAR expression plasmid construction and viral packaging are successful.
Sixth, CAR-T cell in vitro expansion
Count 1 × 106Individual T cells were infected and cells were counted every 2 days post infection and CAR-T cell proliferation was examined. As shown in FIG. 5, the CAR-T cells were all able to proliferate efficiently (about 10-fold expansion within 14 days), with no significant difference in proliferation of 3 CAR-T cells compared to normal T cells (UTD).
Seventhly, detection of target antigen
To validate the CAR-T cell killing effect, we first examined CD276 expression on tumor cells. As shown in FIG. 6, the tumor cells TE-1 and KYSE150 highly expressed CD276, while the cells prepared by the gene knockout technique (KYSE150-CD276KO) did not express CD 276.
Eight, CAR-T cells can kill CD276 positive tumor cells
After 14 days of T cell infection, T cells were counted against target cells and then T cells (effector cells) were incubated with CD276 positive target cells (TE-1 and KYSE150) or CD276 negative target cells (KYSE150-CD276KO) at a ratio of 1:1, 1:5, 1:10 for 16 hours at the effective target ratio (effector cells: target cells, E: T). After the end of the co-incubation, CAR-T cells were analyzed for killing activity on tumor cells using a small animal imaging system (fig. 7). The results show that CAR-T cells are able to kill CD276 positive cells with high efficiency, while the effect on CD276 negative cells is minimal, indicating that CAR-T cells have antigen-dependent killing activity.
Nine study on cytokine secretion by CAR-T cells
To further validate the killing ability of CD 276-specific CAR-T cells, we analyzed the ability of antigen-activated CAR-T cells to secrete cytokines. After incubating T cells (UTD, CD276-28z, CD276-BBz or CD276-28BBz) with target cells at a ratio of E: T1: 1 for 24 hours, Interferon (IFN) and interleukin-2 (IL-2) concentrations in the supernatant were measured by ELISA. As shown in FIG. 8, the CAR-T cells were able to secrete large amounts of cytokines upon contact with antigen-positive tumor cells, whereas the surface of the cell molecules was not significantly upregulated upon incubation with antigen-negative cells, indicating that CAR-T cells could be effectively activated following specific antigen stimulation.
Comparative example 1
The huCD276-CAR structure is constructed, and the structure is different from the structure in the example 1 in that a humanized CD276 single-chain antibody is used as a target, the sequences of the rest fragments are identical, and the schematic structure diagram of the huCD276-CAR structure is shown in FIG. 9. The amino acid sequence of the huCD276-CAR structure is shown as SEQ ID NO.10, and the amino acid sequence of the humanized CD276 single-chain antibody is shown as SEQ ID NO. 11.
Analysis of results
The high affinity CD276-28z-CAR (CD276-CAR) constructed in example 1 and the humanized (huCD276-CAR) CAR constructed in comparative example 1 were transduced into Jurkat-NFAT _ luc2 cells and then co-incubated with antigen positive or negative cells for 24 hours. CAR signal transduction intensity can be determined by detecting fluorescence intensity. The results show that CD276-28z-CAR is able to better transmit T cell activation signals (fig. 10), suggesting that the structure of example 1 is able to more strongly activate T cells.
After incubating the high affinity CD276-28z-CAR (CD276-CAR) constructed in example 1 and the humanized (huCD276) antibody CAR-T cell constructed in comparative example 1 with the target cells for 24 hours, respectively, the activity of the target cells was detected. The results show that high affinity CAR-T cell CD276-28z-CAR has better tumor cell killing activity (fig. 11), suggesting that the structure of example 1 can effectively improve the anti-tumor activity of CAR-T cells.
Various other modifications and changes may be made by those skilled in the art based on the above-described technical solutions and concepts, and all such modifications and changes should fall within the scope of the claims of the present invention.
Sequence listing
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Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
405 410 415
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
420 425 430
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
435 440 445
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
450 455 460
Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
465 470 475 480
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 4045
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 280285
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 tcacatgcag1440
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
agcggctctggcagcgagtt 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 chimeric antigen receptor based on a CD276 antibody, characterized by: the chimeric antigen receptor is a CD276-28z-CAR chimeric antigen receptor, a CD276-BBz-CAR or a CD276-28BBz-CAR chimeric antigen receptor, the amino acid sequence of the CD276-28z-CAR chimeric antigen receptor is shown as SEQ ID NO.1, the amino acid sequence of the CD276-BBz-CAR chimeric antigen receptor is shown as SEQ ID NO.2, and the amino acid sequence of the CD276-28BBz-CAR chimeric antigen receptor is shown as SEQ ID NO. 3.
2. The chimeric antigen receptor according to claim 1, wherein: the CD276-28z-CAR chimeric antigen receptor is formed by connecting a human CD8a molecular signal peptide, a CD276 single-chain antibody, a human CD8a molecular flexible fragment, a human CD28 molecular transmembrane region, an intracellular region and a human CD3z molecular intracellular region in series;
wherein, the amino acid sequence of the signal peptide of the human CD8a molecule is shown as SEQ ID NO.4, the amino acid sequence of the CD276 single-chain antibody is shown as SEQ ID NO.5, the amino acid sequences of the transmembrane region and the intracellular region of the human CD28 molecule are shown as SEQ ID NO.6, and the amino acid sequence of the intracellular region of the human CD3z molecule is shown as SEQ ID NO. 7.
3. The chimeric antigen receptor according to claim 1, wherein: the CD276-BBz-CAR chimeric antigen receptor is formed by connecting a human CD8a molecule signal peptide, a CD276 single-chain antibody, a human CD8a molecule flexible fragment, a transmembrane region, a human 41BB molecule intracellular region and a human CD3z molecule intracellular region in series;
wherein, the amino acid sequence of the signal peptide of the human CD8a molecule is shown as SEQ ID NO.4, the amino acid sequence of the single-chain antibody of the CD276 is shown as SEQ ID NO.5, the amino acid sequences of the transmembrane region and the intracellular region of the human 41BB molecule are shown as SEQ ID NO.8, and the amino acid sequence of the intracellular region of the human CD3z molecule is shown as SEQ ID NO. 7.
4. The chimeric antigen receptor according to claim 1, wherein: the CD276-28BBz-CAR chimeric antigen receptor is formed by connecting a human CD8a molecule signal peptide, a CD276 single-chain antibody, a human CD8a molecule flexible fragment, a transmembrane region, human CD28 and 41BB molecule intracellular regions and a human CD3z molecule intracellular region in series;
wherein, the amino acid sequence of the signal peptide of the human CD8a molecule is shown as SEQ ID NO.4, the amino acid sequence of the single-chain antibody of the CD276 is shown as SEQ ID NO.5, the amino acid sequences of the transmembrane region and the intracellular regions of the CD28 and 41BB are shown as SEQ ID NO.9, and the amino acid sequence of the intracellular region of the human CD3z molecule is shown as SEQ ID NO. 7.
5. A lentiviral expression vector, comprising: the vector carries the chimeric antigen receptor of claim 1.
6. The lentiviral expression vector of claim 5, wherein: the chimeric antigen receptor is expressed in pCDH-EF1-MSC plasmid to form pCDH-EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz or pCDH-EF1-CAR-CD276-28BBz plasmid.
7. A lentivirus, characterized by: the lentivirus is obtained by co-transfecting the lentivirus expression vector of claim 5 or 6 with the packaging plasmids psPAX2 and pMD2.G into a target cell.
8. A method of constructing a CAR-T cell, comprising: the method comprises the following steps:
(1) CAR sequence synthesis: constructing a CD276-28z-CAR chimeric antigen receptor, a CD276-BBz-CAR or a CD276-28BBz-CAR chimeric antigen receptor;
(2) construction of an expression vector: expressing the chimeric antigen receptor in a pCDH-EF1-MSC plasmid to form a pCDH-EF1-CAR-CD276-28z, pCDH-EF1-CAR-CD276-BBz, or pCDH-EF1-CAR-CD276-28BBz plasmid;
(3) and (3) slow virus packaging: mixing the plasmid in the step (2) with a packaging plasmid, and packaging to form a lentivirus;
(4) t cell purification and infection: purifying the T cells, and transfecting the T cells with lentiviruses to obtain CAR-T cells;
(5) t cell CAR expression efficiency assay: detecting the expression of the CAR on the surface of the T cell;
(6) CAR-T cell expansion in vitro: CAR-T cells were expanded.
9. Use of the chimeric antigen receptor of any one of claims 1-4 in the preparation of a CAR-T cell that recognizes a specific target CD 276.
CN201910243041.6A 2019-03-28 2019-03-28 Chimeric antigen receptor, Lentiviral and its application based on CD276 antibody Pending CN109929039A (en)

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WO2021027687A1 (en) * 2019-08-14 2021-02-18 博源润生医药（杭州）有限公司 Chimeric antigen receptor and immune effector cell expressing chimeric antigen receptor
CN112521512A (en) * 2020-12-14 2021-03-19 广州百暨基因科技有限公司 anti-B7H3 chimeric antigen receptor and application thereof
CN112851826A (en) * 2021-02-10 2021-05-28 上海斯丹赛生物技术有限公司 Treatment of UPK2 chimeric antigen receptor and its urethral cancer
CN113402610A (en) * 2021-06-09 2021-09-17 东大生物技术(苏州)有限公司 Group of B7H3 monoclonal antibodies and medical application thereof
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
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
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
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
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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