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

Abstract

The invention discloses a chimeric antigen receptor targeting CD276, a chimeric antigen receptor T cell, a preparation method and a pharmaceutical application, and belongs to the technical field of biomedicine. The chimeric antigen receptor T cells targeting CD276 disclosed in the present invention can target tumor cells expressing CD276, activate T cells to exert cellular immunity, efficiently and specifically kill CD276-positive tumor cells, and better maintain chimeric antigens The vitality and lethality of recipient T cells are potential immune cell therapy for hepatocellular carcinoma. The present invention has been tested in vitro, and the CD276-targeted chimeric antigen receptor T cells have killing activity on CD276-positive liver cancer cell lines.

Description

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

technical field

The invention belongs to the technical field of biomedicine, and relates to a CD276-targeted chimeric antigen receptor, chimeric antigen receptor T cells, a preparation method and a pharmaceutical application.

Background technique

Hepatocellular carcinoma is a malignant tumor that seriously threatens human health, accounting for 90% of all primary liver cancers, and is the second most deadly cancer in the world. Current treatments for hepatocellular carcinoma such as chemotherapy, radiotherapy, hepatectomy, ablation therapy, targeted therapy such as sorafenib, antibody therapy such as immune checkpoint inhibitors, vascular endothelial growth factor receptor inhibitors, however, these therapies The curative effect is not enough (Lee, Y.H., et al., Combinational Immunotherapy for Hepatocellular Carcinoma: Radiotherapy, Immune Checkpoint Blockade and Beyond. Frontiers in Immunology, 2020.11.), causing irreparable damage to the patient and the patient's family. Therefore, new drugs for the treatment of HCC are urgently needed.

Chimeric antigen receptor T cell (CAR-T) therapy is a new type of immune cell therapy, which has the function of specifically and efficiently killing tumor cells expressing corresponding antigens. On the surface of T cells, chimeric antigen receptor T cells are prepared and expanded in vitro, and then reinfused into the human body to activate the autoimmune system and effectively kill 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).) Chimeric antigen receptor T cell technology is considered to be the most likely therapy to overcome cancer, and CAR-T cells targeting CD19 have produced good results in blood tumors . At present, 6 CAR-T drugs have been approved for marketing in the world, showing the great application prospects of CAR-T cell therapy in the treatment of cancer.

CD276, also known as B7-H3, is a type of transmembrane protein belonging to the B7 family. CD276 is highly expressed in a variety of cancers, including lung adenocarcinoma, glioma, neuroblastoma, pancreatic cancer, and ovarian cancer, and is almost not expressed in normal tissues. Monoclonal antibodies targeting CD276 mediate efficient tumor clearance in various tumor models. CD276 is highly expressed in HCC and is associated with the progression of HCC and the poor prognosis of tumor patients. This suggests that CD276 can be used as a potential target for the treatment of HCC.

Currently, studies on the killing activity of CAR-T cells targeting CD276 on hepatocellular carcinoma and other malignant tumors with high expression of CD276 are limited.

Contents of the invention

In order to overcome the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a CD276-targeting chimeric antigen receptor, chimeric antigen receptor T cells, preparation method and pharmaceutical application.

In order to achieve the above object, the present invention adopts the following technical solutions to achieve:

One of the contents of the present invention is to provide a CD276-targeting chimeric antigen receptor, said CD276-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 includes the nucleotide sequence shown in SEQ ID NO:2.

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

Further preferably, the amino acid sequence of the single-chain antibody targeting CD276 includes the amino acid sequence shown in SEQ ID NO:3.

Still further preferably, the gene encoding the single-chain antibody targeting CD276 includes the nucleotide sequence shown in SEQ ID NO:4.

Further preferably, the extracellular hinge region is a CD8α hinge region.

Still further preferably, the amino acid sequence of the CD8α hinge region includes the amino acid sequence shown in SEQ ID NO:5.

Still further preferably, the gene encoding the hinge region of CD8α includes the nucleotide sequence shown in SEQ ID NO:6.

Of course, the extracellular hinge region can also use the CD28 hinge region, IgG1 hinge region or IgG4 hinge region.

Further preferably, the transmembrane region is CD8α transmembrane region.

Still further preferably, the amino acid sequence of the CD8α transmembrane region includes the amino acid sequence shown in SEQ ID NO:7.

Still further preferably, the gene encoding the CD8α transmembrane region includes a nucleotide sequence as shown in SEQ ID NO:8.

Of course, the transmembrane region can also use CD4 transmembrane region, CD28 transmembrane region, CD3ζ transmembrane region or ICOS transmembrane region.

Further preferably, the intracellular structural region is composed of a 4-1BB co-stimulatory structural region and a CD3ζ signaling structural region sequentially in series.

Still further preferably, the amino acid sequence of the 4-1BB co-stimulatory structural region includes the amino acid sequence shown in SEQ ID NO:9.

Still further preferably, the coding gene of the 4-1BB co-stimulatory structural region includes the nucleotide sequence shown in SEQ ID NO:10.

Further preferably, the amino acid sequence of the CD3ζ signal domain includes the amino acid sequence shown in SEQ ID NO: 11.

Still further preferably, the coding gene of the CD3ζ signal structural region includes the nucleotide sequence shown in SEQ ID NO: 12.

Of course, the co-stimulatory domain can also use the intracellular domain of CD28, OX40, ICOS or CD27.

The second content of the present invention is to provide a chimeric antigen receptor T cell targeting CD276, including the above-mentioned chimeric antigen receptor targeting CD276; the amino acid sequence of the chimeric antigen receptor targeting CD276 includes: The amino acid sequence shown in SEQ ID NO:1.

Further, the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 includes the nucleotide sequence shown in SEQ ID NO:2.

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

1) Provide the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276, the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 has the nucleotide sequence shown in SEQ ID NO:2.

2) inserting the gene encoding CAR-CD276 into the pWPXLd vector to obtain the pWPXLd-CAR-CD276 recombinant plasmid;

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

4) Transfect CD3-positive T lymphocytes with the recombinant lentivirus, and obtain chimeric antigen receptor T cells targeting CD276 through separation.

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

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

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

The fourth content of the present invention is to provide a recombinant virus vector, which includes the coding gene of the CD276-targeting chimeric antigen receptor CAR-CD276 as described above.

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

Compared with the nucleotide sequence shown in SEQ ID NO: 2, the nucleotide sequence shown in SEQ ID NO: 13 has more coding genes for signal peptides. The gene encoding the signal peptide can better guide the expression of the chimeric antigen receptor CAR-CD276 to the cell surface.

Preferably, the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 includes the coding gene of the CD8α signal peptide and the coding gene of the CD276-targeting single-chain antibody sequentially connected from the 5' end to the 3' end , the coding gene of the CD8α extracellular hinge region, the coding gene of the CD8α transmembrane region and the coding gene of the intracellular signal region composed of the 4-1BB co-stimulatory structure region and the CD3ζ signal structure region in sequence.

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

Further preferably, the gene encoding the CD8α signal peptide includes the nucleotide sequence shown in SEQ ID NO:15.

Further preferably, the amino acid sequence of the CD8α signal peptide includes the amino acid sequence shown in SEQ ID NO: 16.

Further preferably, the coding gene of the single-chain antibody targeting CD276 includes the nucleotide sequence shown in SEQ ID NO: 4, and the coding gene of the CD8α extracellular hinge region includes the nucleotide sequence shown in SEQ ID NO: 6. The nucleotide sequence of the nucleotide sequence, the coding gene of the CD8α transmembrane region includes the nucleotide sequence shown in SEQ ID NO: 8, the coding gene of the 4-1BB co-stimulatory structural region includes the coding gene shown in SEQ ID NO: 10 The nucleotide sequence shown, the coding gene of the CD3ζ signal structure region include the nucleotide sequence shown in SEQ ID NO:12.

The viral vector is pWPXLd lentiviral vector.

The fifth content of the present invention is to provide the above CD276-targeting chimeric antigen receptor, CD276-targeting chimeric antigen receptor T cell or recombinant virus vector in the preparation of drugs for treating liver cancer.

Preferably, the drug has strong killing activity on positive tumor cells expressing CD276.

The sixth object of the present invention is to provide a drug for treating hepatocellular carcinoma, which includes the above CD276-targeted chimeric antigen receptor, CD276-targeted chimeric antigen receptor T cell or recombinant virus vector.

Of course, the chimeric antigen receptor T cells targeting CD276 are also applicable to the treatment of other malignant tumors that highly express CD276.

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

The present invention provides a chimeric antigen receptor targeting CD276 and T cells including the chimeric antigen receptor targeting CD276. Chimeric antigen receptor T cells targeting CD276 can target tumor cells expressing CD276, activate T cells to play a role in cellular immunity, efficiently and specifically kill CD276-positive tumor cells, and better maintain chimeric antigen receptor T cells Its vitality and lethality are potential immune cell therapy for hepatocellular carcinoma.

The chimeric antigen receptor T cells targeting CD276 disclosed in the present invention use 4-1BB as a co-stimulatory structural region, so that the chimeric antigen receptor T cells targeting CD276 have longer persistence in the body, which is beneficial to the memory table The production of type T cells has strong killing activity against positive tumor cell lines expressing CD276. The present invention has been tested in vitro, and the CD276-targeted chimeric antigen receptor T cells have killing activity on CD276-positive liver cancer cell lines.

Description of drawings

Figure 1 is a plasmid map of the pWPXLd-CAR-CD276 recombinant plasmid;

Figure 2 is a diagram showing the expression results of chimeric antigen receptors targeting CD276 on the surface of T cells; wherein, (a) is T cells with UTD non-transduced virus; (b) is T cells transduced with CAR-CD276 virus;

Figure 3 is a graph showing the killing results of the hepatocellular carcinoma cell line Huh7 by chimeric antigen receptor T cells targeting CD276.

Detailed ways

In order to enable those skilled in the art to better understand the solutions of the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only It is an embodiment of a part of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

It should be noted that the terms "first" and "second" in the description and claims of the present invention and the above drawings are used to distinguish similar objects, but not necessarily used to describe a specific sequence or sequence. It is to be understood that the data so used are interchangeable under appropriate circumstances such that the embodiments of the invention described herein can be practiced in sequences other than those illustrated or described herein. Furthermore, the terms "comprising" and "having", as well as any variations thereof, are intended to cover a non-exclusive inclusion, for example, a process, method, system, product or device comprising a sequence of steps or elements is not necessarily limited to the expressly listed instead, may include other steps or elements not explicitly listed or inherent to the process, method, product or apparatus.

The present invention is described in further detail below in conjunction with accompanying drawing:

One, a kind of preparation method of the chimeric antigen receptor T cell targeting CD276, comprises the following steps:

(1) Prepare the gene sequence of chimeric antigen receptor CAR-CD276 targeting CD276

The CD276-targeting chimeric antigen receptor CAR-CD276 structure includes CD8α signal peptide, CD276-targeting single-chain antibody (CD276-scFv), CD8α hinge region, CD8α transmembrane region, 4-1BB co-stimulatory domain and CD3ζ signal The coding gene of the structural region, the coding gene of the signal peptide is shown in SEQ ID NO: 15, and the coding gene of the single-chain antibody targeting CD276 includes the nucleotide sequence shown in SEQ ID NO: 4, the The coding gene of the CD8α extracellular hinge region includes the nucleotide sequence shown in SEQ ID NO:6, the coding gene of the CD8α transmembrane region includes the nucleotide sequence shown in SEQ ID NO:8, the 4 - The coding gene of the costimulatory structural region of 1BB includes the nucleotide sequence shown in SEQ ID NO:10, and the coding gene of the CD3ζ signal structural region includes the nucleotide sequence shown in SEQ ID NO:12.

The chimeric antigen receptor CAR-CD276 structure targeting CD276 was gene-synthesized by Jiangsu Jinweizhi Biotechnology Co., Ltd., and the coding gene of the chimeric antigen receptor CAR-CD276 targeting CD276 was obtained. The CAR-CD276 The coding gene includes the nucleotide sequence shown in SEQ ID NO:13.

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

The synthetic CAR-CD276 coding gene was inserted between the BamH1 and EcoR1 restriction sites of the pWPXLd vector, and then transformed into Escherichia coli competent cells DH5α for positive clone PCR identification and sequencing identification. Figure 1 shows the pWPXLd-CAR-CD276 recombinant plasmid map.

(3) Construction of recombinant lentivirus

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 48 hours, first centrifuged at 2000 rpm for 5 minutes at room temperature, the supernatant was taken, and then filtered through a 0.45 μm filter membrane, and the obtained recombinant lentivirus 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)

PBMC were derived from autologous venous blood of healthy volunteers.

The blood of the healthy volunteers was extracted, the peripheral blood mononuclear cells were collected by Ficoll, and the middle layer cells were collected after centrifugation; PBMC were obtained after washing and counting with PBS.

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

Take the above PBMCs, add KBM581 medium to make a cell suspension; add CD3/CD28 immunomagnetic beads according to the ratio of magnetic beads to cells at 1:1, and incubate on a shaker at room temperature; The cells were screened; after removing the unadsorbed cell suspension, the above-mentioned KBM581 medium was added to resuspend the magnetic bead-cell mixture to obtain CD3-positive T lymphocytes, which were then used for lentivirus infection after continuing to culture for 24 hours.

c) Preparation of antigen-specific T lymphocytes by virus transfection

The above-mentioned CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method were added to the recombinant lentivirus corresponding to the number of CD3 positive cells for cultivation.

On the third day of culture, collect an appropriate number of T cells infected with lentivirus, and analyze the expression of CAR on the surface by flow cytometry. (b) T cells transduced with CAR-CD276 virus. Biotinylated anti-fab primary antibody was used for staining, followed by streptavidin-conjugated APC secondary antibody for flow cytometric analysis. The results in Figure 2 show that CAR-CD276 has 26% surface expression after subtracting the UTD background value, indicating that chimeric antigen receptor T cells targeting CD276 were successfully prepared. Continue to culture for 48 hours, collect chimeric antigen receptor T cells targeting CD276 for killing assay analysis or store in cell cryopreservation solution, place in a programmed cooling box at -80°C for 24 hours, and then transfer to liquid nitrogen irrigation Long-term preservation.

2. In vitro function test

The present invention has been tested in vitro, and the CD276-targeted chimeric antigen receptor T cells have killing activity on CD276-positive liver cancer cell lines. The specific methods and results are as follows:

(1) RTCA system was used to analyze the killing of CD276-positive tumor cell lines by T cells targeting CD276 chimeric antigen receptor.

First use 50ul DMEM medium to balance the RTCA single plate, then collect the cultured target cells Huh7, count the cells, add 50ul cell suspension containing 5000 Huh7 cells to each well, and place in a 37°C incubator for 15 minutes , and then placed in the RTCA resistance system, after 24 hours, collect the chimeric antigen receptor T cells targeting CD276, and count the cells according to the ratio of different effector cells to target cells (E:T) 10:1, 5: 1, 2.5:1 Add 100ul of chimeric antigen receptor T cell suspension corresponding to the number of cells into Huh7 cells, set up control group (control, plus culture medium group), UTD group (plus non-transduced virus T cells) , CAR-T group (plus chimeric antigen receptor T cell group targeting CD276), each group was set up with 3 duplicate wells, put into RTCA resistance system, and the killing activity was analyzed after 24 hours.

(2) Analysis of results

The UTD or CAR-T killing rate analysis was performed using the cell index at the specified time point.

The formula for calculating the UTD kill rate is:

UTD killing rate=(cell index value of control group-cell index value of UTD group)/cell index value of control group×100.

The formula for calculating the CAR-T killing rate is:

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

The results are shown in Figure 3. Compared with the UTD group, CAR-T has a stronger killing activity on Huh7 cells (Figure 3). It shows that the constructed CD276-targeting chimeric antigen receptor T cells have strong killing activity on positive tumor cell lines expressing CD276, which indicates that the CD276-targeting chimeric antigen receptor T cells are effective in the treatment of hepatocellular carcinoma. has a great application prospect.

The above content is only to illustrate the technical ideas of the present invention, and cannot limit the protection scope of the present invention. Any changes made on the basis of the technical solutions according to the technical ideas proposed in the present invention shall fall within the scope of the claims of the present invention. within the scope of protection.

sequence listing

<110> Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences

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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 gggtggagg atctggaggt gggggctctg gggtggggg 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 gcgccccagag 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 aggacccta 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 (10)

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.

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