CN113999320A - CD 276-targeted chimeric antigen receptor taking CD28 and 4-1BB as co-stimulation domains and application thereof - Google Patents

Abstract

The invention discloses a CD 276-targeted chimeric antigen receptor taking CD28 and 4-1BB as co-stimulation structural domains and application thereof, belonging to the technical field of biological medicines. The CD 276-targeted chimeric antigen receptor T cell disclosed by the invention takes CD28 and 4-1BB as co-stimulation structural domains, so that the CD 276-targeted chimeric antigen receptor T cell has strong killing activity on a positive tumor cell line expressing CD276, has stronger killing activity and durability in vivo, and is favorable for the generation of memory phenotype T cells. Through in vitro functional tests, the chimeric antigen receptor T cell targeting CD276 has killing activity on a CD276 positive hepatoma cell line.

Description

CD 276-targeted chimeric antigen receptor taking CD28 and 4-1BB as co-stimulation domains and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a chimeric antigen receptor and a chimeric antigen receptor T cell of a targeted CD276 with CD28 and 4-1BB as co-stimulation structural domains, and a preparation method and application thereof.

Background

Hepatocellular carcinoma is a malignant tumor seriously threatening human health, and the current treatment methods aiming at hepatocellular carcinoma, such as chemotherapy, radiotherapy, hepatectomy, ablation therapy, targeted therapy, such as sorafenib, antibody therapy, such as immune checkpoint inhibitor and vascular endothelial growth factor receptor inhibitor, cannot achieve good treatment effect.

The chimeric antigen receptor T cell therapy is a novel immune cell therapy method, and has the function of specifically and efficiently killing tumor cells expressing corresponding antigens. CD276, also known as B7-H3, is a transmembrane protein belonging to the B7 family. CD276 is highly expressed in a variety of cancers including lung adenocarcinoma, brain glioma, neuroblastoma, pancreatic carcinoma, ovarian carcinoma, and is rarely 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-targeted CAR-T cells have limited research on killing activity against hepatocellular carcinoma and other CD 276-highly expressing malignancies.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a chimeric antigen receptor targeting CD276 and using CD28 and 4-1BB as co-stimulation domains, a chimeric antigen receptor T cell, a preparation method and application.

In order to achieve the purpose, the invention adopts the following technical scheme to realize the purpose:

one aspect of the present invention is to provide a CD 276-targeted chimeric antigen receptor with CD28 and 4-1BB as co-stimulation domains, wherein the CD 276-targeted chimeric antigen receptor with CD28 and 4-1BB as co-stimulation domains comprises an amino acid sequence shown in SEQ ID NO. 1.

Preferably, the encoding gene of the CD 276-targeted chimeric antigen receptor taking CD28 and 4-1BB as co-stimulation domains comprises a nucleotide sequence shown as SEQ ID NO. 2.

Preferably, the CD276 targeting chimeric antigen receptor with CD28 and 4-1BB as co-stimulatory domains comprises the amino acid sequences of a single chain antibody targeting CD276, an extracellular hinge region, a transmembrane region, a co-stimulatory domain, a signal transduction region, P2A and the fluorescent protein EGFP, in order from the amino terminus to the 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 encoding gene of the single chain antibody targeting CD276 comprises a nucleotide sequence shown as SEQ ID NO. 4.

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

Still further preferably, the amino acid sequence of the CD8 a hinge region comprises the amino acid sequence shown as SEQ ID NO. 5.

Still more preferably, the gene encoding the CD8 a hinge region comprises the nucleotide sequence shown in 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 the CD8 α transmembrane region.

Still more preferably, the amino acid sequence of the CD8 a transmembrane region comprises the amino acid sequence shown in SEQ ID NO. 7.

Still more preferably, the gene encoding the transmembrane region of CD8 α comprises the nucleotide sequence shown in SEQ ID NO. 8.

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

Further preferably, the costimulatory domain consists of the intracellular domain of CD28, the intracellular domain of 4-1BB, connected in series.

Still more preferably, the amino acid sequence of said intracellular domain of CD28 is represented by SEQ ID NO. 9, and the nucleotide sequence encoding said intracellular domain of CD28 is represented by SEQ ID NO. 10.

More preferably, the amino acid sequence of the 4-1BB intracellular domain is represented by SEQ ID NO. 11 and the nucleotide sequence encoding the 4-1BB intracellular domain is represented by SEQ ID NO. 12.

Further preferably, the signaling region is the intracellular region CD3 ζ; the amino acid sequence of the intracellular domain of CD3 ζ is shown in SEQ ID NO 13.

Even more preferably, the gene encoding the intracellular domain of CD3 ζ comprises the nucleotide sequence set forth in SEQ ID NO. 14.

Further preferably, the amino acid sequence of P2A is shown as SEQ ID NO. 15; the nucleotide sequence encoding P2A is shown in SEQ ID NO 16.

Further preferably, the amino acid sequence of the fluorescent protein EGFP is shown as SEQ ID NO. 17; the nucleotide sequence of the coded fluorescent protein EGFP is shown as SEQ ID NO. 18.

The second aspect of the present invention provides a chimeric antigen receptor T cell targeting CD276, comprising the above chimeric antigen receptor targeting CD276 with CD28 and 4-1BB as co-stimulation domains; the amino acid sequence of the chimeric antigen receptor targeting CD276 with CD28 and 4-1BB as co-stimulation domains comprises the amino acid sequence shown as SEQ ID NO. 1.

Further, the gene encoding the chimeric antigen receptor CAR-CD276-28BB zeta of the targeting CD276 comprises a nucleotide sequence shown as SEQ ID NO. 2.

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

1) inserting a gene encoding a chimeric antigen receptor CAR-CD276-28BB zeta of a targeting CD276 with CD28 and 4-1BB as co-stimulation domains into a pWPXld vector to obtain a pWPXld-CAR-CD276-28BB zeta recombinant plasmid;

wherein, the nucleotide sequence of the coding gene of the chimeric antigen receptor CAR-CD276-28BB zeta of the targeting CD276 is shown as SEQ ID NO. 2;

2) co-transfecting the pWPXld-CAR-CD276-28BB zeta recombinant plasmid, an envelope plasmid and a packaging plasmid to a host cell to obtain a recombinant lentivirus;

3) and (3) transfecting the recombinant lentivirus with CD3 positive T lymphocytes, and separating to obtain the chimeric antigen receptor T cells targeting CD 276.

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

Further, in step 3), the CD3 positive T lymphocytes are obtained by separating from human peripheral blood mononuclear cells.

Further, 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 aspect of the present invention is to provide a recombinant viral vector comprising the gene encoding CAR-CD276-28BB ζ of the CD276 targeted chimeric antigen receptor T cell as described above.

The recombinant virus vector comprises a coding gene of CAR-CD276-28BB zeta of a chimeric antigen receptor T cell targeting CD276, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2 or SEQ ID NO. 19;

compared with the nucleotide sequence shown in SEQ ID NO. 2, the nucleotide sequence shown in SEQ ID NO. 19 has more coding genes of the signal peptide. The gene encoding the signal peptide can better guide the chimeric antigen receptor CAR-CD276-28BB zeta to be expressed on the cell surface.

Preferably, the gene encoding CAR-CD276-28BB zeta of the CD 276-targeted chimeric antigen receptor T cell is connected in series from the 5 'end to the 3' end by a gene encoding a CD8 alpha signal peptide, a gene encoding a single chain antibody targeted to CD276, a gene encoding a CD8 alpha extracellular hinge region, a gene encoding a CD8 alpha transmembrane region, a gene encoding a CD28 intracellular region, a gene encoding a 4-1BB intracellular region, a gene encoding a CD3 zeta intracellular region, a gene encoding a self-splicing element P2A and a gene encoding a fluorescent protein EGFP; wherein:

further preferably, the amino acid sequence of CAR-CD276-28BB ζ is set forth in SEQ ID NO: 20;

further preferably, the nucleotide sequence of the coding gene of the CD8 alpha signal peptide is shown as SEQ ID NO. 21;

further preferably, the amino acid sequence of the CD8 alpha signal peptide is shown in SEQ ID NO. 22.

The virus vector is pWPXld slow virus vector.

The fifth aspect of the present invention is to provide an application of the chimeric antigen receptor targeting CD276, the chimeric antigen receptor T cell targeting CD276 or the recombinant viral vector, which uses CD28 and 4-1BB as co-stimulation domains, in the preparation of a medicament for treating liver cancer.

Preferably, the agent has a strong killing activity against CD 276-expressing positive tumor cells, a long-lasting effect, and an increased production of T cells of the memory phenotype.

The invention also aims to provide a medicine for treating hepatocellular carcinoma, which comprises the CD 276-targeted chimeric antigen receptor taking CD28 and 4-1BB as co-stimulation domains, the CD 276-targeted chimeric antigen receptor T cell 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 targeting CD276, and T cells comprising the chimeric antigen receptor targeting CD 276. The chimeric antigen receptor T cell targeting CD276 can target tumor cells expressing CD276, activate the T cell to play a role in cellular immunity, efficiently and specifically kill CD276 positive tumor cells, better maintain the vitality and the lethality of the chimeric antigen receptor T cell, and is a potential immune cell therapy for radically treating hepatocellular carcinoma.

The CD 276-targeted chimeric antigen receptor T cell disclosed by the invention takes CD28 and 4-1BB as co-stimulation structural domains, so that the CD 276-targeted chimeric antigen receptor T cell has strong killing activity on a positive tumor cell line expressing CD276, has stronger killing activity and durability in vivo, and is favorable for the generation of memory phenotype T cells. Through in vitro functional tests, the chimeric antigen receptor T cell targeting CD276 has killing activity on a CD276 positive hepatoma cell line.

Drawings

FIG. 1 is a plasmid map of pWPXld-CAR-CD276-28BB zeta recombinant plasmid;

FIG. 2 is a graph showing the results of expression of chimeric antigen receptor CAR-CD276-28BB ζ on the surface of T cells; wherein (a) is a T cell of a UTD non-transduced virus; (b) (ii) a T cell that transduces the chimeric antigen receptor CAR-CD276-28BB ζ to CAR-T;

FIG. 3 is a graph showing the results of killing of a hepatocellular carcinoma cell line Huh7 by chimeric antigen receptor T cells, CAR-CD276-28BB ζ T cells.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that the terms "first," "second," and the like in the description and claims of the present invention and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or 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 accompanying drawings:

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

(1) gene sequence for preparing chimeric antigen receptor CAR-CD276-28BB zeta of targeting CD276

The CD276 targeting chimeric antigen receptor CAR-CD276-28BB zeta structure comprises a CD8 alpha signal peptide, a CD276 targeting single-chain antibody (CD276-scFv), a CD8 alpha hinge region, a CD8 alpha transmembrane region, a CD28 intracellular region, a 4-1BB intracellular region, a CD3 zeta signal region, a P2A and EGFP encoding genes, wherein the encoding genes of the signal peptides are shown as SEQ ID NO:21, the encoding genes of the CD276 targeting single-chain antibody comprise a nucleotide sequence shown as SEQ ID NO:4, the encoding genes of the CD8 alpha extracellular hinge region comprise a nucleotide sequence shown as SEQ ID NO:6, the encoding genes of the CD8 alpha transmembrane region comprise a nucleotide sequence shown as SEQ ID NO:8, the encoding genes of the CD28 intracellular region comprise a nucleotide sequence shown as SEQ ID NO:10, the encoding genes of the 4-1BB intracellular region comprise a nucleotide sequence shown as SEQ ID NO:12, and the encoding genes of the 4-1BB intracellular region comprise a nucleotide sequence shown as SEQ ID NO:12, The coding gene of the intracellular region of CD3 zeta comprises a nucleotide sequence shown as SEQ ID NO. 14, the coding gene of P2A comprises a nucleotide sequence shown as SEQ ID NO. 16, and the coding gene of the fluorescent protein EGFP comprises a nucleotide sequence shown as SEQ ID NO. 18.

The CD 276-targeted chimeric antigen receptor CAR-CD276-28BB zeta encoding gene is synthesized by Jiangsu Jinzhi biotechnology limited to obtain the CD 276-targeted chimeric antigen receptor CAR-CD276-28BB zeta encoding gene, and the CAR-CD276-28BB zeta encoding gene comprises a nucleotide sequence shown in SEQ ID NO. 19.

(2) Construction of pWPXld-CAR-CD276-28BB zeta recombinant plasmid

Inserting the coding gene of CAR-CD276-28BB zeta between BamH1 and EcoR1 enzyme cutting sites of pWPXld vector, transferring into Escherichia coli competent cell DH5 alpha, and performing positive clone PCR identification and sequencing identification. The map of the pWPXld-CAR-CD276-28BB zeta recombinant plasmid is shown in FIG. 1.

(3) Recombinant lentivirus construction

The pWPXld-CAR-CD276-28BB zeta recombinant plasmid, the packaging plasmid psPAX2, and the envelope plasmid pMD2G were co-transfected into cultured HEK293T cells using lipofectamine3000 transfection reagent. And collecting the supernatant containing the virus at 48h, firstly centrifuging at 2000rpm at room temperature for 5 minutes, taking the supernatant, and then filtering through a 0.45-micron filter membrane to obtain the recombinant lentivirus supernatant for T cell infection.

(4) Preparation of CD 276-targeted chimeric antigen receptor T cells

a) Isolation of PBMC (peripheral blood mononuclear cells)

PBMCs were derived from autologous venous blood of healthy volunteers.

Drawing blood of a volunteer, and sending the blood to a blood separation chamber; collecting peripheral blood mononuclear cells by using Ficoll, and taking middle layer cells after centrifugal separation; PBMC were obtained after counting by PBS wash.

b) Separation of antigen specific T lymphocyte by immunomagnetic bead method

Taking the PBMC, adding a KBM581 culture medium to prepare a 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 the cells incubated with the magnetic beads by using a magnet; after removal of the non-adsorbed liquid, the magnetic bead-cell mixture was resuspended by adding the above-mentioned KBM581 medium to obtain CD 3-positive T lymphocytes, which were further cultured for 24 hours before being used for lentivirus infection.

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

And (3) 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, appropriate numbers of lentivirus-infected T cells were collected and surface CAR expression was analyzed by flow cytometry, and the results are shown in figure 2, where (a) is UTD non-transduced viral T cells and (b) is CAR-T transduced chimeric antigen receptor CAR-CD276-28BB ζ T cells. Flow analysis of EGFP fluorescent protein expression. The results showed that CAR-CD276-28BB ζ was 19% surface expressed, indicating successful preparation of chimeric antigen receptor T cells targeting CD 276. Culturing is continued for 48 hours, and the chimeric antigen receptor T cells targeting CD276 are collected for analysis of effect examples or stored in cell freezing solution special for reinfusion, placed in a temperature-programmed box for storage at-80 ℃ for 24 hours, and then transferred to a liquid nitrogen tank for storage.

Second, in vitro functional test

Through in vitro functional tests, the chimeric antigen receptor T cell targeting CD276 has killing activity on a CD276 positive hepatoma cell line. The specific methods and results are as follows:

(1) the RTCA system was used to analyze killing of CD 276-targeted chimeric antigen receptor T cells against CD 276-positive tumor cell lines.

Firstly, 50ul of DMEM culture medium is used for balancing RTCA veneer, then cultured target cells Huh7 are collected, the cells are counted, 50ul of cell suspension containing 5000 Huh7 cells was added to each well, placed in an incubator at 37 ℃ for 15 minutes, then placing the cells in an RTCA resistance system, collecting chimeric antigen receptor T cells targeting CD276 after 24 hours, counting the cells, adding 100ul of chimeric antigen receptor T cell suspension with the corresponding cell number into Huh7 cells according to different ratios of effector cells to target cells (E: T) of 20:1, 10:1, 5:1, 2.5:1, setting a control group (control, culture medium group), a UTD group (T cells without virus transduction), a CAR-T group (CD 276-targeted chimeric antigen receptor T cell group), setting 3 multiple wells in each group, placing the cells in the RTCA resistance system, and performing killing activity analysis after 24 hours.

(2) Analysis of results

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

The UTD killing rate calculation formula is as follows:

UTD killing rate (cell index value of control group-cell index value of UTD group)/cell index value of control group x 100

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

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

The result is shown in fig. 3, CAR-T has stronger killing activity to Huh7 cells compared with UTD group, which indicates that the constructed chimeric antigen receptor T cell targeting CD276 has stronger killing activity to the positive tumor cell line expressing CD276, indicating that the chimeric antigen receptor T cell targeting CD276 has a greater application prospect in the treatment of hepatocellular carcinoma.

The above-mentioned contents are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and any modification made on the basis of 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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<213> Artificial Sequence (Artificial Sequence)

<400> 4

gaggtacaac tggtggagtc tggggggggc ctggttcagc ctgggggctc tctgagactg 60

agctgtgctg cctctggctt caccttcagc agctttggca tgcactgggt gagacaagcc 120

cctggcaagg gcctggagtg ggtggcctac atcagctctg acagctctgc catctactat 180

gctgacacag tgaagggcag attcaccatc agcagagaca atgccaagaa cagcctgtac 240

ctgcagatga acagcctgag agatgaggac acagctgtgt actactgtgg cagaggcaga 300

gagaacatct actatggcag cagactggac tattggggcc aaggcacaac agtgacagtc 360

agctctgggg gtggaggatc tggaggtggg ggctctgggg gtgggggatc tgacattcag 420

ctgacacaga gccctagctt cctgtctgcc tctgtggggg acagagtgac catcacctgc 480

aaggcttctc agaatgtgga caccaatgtg gcctggtatc agcagaagcc tggcaaggcc 540

cccaaggccc tgatctactc tgctagctac agatactctg gggtgcctag cagattctct 600

ggctctggct ctggcacaga cttcaccctg accatcagca gcctgcagcc tgaggacttt 660

gccacctact actgtcagca gtacaacaac taccccttca cctttggcca aggcactaag 720

ctggaaatca ag 732

<210> 5

<211> 45

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 6

<211> 135

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

accactaccc ctgcccctag accacctacc cctgccccaa caattgcatc tcagcccctg 60

agcttgagac ctgaagcatg cagacctgct gctggggggg ctgtgcacac aagaggcctg 120

gactttgcct gtgac 135

<210> 7

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210> 8

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atctacatct gggcccccct ggctggcacc tgtggggtgc tgctgctgag cctggtgatc 60

accctgtact gc 72

<210> 9

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 10

<211> 123

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

agaagcaaaa gaagtagact gctgcactct gactacatga acatgacccc tagaagacct 60

ggccccacaa gaaagcacta tcagccctat gcccccccta gagactttgc tgcctacaga 120

agc 123

<210> 11

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 12

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aagagaggga gaaagaagct gctgtacatc ttcaagcagc ccttcatgag acctgtgcag 60

accacccaag aggaggatgg ctgcagctgc agattccctg aggaggagga ggggggctgt 120

gagctg 126

<210> 13

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

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> 14

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

agagtgaagt tcagcagatc tgctgatgcc cctgcctaca agcaagggca gaatcagctc 60

tacaatgagc tgaacctggg cagaagagag gagtatgatg tgctggacaa gagaagaggc 120

agagaccctg agatgggggg caagcctaga agaaagaacc cccaagaggg cctgtataat 180

gagttgcaaa aggacaagat ggctgaggcc tactctgaga ttggcatgaa gggggagaga 240

agaagaggca agggccatga tggcctgtac caaggcctga gcacagccac caaggacacc 300

tatgatgccc tgcacatgca agccctgccc cctaga 336

<210> 15

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 16

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gccaccaact tcagcctgct gaagcaagct ggggatgtgg aggagaaccc tggcccc 57

<210> 17

<211> 239

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 18

<211> 720

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atggtgagca agggggagga gctgttcact ggggtggtgc ccatcctggt ggagctggat 60

ggggatgtga atggccacaa gttctctgtg tctggggagg gggaggggga tgccacctat 120

ggcaagctga ccctgaagtt catctgcacc actggcaagc tgcctgtgcc ctggcccacc 180

ctggtgacca ccctgaccta tggggtgcag tgcttcagca gataccctga ccacatgaag 240

cagcatgact tcttcaagtc tgccatgcct gagggctatg tgcaagagag aaccatcttc 300

ttcaaggatg atggcaacta caagacaaga gctgaggtga agtttgaggg ggacaccctg 360

gtgaacagaa ttgagctgaa gggcattgac ttcaaggagg atggcaacat cctgggccac 420

aagctggagt acaactacaa cagccacaat gtgtacatca tggctgacaa gcagaagaat 480

ggcatcaagg tgaacttcaa gatcagacac aacattgagg atggctctgt gcagctggct 540

gaccactatc agcagaacac ccccattggg gatggccctg tgctgctgcc tgacaaccac 600

tacctgagca cacagtctgc cctgagcaag gaccccaatg agaagagaga ccacatggtg 660

ctgctggagt ttgtgacagc tgctggcatc accctgggca tggatgagct gtacaagtga 720

<210> 19

<211> 2364

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

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 tacccctgcc cctagaccac ctacccctgc cccaacaatt 840

gcatctcagc ccctgagctt gagacctgaa gcatgcagac ctgctgctgg gggggctgtg 900

cacacaagag gcctggactt tgcctgtgac atctacatct gggcccccct ggctggcacc 960

tgtggggtgc tgctgctgag cctggtgatc accctgtact gcagaagcaa aagaagtaga 1020

ctgctgcact ctgactacat gaacatgacc cctagaagac ctggccccac aagaaagcac 1080

tatcagccct atgccccccc tagagacttt gctgcctaca gaagcaagag agggagaaag 1140

aagctgctgt acatcttcaa gcagcccttc atgagacctg tgcagaccac ccaagaggag 1200

gatggctgca gctgcagatt ccctgaggag gaggaggggg gctgtgagct gagagtgaag 1260

ttcagcagat ctgctgatgc ccctgcctac aagcaagggc agaatcagct ctacaatgag 1320

ctgaacctgg gcagaagaga ggagtatgat gtgctggaca agagaagagg cagagaccct 1380

gagatggggg gcaagcctag aagaaagaac ccccaagagg gcctgtataa tgagttgcaa 1440

aaggacaaga tggctgaggc ctactctgag attggcatga agggggagag aagaagaggc 1500

aagggccatg atggcctgta ccaaggcctg agcacagcca ccaaggacac ctatgatgcc 1560

ctgcacatgc aagccctgcc ccctagagcc accaacttca gcctgctgaa gcaagctggg 1620

gatgtggagg agaaccctgg ccccatggtg agcaaggggg aggagctgtt cactggggtg 1680

gtgcccatcc tggtggagct ggatggggat gtgaatggcc acaagttctc tgtgtctggg 1740

gagggggagg gggatgccac ctatggcaag ctgaccctga agttcatctg caccactggc 1800

aagctgcctg tgccctggcc caccctggtg accaccctga cctatggggt gcagtgcttc 1860

agcagatacc ctgaccacat gaagcagcat gacttcttca agtctgccat gcctgagggc 1920

tatgtgcaag agagaaccat cttcttcaag gatgatggca actacaagac aagagctgag 1980

gtgaagtttg agggggacac cctggtgaac agaattgagc tgaagggcat tgacttcaag 2040

gaggatggca acatcctggg ccacaagctg gagtacaact acaacagcca caatgtgtac 2100

atcatggctg acaagcagaa gaatggcatc aaggtgaact tcaagatcag acacaacatt 2160

gaggatggct ctgtgcagct ggctgaccac tatcagcaga acacccccat tggggatggc 2220

cctgtgctgc tgcctgacaa ccactacctg agcacacagt ctgccctgag caaggacccc 2280

aatgagaaga gagaccacat ggtgctgctg gagtttgtga cagctgctgg catcaccctg 2340

ggcatggatg agctgtacaa gtga 2364

<210> 20

<211> 787

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

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 Arg Ser

325 330 335

Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg

340 345 350

Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg

355 360 365

Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr

370 375 380

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

385 390 395 400

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Arg Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu

530 535 540

Asn Pro Gly Pro Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val

545 550 555 560

Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe

565 570 575

Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr

580 585 590

Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr

595 600 605

Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro

610 615 620

Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly

625 630 635 640

Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys

645 650 655

Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile

660 665 670

Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His

675 680 685

Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp

690 695 700

Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile

705 710 715 720

Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro

725 730 735

Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr

740 745 750

Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val

755 760 765

Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu

770 775 780

Leu Tyr Lys

785

<210> 21

<211> 63

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

atggccctgc ctgtgacagc cctgttactt cccctggccc tgttactgca tgctgctaga 60

cct 63

<210> 22

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

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 (17)

1. The CD 276-targeting chimeric antigen receptor taking CD28 and 4-1BB as co-stimulation domains is characterized in that the amino acid sequence of the CD 276-targeting chimeric antigen receptor is shown as SEQ ID NO. 1.

2. The CD 276-targeted chimeric antigen receptor taking CD28 and 4-1BB as co-stimulation domains as claimed in claim 1, wherein the nucleotide sequence encoding the CD 276-targeted chimeric antigen receptor is shown as SEQ ID NO. 2.

3. The CD 276-targeted chimeric antigen receptor with CD28 and 4-1BB as co-stimulation domains according to claim 1, wherein the CD 276-targeted chimeric antigen receptor comprises the amino acid sequences of a CD 276-targeted single-chain antibody, an extracellular hinge region, a transmembrane region, a co-stimulation domain, a signal transduction region, P2A and a fluorescent protein EGFP, which are sequentially connected from an amino terminal to a carboxyl terminal.

4. The CD 276-targeting chimeric antigen receptor using CD28 and 4-1BB as co-stimulatory domains according to claim 3, wherein the amino acid sequence of the CD 276-targeting single-chain antibody is shown as SEQ ID NO. 3, and the nucleotide sequence of the CD 276-targeting single-chain antibody is shown as SEQ ID NO. 4.

5. The chimeric antigen receptor targeting CD276 with CD28 and 4-1BB as co-stimulatory domains according to claim 3, wherein the extracellular hinge region is a CD8 α hinge region, the amino acid sequence of which is shown in SEQ ID NO. 5, and the nucleotide sequence encoding the CD8 α hinge region is shown in SEQ ID NO. 6.

6. The chimeric antigen receptor targeting CD276 with CD28 and 4-1BB as co-stimulation domains of claim 3, wherein the transmembrane region is CD8 α transmembrane region, the amino acid sequence of which is shown in SEQ ID NO. 7, and the nucleotide sequence coding for the CD8 α transmembrane region is shown in SEQ ID NO. 8.

7. The CD 276-targeted chimeric antigen receptor with CD28 and 4-1BB as co-stimulatory domains according to claim 3, wherein the co-stimulatory domain consists of a CD28 intracellular domain and a 4-1BB intracellular domain connected in series; wherein:

the amino acid sequence of the CD28 intracellular region is shown as SEQ ID NO. 9, and the nucleotide sequence for coding the CD28 intracellular region is shown as SEQ ID NO. 10;

the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO. 11, and the nucleotide sequence encoding the 4-1BB intracellular domain is shown in SEQ ID NO. 12.

8. The chimeric antigen receptor targeting CD276 with CD28 and 4-1BB as co-stimulatory domains according to claim 3, wherein the signal transduction region is CD3 ζ intracellular domain, and the amino acid sequence of CD3 ζ intracellular domain is shown in SEQ ID NO 13; the nucleotide sequence encoding the intracellular domain of CD3 ζ is shown in SEQ ID NO. 14.

9. The CD 276-targeted chimeric antigen receptor with CD28 and 4-1BB as co-stimulation domains according to claim 3, wherein the P2A amino acid sequence is shown as SEQ ID NO. 15; the nucleotide sequence for coding P2A is shown as SEQ ID NO. 16; the amino acid sequence of the fluorescent protein EGFP is shown as SEQ ID NO. 17; the nucleotide sequence of the coded fluorescent protein EGFP is shown as SEQ ID NO. 18.

10. A CD 276-targeted chimeric antigen receptor T cell comprising the CD 276-targeted chimeric antigen receptor of any one of claims 1 to 9, which has CD28 and 4-1BB as co-stimulatory domains.

11. A method for preparing a CD276 targeted chimeric antigen receptor T cell, comprising the steps of:

1) inserting a gene encoding a chimeric antigen receptor CAR-CD276-28BB zeta of a targeting CD276 with CD28 and 4-1BB as co-stimulation domains into a pWPXld vector to obtain a pWPXld-CAR-CD276-28BB zeta recombinant plasmid;

wherein, the nucleotide sequence of the coding gene of the chimeric antigen receptor CAR-CD276-28BB zeta of the targeting CD276 is shown as SEQ ID NO. 2;

2) co-transfecting the pWPXld-CAR-CD276-28BB zeta recombinant plasmid, an envelope plasmid and a packaging plasmid to a host cell to obtain a recombinant lentivirus;

3) and (3) transfecting the recombinant lentivirus with CD3 positive T lymphocytes, and separating to obtain the chimeric antigen receptor T cells targeting CD 276.

12. The method for producing a CD276 targeted chimeric antigen receptor T cell according to claim 11, 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 CD3 positive T lymphocyte is separated from human peripheral blood mononuclear cells.

13. A recombinant viral vector comprising a gene encoding CAR-CD276-28BB ζ of a chimeric antigen receptor T cell targeting CD276, wherein the nucleotide sequence of the gene is set forth in SEQ ID NO. 2 or SEQ ID NO. 19.

14. The recombinant viral vector according to claim 13, wherein the gene encoding CAR-CD276-28BB ζ of CD 276-targeted chimeric antigen receptor T cells is linked in tandem from 5 'end to 3' end by a gene encoding CD8 α signal peptide, a gene encoding single chain antibody targeting CD276, a gene encoding CD8 α extracellular hinge region, a gene encoding CD8 α transmembrane region, a gene encoding CD28 intracellular region, a gene encoding 4-1BB intracellular region, a gene encoding CD3 ζ intracellular region, a gene encoding self-cleaving element P2A, and a gene encoding fluorescent protein EGFP; wherein:

the amino acid sequence of CAR-CD276-28BB ζ is shown as SEQ ID NO: 20;

the nucleotide sequence of the coding gene of the CD8 alpha signal peptide is shown as SEQ ID NO. 21;

the amino acid sequence of the CD8 alpha signal peptide is shown in SEQ ID NO. 22.

15. Use of the CD276 targeting chimeric antigen receptor according to any one of claims 1 to 9, which uses CD28 and 4-1BB as co-stimulatory domains, the CD276 targeting chimeric antigen receptor T cell according to claim 10, or the recombinant viral vector according to claim 13 for the preparation of a medicament for the treatment of liver cancer.

16. The use of claim 13, wherein the medicament has a potent and durable killing activity against CD 276-expressing positive tumor cells and an enhanced production of T cells of a memory phenotype.

17. A drug for treating hepatocellular carcinoma, which comprises the CD 276-targeted chimeric antigen receptor with CD28 and 4-1BB as co-stimulatory domains according to any one of claims 1 to 9, the CD 276-targeted chimeric antigen receptor T-cell according to claim 10, or the recombinant viral vector according to claim 13.

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