CN113980143B

CN113980143B - Chimeric antigen receptor, chimeric antigen receptor T cell targeting CD276, preparation method and pharmaceutical application

Info

Publication number: CN113980143B
Application number: CN202111290880.7A
Authority: CN
Inventors: 万晓春; 曹国政
Original assignee: Shenzhen Institute of Advanced Technology of CAS; Shenzhen Technology University
Current assignee: Shenzhen Institute of Advanced Technology of CAS; Shenzhen Technology University
Priority date: 2021-11-02
Filing date: 2021-11-02
Publication date: 2023-04-25
Anticipated expiration: 2041-11-02
Also published as: CN113980143A

Abstract

The invention discloses a chimeric antigen receptor targeting CD276, a chimeric antigen receptor T cell, a preparation method and pharmaceutical application, and belongs to the technical field of biological medicines. The chimeric antigen receptor T cell targeting CD276 disclosed by the invention can target tumor cells expressing CD276, activate the T cells to play a role of cellular immunity, efficiently and specifically kill CD276 positive tumor cells, better maintain the activity and killing power of the chimeric antigen receptor T cells, and is a potential immune cell therapy for radically treating hepatocellular carcinoma. Through in vitro functional tests, the chimeric antigen receptor T cells targeting CD276 have killing activity on a CD276 positive liver cancer cell line.

Description

Chimeric antigen receptor, chimeric antigen receptor T cell targeting CD276, preparation method and pharmaceutical application

Technical Field

The invention belongs to the technical field of biological medicines, and relates to a chimeric antigen receptor, a chimeric antigen receptor T cell targeting CD276, a preparation method and pharmaceutical application.

Background

Hepatocellular carcinoma is a malignant tumor that severely threatens human health, accounting for 90% of all primary liver cancers, the second most lethal cancer worldwide. Current treatments for hepatocellular carcinoma such as chemotherapy, radiation therapy, hepatectomy, ablative therapy, targeted therapies such as sorafenib, antibody therapies such as immune checkpoint inhibitors, vascular endothelial growth factor receptor inhibitors, however, the efficacy of these therapies is inadequate (Lee, y.h., et al Combinational Immunotherapy for Hepatocellular Carcinoma: radiation, immune Checkpoint Blockade and beyond. Thus, there is an urgent need for new drugs for treating hepatocellular carcinoma.

Chimeric antigen receptor T cell (CAR-T) therapy is a novel immune cell therapy method, has a specific and efficient function of killing tumor cells expressing corresponding antigens, enables chimeric antigen receptors to be expressed on the surfaces of T cells through genetic engineering means, activates an autoimmune system by preparing and amplifying chimeric antigen receptor T cells in vitro and then back into the human body, exerts efficient killing of tumor cells (1.rafiq, s., c.s. hackett, and r.j. Brentjens, engineering strategies to overcome the current roadblocks in CAR T cell therapy.nature Reviews Clinical Oncology,2020.17 (3): p.147-167.;2.benmebarek, m.r..et al, killing Mechanisms of Chimeric Antigen Receptor (CAR) T cells.international Journal of Molecular Sciences,2019.20 (6)), is considered as the most likely therapy for cancer, and CAR-T cells targeting CD19 produce good effects in blood tumors. Currently, 6 types of CAR-T drugs are marketed in batches worldwide, and the huge application prospect of CAR-T cell therapy in cancer treatment is shown.

CD276, also known as B7-H3, is a class of transmembrane proteins belonging to the B7 family. CD276 is highly expressed in various cancers including lung adenocarcinoma, brain glioma, neuroblastoma, pancreatic cancer, ovarian cancer, etc., and is hardly expressed in normal tissues. Monoclonal antibodies targeting CD276 mediate efficient tumor clearance in a variety of tumor models. CD276 is highly expressed in hepatocellular carcinoma and is associated with the progression of liver cancer and poor prognosis in tumor patients. This suggests that CD276 may be a potential target for the treatment of hepatocellular carcinoma.

Currently, CD 276-targeting CAR-T cells have limited studies on the killing activity of hepatocellular carcinoma and other malignancies that highly express CD 276.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a chimeric antigen receptor, a chimeric antigen receptor T cell targeting CD276, a preparation method and pharmaceutical application.

In order to achieve the above purpose, the invention is realized by adopting the following technical scheme:

one of the aspects of the present invention is to provide a CD 276-targeting chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID No. 1.

Preferably, the coding gene of the chimeric antigen receptor targeting CD276 comprises a nucleotide sequence as shown in SEQ ID NO. 2.

Preferably, the chimeric antigen receptor targeting CD276 comprises an amino acid sequence of a single chain antibody targeting CD276, an extracellular hinge region, a transmembrane region, and an intracellular structural region in order from amino terminus to carboxy terminus.

Further preferably, the amino acid sequence of the single chain antibody targeting CD276 comprises the amino acid sequence shown as SEQ ID NO. 3.

Still more preferably, the gene encoding the CD 276-targeting single-chain antibody comprises a nucleotide sequence as shown in SEQ ID NO. 4.

Further preferably, the extracellular hinge region is a CD8 a hinge region.

Still more preferably, the amino acid sequence of the CD 8. Alpha. Hinge region comprises the amino acid sequence shown in SEQ ID NO. 5.

Still more preferably, the coding gene of the CD 8. Alpha. Hinge region comprises the nucleotide sequence shown as SEQ ID NO. 6.

Of course, the extracellular hinge region may also use a CD28 hinge region, an IgG1 hinge region, or an IgG4 hinge region.

Further preferably, the transmembrane region is a CD8 a transmembrane region.

Still more preferably, the amino acid sequence of the CD 8. Alpha. Transmembrane region comprises the amino acid sequence shown in SEQ ID NO. 7.

Still more preferably, the coding gene of the CD 8. Alpha. Transmembrane region comprises the nucleotide sequence shown in SEQ ID NO. 8.

Of course, the transmembrane region may also be a CD4 transmembrane region, a CD28 transmembrane region, a CD3 zeta transmembrane region or a transmembrane region of ICOS.

Further preferably, the intracellular domain is comprised of a 4-1BB costimulatory domain and a CD3ζ signaling domain in series.

Still more preferably, the amino acid sequence of the 4-1BB costimulatory domain comprises the amino acid sequence shown as SEQ ID NO. 9.

Still more preferably, the coding gene of the 4-1BB costimulatory structural region comprises the nucleotide sequence shown as SEQ ID NO. 10.

Further preferably, the amino acid sequence of the CD3ζ signaling domain comprises the amino acid sequence shown in SEQ ID NO. 11.

Still more preferably, the coding gene for the CD3ζ signaling domain comprises a nucleotide sequence as depicted in SEQ ID NO. 12.

Of course, the co-stimulatory domain may also use the intracellular domains of CD28, OX40, ICOS or CD 27.

The second aspect of the present invention is to provide a CD 276-targeting chimeric antigen receptor T cell comprising the CD 276-targeting chimeric antigen receptor described above; the amino acid sequence of the chimeric antigen receptor targeting CD276 comprises the amino acid sequence shown as SEQ ID NO. 1.

Further, the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 comprises a nucleotide sequence shown as SEQ ID NO. 2.

The third aspect of the present invention is to provide a method for preparing a chimeric antigen receptor T cell targeting CD276, comprising the steps of:

1) The coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 is provided, and the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 is shown as a nucleotide sequence shown as SEQ ID NO. 2.

2) Inserting the coding gene of the CAR-CD276 into a pWPXLD vector to obtain a pWPXLD-CAR-CD276 recombinant plasmid;

3) Co-transfecting the pWPXLd-CAR-CD276 recombinant plasmid, envelope plasmid and packaging plasmid into a host cell to obtain recombinant lentivirus;

4) The recombinant lentivirus is transfected into CD3 positive T lymphocytes, and chimeric antigen receptor T cells targeting CD276 are obtained through separation.

Further, the envelope plasmid is PMD2G, the packaging plasmid is psPAX2, and the host cell is HEK293T cell.

Further, in step 4), the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells.

Further, the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood and the like.

The fourth object of the present invention is to provide a recombinant viral vector comprising a gene encoding the chimeric antigen receptor CAR-CD276 targeting CD276 as described above.

The recombinant virus vector comprises a chimeric antigen receptor CAR-CD276 coding gene targeting CD276, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2 or SEQ ID NO. 13;

the nucleotide sequence shown in SEQ ID NO. 13 has more coding genes of the signal peptide than the nucleotide sequence shown in SEQ ID NO. 2. The coding gene of the signal peptide can better guide the chimeric antigen receptor CAR-CD276 to reach the cell surface.

Preferably, the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 comprises a coding gene of a CD8 alpha signal peptide, a coding gene of a single-chain antibody targeting CD276, a coding gene of a CD8 alpha extracellular hinge region, a coding gene of a CD8 alpha transmembrane region and a coding gene of an intracellular signal region formed by sequentially connecting a 4-1BB costimulatory structural region and a CD3 zeta signal structural region in series from the 5 'end to the 3' end.

Further preferably, the amino acid sequence of CAR-CD276 includes the amino acid sequence shown as SEQ ID NO. 14.

Further preferably, the coding gene of the CD8 alpha signal peptide comprises a nucleotide sequence shown as SEQ ID NO. 15.

Further preferably, the amino acid sequence of the CD8 alpha signal peptide comprises the amino acid sequence shown as SEQ ID NO. 16.

Further preferably, the coding gene of the single-chain antibody targeting CD276 comprises a nucleotide sequence shown as SEQ ID NO. 4, the coding gene of the extracellular hinge region of CD8 alpha comprises a nucleotide sequence shown as SEQ ID NO. 6, the coding gene of the transmembrane region of CD8 alpha comprises a nucleotide sequence shown as SEQ ID NO. 8, the coding gene of the costimulatory structural region of 4-1BB comprises a nucleotide sequence shown as SEQ ID NO. 10, and the coding gene of the signal structural region of CD3 zeta comprises a nucleotide sequence shown as SEQ ID NO. 12.

The virus vector is a pWPXLd lentiviral vector.

The fifth aspect of the present invention provides the use of the above-mentioned chimeric antigen receptor targeting CD276, chimeric antigen receptor T cell targeting CD276 or recombinant viral vector for preparing a medicament for treating liver cancer.

Preferably, the drug has strong killing activity against positive tumor cells expressing CD 276.

The sixth object of the present invention is to provide a medicament for treating hepatocellular carcinoma, which comprises the chimeric antigen receptor targeting CD276, the chimeric antigen receptor T cell targeting CD276 or a recombinant viral vector.

Of course, the chimeric antigen receptor T cells targeting CD276 are also suitable for use in the treatment of other malignancies that highly express CD 276.

Compared with the prior art, the invention has the following beneficial effects:

the present invention provides a chimeric antigen receptor that targets CD276, and chimeric antigen receptor T cells comprising a CD 276-targeting chimeric antigen receptor. The chimeric antigen receptor T cells targeting CD276 can target tumor cells expressing CD276, activate the T cells to play a role of cellular immunity, efficiently and specifically kill CD276 positive tumor cells, better maintain the activity and the killing power of the chimeric antigen receptor T cells, and are an immune cell therapy with potential for radically treating hepatocellular carcinoma.

The chimeric antigen receptor T cells targeting the CD276 disclosed by the invention take 4-1BB as a co-stimulatory structural region, so that the chimeric antigen receptor T cells targeting the CD276 have longer persistence in vivo, are beneficial to the generation of memory phenotype T cells, and have strong killing activity on a positive tumor cell line expressing the CD 276. Through in vitro functional tests, the chimeric antigen receptor T cells targeting CD276 have killing activity on a CD276 positive liver cancer cell line.

Drawings

FIG. 1 is a plasmid map of a pWPXLd-CAR-CD276 recombinant plasmid;

FIG. 2 is a graph showing the results of expression of a CD 276-targeted chimeric antigen receptor on the surface of T cells; wherein (a) is a T cell of a UTD non-transduced virus; (b) T cells transduced with CAR-CD 276;

FIG. 3 is a graph showing the killing results of CD 276-targeted chimeric antigen receptor T cells against the hepatocellular carcinoma cell line Huh 7.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.

The invention is described in further detail below with reference to the attached drawing figures:

1. a method of making a CD 276-targeted chimeric antigen receptor T cell comprising the steps of:

(1) Preparation of a chimeric antigen receptor for CD276 the gene sequence of CAR-CD276 targeting CD276

The chimeric antigen receptor CAR-CD276 structure targeting CD276 comprises a CD8 alpha signal peptide, a single chain antibody (CD 276-scFv) targeting CD276, a CD8 alpha hinge region, a CD8 alpha transmembrane region, a 4-1BB costimulatory structural region and a coding gene of a CD3 zeta signal structural region, wherein the coding gene of the signal peptide is shown as SEQ ID NO. 15, the coding gene of the single chain antibody targeting CD276 comprises a nucleotide sequence shown as SEQ ID NO. 4, the coding gene of the extracellular hinge region of CD8 alpha comprises a nucleotide sequence shown as SEQ ID NO. 6, the coding gene of the CD8 alpha transmembrane region comprises a nucleotide sequence shown as SEQ ID NO. 8, the coding gene of the 4-1BB costimulatory structural region comprises a nucleotide sequence shown as SEQ ID NO. 10, and the coding gene of the CD3 zeta signal structural region comprises a nucleotide sequence shown as SEQ ID NO. 12.

The chimeric antigen receptor CAR-CD276 structure of the targeting CD276 is synthesized by Jiangsu Jiujingzhi biotechnology Co., ltd, so as to obtain the encoding gene of the chimeric antigen receptor CAR-CD276 of the targeting CD276, wherein the encoding gene of the CAR-CD276 comprises a nucleotide sequence shown as SEQ ID NO. 13.

(2) Construction of pWPXLd-CAR-CD276 recombinant plasmid

The coding gene of the synthesized CAR-CD276 is inserted between BamH1 and EcoR1 restriction sites of a pWPXLD vector, and then transferred into competent cells DH5 alpha of escherichia coli for positive clone PCR identification and sequencing identification. The pWPXLd-CAR-CD276 recombinant plasmid map is shown in FIG. 1.

(3) Recombinant lentivirus construction

The pWPXLd-CAR-CD276 recombinant plasmid, packaging plasmid psPAX2 and envelope plasmid pMD2G were co-transfected into cultured HEK293T cells using lipofectamine3000 transfection reagent. The virus-containing supernatant was collected at 48h, centrifuged at 2000rpm at room temperature for 5 minutes, the supernatant was collected and filtered through a 0.45 μm filter, and the resulting recombinant lentiviral supernatant was used for T cell infection.

(4) Preparation of chimeric antigen receptor T cells targeting CD276

a) Isolation of PBMC (peripheral blood mononuclear cells)

PBMCs were derived from autologous venous blood from healthy volunteers.

Extracting blood of the healthy volunteers, collecting peripheral blood mononuclear cells by using Ficoll, and taking middle layer cells after centrifugal separation; PBMCs were obtained after washing and counting with PBS.

b) Isolation of antigen-specific T lymphocytes by immunomagnetic bead method

Adding KBM581 culture medium into the PBMC to prepare cell suspension; adding CD3/CD28 immunomagnetic beads according to the ratio of the magnetic beads to the cells being 1:1, and incubating in a shaking table at room temperature; screening cells incubated with the magnetic beads by using a magnet; after removing the unadsorbed cell suspension, the bead-cell mixture was resuspended in KBM581 medium described above to give CD3 positive T lymphocytes which were used for lentiviral infection after further incubation for 24 hours.

c) Method for preparing antigen-specific T lymphocyte by virus transfection method

And adding the recombinant lentivirus with the number corresponding to that of the CD3 positive cells into the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method for culture.

On day 3 of culture, a suitable number of lentiviral infected T cells were collected and flow cytometry analyzed for surface CAR expression, as shown in fig. 2, where (a) is UTD non-transduced viral T cells and (b) is CAR-CD276 transduced viral T cells. Staining was performed using biotin-labeled anti-fab primary antibodies, followed by flow analysis using streptavidin-coupled APC secondary antibodies. The results in figure 2 show that, minus the UTD background value, CAR-CD276 has 26% surface expression, indicating successful production of CD 276-targeted chimeric antigen receptor T cells. And culturing for 48 hours, collecting chimeric antigen receptor T cells targeting CD276 for killing experimental analysis or storing in cell cryopreservation liquid, storing in a program cooling box at-80 ℃ for 24 hours, and then transferring to liquid nitrogen tank for long-term storage.

2. In vitro functional test

Through in vitro functional tests, the chimeric antigen receptor T cells targeting CD276 have killing activity on a CD276 positive liver cancer cell line. The specific method and the results are as follows:

(1) Targeting CD276 chimeric antigen receptor T cells was analyzed for killing of CD276 positive tumor cell lines using the RTCA system.

The balancing of RTCA single plates is firstly carried out by using 50ul DMEM culture medium, then cultured target cells Huh7 are collected, cell counts are carried out, 50ul cell suspension containing 5000 Huh7 cells is added to each hole, the cells are placed in a 37 ℃ incubator for 15 minutes and then placed in an RTCA resistance system, after 24 hours, chimeric antigen receptor T cells targeting CD276 are collected, the cell counts are carried out, 100ul chimeric antigen receptor T cell suspension with corresponding cell numbers are added to the Huh7 cells according to different ratios (E: T) of 10:1,5:1,2.5:1, a control group (control, culture medium group) is arranged, a UTD group (T cells added with untransduced viruses), a CAR-T group (T cell group added with targeting CD276 chimeric antigen receptor) are arranged in each group, 3 compound holes are arranged in the RTCA resistance system, and killing activity analysis is carried out after 24 hours.

(2) Analysis of results

UTD or CAR-T killing rate analysis was performed using cell index (cell index) at the indicated time points.

The UTD killing rate calculation formula is as follows:

UTD kill rate= (control cell index value-UTD cell index value)/control cell index value x 100.

The CAR-T killing rate calculation formula is as follows:

CAR-T killing rate= (control group cell index value-CAR-T group cell index value)/control group cell index value x 100.

The results are shown in figure 3, which shows that CAR-T has a stronger killing activity against Huh7 cells than UTD group (figure 3). The constructed chimeric antigen receptor T cells targeting the CD276 have strong killing activity on a positive tumor cell line expressing the CD276, and the chimeric antigen receptor T cells targeting the CD276 are predicted to have a great application prospect in the treatment of hepatocellular carcinoma.

The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.

Sequence listing

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<210> 11

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 11

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 12

<211> 339

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgctga 339

<210> 13

<211> 1467

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

atggccctgc ctgtgacagc cctgttactt cccctggccc tgttactgca tgctgctaga 60

cctgaggtac aactggtgga gtctgggggg ggcctggttc agcctggggg ctctctgaga 120

ctgagctgtg ctgcctctgg cttcaccttc agcagctttg gcatgcactg ggtgagacaa 180

gcccctggca agggcctgga gtgggtggcc tacatcagct ctgacagctc tgccatctac 240

tatgctgaca cagtgaaggg cagattcacc atcagcagag acaatgccaa gaacagcctg 300

tacctgcaga tgaacagcct gagagatgag gacacagctg tgtactactg tggcagaggc 360

agagagaaca tctactatgg cagcagactg gactattggg gccaaggcac aacagtgaca 420

gtcagctctg ggggtggagg atctggaggt gggggctctg ggggtggggg atctgacatt 480

cagctgacac agagccctag cttcctgtct gcctctgtgg gggacagagt gaccatcacc 540

tgcaaggctt ctcagaatgt ggacaccaat gtggcctggt atcagcagaa gcctggcaag 600

gcccccaagg ccctgatcta ctctgctagc tacagatact ctggggtgcc tagcagattc 660

tctggctctg gctctggcac agacttcacc ctgaccatca gcagcctgca gcctgaggac 720

tttgccacct actactgtca gcagtacaac aactacccct tcacctttgg ccaaggcact 780

aagctggaaa tcaagaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc 840

gcgtcgcagc ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg 900

cacacgaggg ggctggactt cgcctgtgat atctacatct gggcgccctt ggccgggact 960

tgtggggtcc ttctcctgtc actggttatc accctttact gcaaacgggg cagaaagaaa 1020

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1080

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgag agtgaagttc 1140

agcaggagcg cagacgcccc cgcgtacaag cagggccaga accagctcta taacgagctc 1200

aatctaggac gaagagagga gtacgatgtt ttggacaaga gacgtggccg ggaccctgag 1260

atggggggaa agccgagaag gaagaaccct caggaaggcc tgtacaatga actgcagaaa 1320

gataagatgg cggaggccta cagtgagatt gggatgaaag gcgagcgccg gaggggcaag 1380

gggcacgatg gcctttacca gggtctcagt acagccacca aggacaccta cgacgccctt 1440

cacatgcagg ccctgccccc tcgctga 1467

<210> 14

<211> 488

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 14

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 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Phe Gly Met His Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ala Tyr Ile Ser Ser Asp Ser Ser Ala Ile Tyr

65 70 75 80

Tyr Ala Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Gly Arg Gly Arg Glu Asn Ile Tyr Tyr Gly Ser

115 120 125

Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile

145 150 155 160

Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg

165 170 175

Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Thr Asn Val Ala

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ala Leu Ile Tyr Ser

195 200 205

Ala Ser Tyr Arg Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

225 230 235 240

Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Asn Tyr Pro Phe Thr Phe

245 250 255

Gly Gln Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg

260 265 270

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg

275 280 285

Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly

290 295 300

Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr

305 310 315 320

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg

325 330 335

Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro

340 345 350

Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu

355 360 365

Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala

370 375 380

Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu

385 390 395 400

Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly

405 410 415

Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu

420 425 430

Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser

435 440 445

Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly

450 455 460

Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu

465 470 475 480

His Met Gln Ala Leu Pro Pro Arg

485

<210> 15

<211> 63

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

atggccctgc ctgtgacagc cctgttactt cccctggccc tgttactgca tgctgctaga 60

cct 63

<210> 16

<211> 21

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 16

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

20

Claims

1. An application of a chimeric antigen receptor targeting CD276, a chimeric antigen receptor T cell targeting CD276 or a recombinant viral vector in preparing a medicament for treating liver cancer, which is characterized in that:

the amino acid sequence of the chimeric antigen receptor targeting CD276 is shown as SEQ ID NO. 1;

the CD 276-targeting chimeric antigen receptor T cells include the CD 276-targeting chimeric antigen receptor described above;

the recombinant virus vector comprises a coding gene of a chimeric antigen receptor CAR-CD276 targeting CD276, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2 or SEQ ID NO. 13.

2. The use of claim 1, wherein the medicament has strong killing activity against positive tumor cells expressing CD 276.

3. The use according to claim 1, wherein the nucleotide sequence encoding the CD 276-targeting chimeric antigen receptor is as set forth in seq id No. 2.

4. The use of claim 1, wherein the CD 276-targeting chimeric antigen receptor comprises the amino acid sequence of a CD 276-targeting single chain antibody, an extracellular hinge region, a transmembrane region, and an intracellular structural region, sequentially linked from amino-terminus to carboxy-terminus.

5. The use of claim 4, wherein the single-chain antibody targeting CD276 has an amino acid sequence shown in SEQ ID No. 3 and the nucleotide sequence encoding the single-chain antibody targeting CD276 is shown in SEQ ID No. 4.

6. The use according to claim 4, wherein the extracellular hinge region is a CD8 alpha hinge region having an amino acid sequence as shown in SEQ ID NO. 5 and a nucleotide sequence encoding the CD8 alpha hinge region as shown in SEQ ID NO. 6.

7. The use according to claim 4, wherein the transmembrane region is a CD 8. Alpha. Transmembrane region having the amino acid sequence shown in SEQ ID NO. 7 and the nucleotide sequence encoding the CD 8. Alpha. Transmembrane region is shown in SEQ ID NO. 8.

8. The use of claim 4, wherein the intracellular domain consists of a 4-1BB costimulatory domain and a cd3ζ signaling domain in series; wherein:

the amino acid sequence of the 4-1BB costimulatory structural region is shown as SEQ ID NO. 9, and the nucleotide sequence for encoding the 4-1BB costimulatory structural region is shown as SEQ ID NO. 10;

the amino acid sequence of the CD3 zeta signal structural region is shown as SEQ ID NO. 11, and the nucleotide sequence of the CD3 zeta signal structural region is shown as SEQ ID NO. 12.

9. The use of claim 1, wherein said CD 276-targeting chimeric antigen receptor T cells are prepared according to the following method:

1) Inserting the encoding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 into a pWPXLD vector to obtain a pWPXLD-CAR-CD276 recombinant plasmid;

wherein, the nucleotide sequence of the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 is shown as SEQ ID NO. 2;

2) Co-transfecting the pWPXLd-CAR-CD276 recombinant plasmid, envelope plasmid and packaging plasmid into a host cell to obtain recombinant lentivirus;

3) The recombinant lentivirus is transfected into CD3 positive T lymphocytes, and chimeric antigen receptor T cells targeting CD276 are obtained through separation.

10. The use according to claim 9, wherein in step 2) the envelope plasmid is PMD2G, the packaging plasmid is psPAX2 and the host cell is HEK293T cell; in step 3), the CD 3-positive T lymphocytes are isolated from human peripheral blood mononuclear cells.