CN107982538B

CN107982538B - Pharmaceutical composition and application thereof

Info

Publication number: CN107982538B
Application number: CN201711432469.2A
Authority: CN
Inventors: 不公告发明人
Original assignee: Shenzhen In Vivo Biomedicine Technology Ltd
Current assignee: Shenzhen In Vivo Biomedicine Technology Ltd
Priority date: 2017-12-26
Filing date: 2017-12-26
Publication date: 2021-10-22
Anticipated expiration: 2037-12-26
Also published as: CN107982538A

Abstract

The invention relates to the field of cellular immunotherapy of tumors, in particular to a pharmaceutical composition and application thereof, wherein the pharmaceutical composition comprises a drug specifically targeting renal tumor GPC3, and the drug specifically targeting renal tumor GPC3 comprises any one or combination of at least two of CAR-T cells, anti-GPC 3 antibodies, TCR-T cells targeting GPC3, DC-CIK targeting GPC3 or siRNA targeting silencing GPC 3. The invention discovers that GPC3 can be used as a novel target antigen of nephroblastoma, and the medicine composition can effectively kill and inhibit the nephroblastoma.

Description

Pharmaceutical composition and application thereof

Technical Field

The invention relates to the field of tumor cell immunotherapy, in particular to a pharmaceutical composition and application thereof.

Background

Nephroblastoma is the most common primary urinary system malignant tumor of children, has the characteristics of difficult early diagnosis, poor prognosis, easy recurrence and the like, accounts for 6-7 percent of malignant tumors of children under 15 years old, wherein about 75 percent of the malignant tumors are developed in children under 5 years old, patients with congenital deformity are usually developed before 1 year old, the average developed age is about 3.5 years old, the development rate is one ten thousandth, and the nephroblastoma is rare in adults.

In recent years, with the development of a comprehensive treatment scheme based on the mutual combination of surgery, chemotherapy and radiotherapy, the survival rate is obviously improved, and the survival rate reaches 87% 5 years after treatment, but a certain number of cases show higher recurrence rate and distant lesion metastasis rate due to tumor cell change, later clinical stage W and tumor rupture, so that the prognosis is poor, and a specific long-term treatment scheme of a nephroblastoma individual also brings higher prognosis risk. Studies have shown that the recurrence rate of nephroblastoma is close to 15%, and the long-term survival rate of relapsed patients is only 50%. However, both the inter-varietal renal blastoma and the chemotherapy drug insensitivity are associated with poor prognosis and, as the patient's survival increases, there is also a risk of secondary tumors. The existing research reports that after nephroblastoma is treated by chemotherapy, radiotherapy and the like, daily physical activity of a secondary Cusing syndrome, a long-term survivor of male nephroblastoma is reduced, non-hodgkin lymphoma, lymphoma cell leukemia, central nervous system leukemia and the like ("research progress of pathogenesis of nephroblastoma, zai huibo, wujian xin, chinese tumor, 2017) indicate that the existing treatment method has large body loss to patients, poor prognosis and reduced life quality after treatment, and a new treatment method with better curative effect and low side effect is urgently needed.

With the development of tumor-targeted immunotherapy such as monoclonal antibodies, CAR T cells, TCR T cells, etc., the discovery of tumor surface markers will provide a new approach for tumor therapy. Compared with the existing tumor chemotherapy, radiotherapy and surgical treatment, the tumor targeted immunotherapy has the advantages of targeted specificity, more obvious curative effect, low side effect, good prognosis, high life quality after healing and the like.

In addition, lack of effective tumor markers may lead to severe complications and adverse consequences from over-treatment of low risk cases and under-treatment of high risk cases. CN 102633864A discloses a GPC3 antigen polypeptide, a polyclonal antibody against GPC3 and application thereof, and the preparation of the polyclonal antibody against GPC3 shows that the polyclonal antibody can specifically recognize GPC3 protein, and is used for detecting GPC3 protein expressed by liver cancer cells, thereby providing help for liver cancer diagnosis. CN 104140974 a discloses a nucleic acid encoding a chimeric antigen receptor protein expressed on the surface of human T lymphocytes, comprising an extracellular binding region, a transmembrane region and an intracellular signaling region connected in sequence, wherein the extracellular binding region comprises a single chain antibody scFv (GPC3) specifically recognizing the C-terminal epitope of GPC3, said GPC3 is also expressed in tumors such as melanoma, clear cell carcinoma of the ovary, yolk sac tumor, neuroblastoma, etc. Therefore, GPC3 is mainly used for detecting liver cancer in the prior art, and cannot detect nephroblastoma.

Therefore, the discovery of new nephroblastoma markers and biological indicators is an important step in the refinement of treatment regimens and in the reduction of adverse reactions, and is also the key to the improvement of the survival rate and quality of life of children.

Disclosure of Invention

Aiming at the current problems, the invention provides a pharmaceutical composition and application thereof, which are used for detecting GPC3 transcription expression and protein expression in serum and/or CTC cells and/or kidney (tumor) tissue samples of patients with nephroblastoma through methods such as qrt-PCR, Eliza, immunohistochemistry, flow cytometry and the like, so as to carry out (early) diagnosis and prognosis evaluation on the nephroblastoma.

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

in one aspect, the invention provides a pharmaceutical composition comprising a drug that specifically targets renal tumor GPC 3.

In the invention, GPC3 is a novel antigen of nephroblastoma found by the inventor, and the inventor finds that CAR T cells, antibodies and the like targeting GPC3 antigen can effectively kill and inhibit nephroblastoma cells, and has the advantages of targeting specificity, more remarkable curative effect, low side effect, good prognosis, high quality of life after healing and the like.

According to the invention, the drug specifically targeting renal tumor GPC3 comprises any one or combination of at least two of CAR-T cells, anti-GPC 3 antibody, TCR-T cells targeting GPC3, DC-CIK targeting GPC3 or siRNA targeting silent GPC 3.

In the invention, the inventor constructs CAR T cells, anti-GPC 3 antibodies, TCR-T cells targeting GPC3, DC-CIK targeting GPC3 or siRNA targeting silencing GPC3 based on the neoantigen GPC3 of the nephroblastoma, and the nephroblastoma can kill and inhibit the nephroblastoma.

In the present invention, the drug may also be used in combination with other therapeutic techniques, including chemotherapy, radiation therapy, surgical resection or antibody therapy.

According to the invention, the GPC 3-targeted CAR-T cells are expressed by transfecting GPC 3-targeted recombinant virus into T cells.

According to the invention, the recombinant virus targeting GPC3 is used for transfecting a chimeric antigen receptor targeting GPC3 into an immune effector cell.

According to the invention, the immune effector cell is any one of T cell, B cell, NK cell, NKT cell, dendritic cell or macrophage or the combination of at least two of the T cell, the B cell, the NK cell, the NKT cell and the macrophage.

According to the invention, the chimeric antigen receptor targeting GPC3 comprises a signal peptide, an anti-GPC 3 antibody extracellular domain, a transmembrane region, an intracellular costimulatory signaling domain, and a CD3 zeta signaling domain.

According to the invention, the signal peptide is a human IgM signal peptide and/or a human CD8 a signal peptide, preferably a human IgM signal peptide.

According to the invention, the transmembrane region is any one of or a combination of at least two of a human CD28 transmembrane region, a human CD8 transmembrane region, a human 4-1BB transmembrane region or a transmembrane region of an intracellular costimulatory signal molecule, preferably a human CD28 transmembrane region and/or a human 4-1BB transmembrane region.

According to the invention, the intracellular co-stimulatory signaling domain further comprises at least one or a combination of two of CD, 4-1BB, CD, OX, CD, PD-1, ICOS, lymphocyte function-associated antigen 1, CD, LIGHT, NKG2, B-H, CDS, ICAM-1, GITR, BAFFR, HVEM, SLAMF, NKp, CD160, CD α, CD β, IL2 γ, IL7 α, ITGA, VLA, CD49, ITGA, IA, CD49, ITGA, VLA-6, CD49, ITGAD, CD11, ITGAE, CD103, ITGAL, CD11, CD-1, ITGAM, CD11, ITGAX, CD11, ITGB, CD GB, CD, ITGB, CD-1, ITGA, TNFR, TRANCEL/KL, LFM, SLF, CD160, ACAM, ACAP, TAMG, TARG, CD, TARG, TAM, TARG, TAM, TARG, TAM, TARG, TAM, TARG, TAM, TARG, preferably any one or a combination of at least two of CD27, OX40, CD40 or ICOS.

In the present invention, the chimeric antigen receptor is applicable as long as it can target GPC3, and targeting to wilms can be achieved by targeting GPC3 on wilms.

According to the invention, the chimeric antigen receptor targeting GPC3 is IgM-GPC3-CD28-OX40-CD3 xi, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO.1, and the amino acid sequence shown as SEQ ID NO.1 is as follows:

MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAAAACVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

according to the invention, the chimeric antigen receptor targeting GPC3 is IgM-GPC3-CD28-ICOS-CD3 xi, the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO.2, and the amino acid sequence shown as SEQ ID NO.2 is as follows:

MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAAAAKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHNGGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHANYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

according to the invention, the chimeric antigen receptor targeting GPC3 is IgM-GPC3-CD28-CD40-CD3 xi, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO.3, and the amino acid sequence shown as SEQ ID NO.3 is as follows:

MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAAAAVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILLVLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

according to the invention, the chimeric antigen receptor targeting GPC3 is IgM-GPC3-41BB-CD27-CD3 ξ, the amino acid sequence of which is shown as SEQ ID NO.4, and the amino acid sequence shown as SEQ ID NO.4 is as follows:

MLLLVTSLLLCELPHPAFLLIPDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSAAAAARPHPWWLCVLGTLVGLSATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSSDFIRILVIFSGMFLVFTLAGALFLHQRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSPKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

according to the present invention, the anti-GPC 3 antibody is an antibody having an antibody-dependent cell injury activity and/or a complement-dependent cell injury activity.

According to the invention, the amino acid sequence of the heavy chain of the anti-GPC 3 antibody is shown in SEQ ID NO.5, and the amino acid sequence shown in SEQ ID NO.5 is as follows:

QVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSA.

according to the invention, the amino acid sequence of the light chain of the anti-GPC 3 antibody is shown in SEQ ID NO.6, and the amino acid sequence shown in SEQ ID NO.6 is as follows:

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIK.

according to the invention, the nucleotide sequence of the siRNA targeting silencing GPC3 is shown in SEQ ID NO.7-16, and the nucleotide sequence of the siRNA of SEQ ID NO.7-16 is as follows:

sequence coding	Initiation site	siRNA sequence (DNA)	Region(s)	GC％
					SEQ ID NO.7	809	CCTGAAAGTATTTGGGAAT	ORF	36.85
SEQ ID NO.8	890	GGCTCTGAATCTTGGAATT	ORF	42.11
					SEQ ID NO.9	995	GGGACTGATGATGGTTAAA	ORF	42.11
SEQ ID NO.10	1178	CCATGATTCTATCCAGTAT	ORF	36.85
					SEQ ID NO.11	1190	CCAGTATGTCCAGAAGAAT	ORF	42.11
SEQ ID NO.12	1316	CCATTCTCAACAACGCCAA	ORF	47.37
					SEQ ID NO.13	1330	GCCAATATAGATCTGCTTA	ORF	36.85
SEQ ID NO.14	1424	CCGAAGAAGGGAACTAATT	ORF	42.11
					SEQ ID NO.15	1634	GCCAGTGGTCAGTCAAATT	ORF	47.37
SEQ ID NO.16	1832	CCTTGCAGAACTGGCCTAT	ORF	52.64

According to the present invention, the pharmaceutical composition is used in combination with any one or a combination of at least two of surgery, chemotherapy, or radiation therapy.

In a second aspect, the present invention provides a use of the pharmaceutical composition according to the first aspect for preparing a medicament for treating renal tumor cells.

According to the invention, the renal tumor cells are any one of or a combination of at least two of renal Squamous cell carcinoma (Squamous cell carcinoma), renin tumor (renomina), renal vascular lipoma (Angiomyolipoma), belini ductal carcinoma (Bellini duct carcinoma), renal Clear cell sarcoma (Clear-cell sarcoma of the kidney), Mesoblastic nephroma (mesoblast), Wilms' tumor (Nephroblastoma), Mixed epithelial stromal tumor (Mixed epithelial tumor), preferably Nephroblastoma.

According to the present invention, the nephroblastoma is an adult nephroblastoma and/or a pediatric nephroblastoma.

In a third aspect, the present invention provides a kit comprising a test substance which specifically detects the level of transcription or/and protein expression of the GPC3 gene.

Preferably, the kit comprises qRT-PCR primers and/or RT-PCR primers.

According to the invention, the nucleotide sequence of the qRT-PCR primer is shown in SEQ ID NO.17-22 or a nucleotide sequence with 80% identity, preferably 90% identity, with the nucleotide sequence shown in SEQ ID NO.17-22 as follows:

sequence coding	Sequences (DNA)
		SEQ ID NO.17 (upstream primer)	CCTTTGAAATTGTTGTTCGCCA
SEQ ID NO.18 (downstream primer)	CCTGGGTTCATTAGCTGGGTA
		Upstream primer of SEQ ID NO.19	CAGTAAGGACTGTGGCCGAAT
SEQ ID NO.20 (downstream primer)	AGCAGTACGTTCTCCATGTCAT
		Upstream primer of SEQ ID NO.21	ATTGGCAAGTTATGTGCCCAT
SEQ ID NO.22 (downstream primer)	TTCGGCTGGATAAGGTTTCTTC

According to the invention, the nucleotide sequence of the RT-PCR primer is shown in SEQ ID NO.23-24 or a nucleotide sequence with 80% identity, preferably 90% identity with the nucleotide sequence.

Sequence coding	Sequences (DNA)
		SEQ ID NO.23 (upstream primer)	GGTGGTGGCGATGCTGCTCAGCTTGGAC
SEQ ID NO.24 (lower)Swimming primer)	AGCTGGGTATAGATGACTGGAAACAG

In a fourth aspect, the present invention provides a use of the kit according to the third aspect for preparing a medicament and/or a reagent for detecting and/or treating renal tumor cells.

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

(1) the invention discovers that GPC3 can be used as a novel targeting antigen of nephroblastoma, and the medicine composition can effectively kill and inhibit the nephroblastoma;

(2) in the invention, the pharmaceutical composition can be a medicament or a reagent constructed by methods of lentivirus, qRT-PCR, Eliza, immunohistochemistry, flow cytometry and the like, is a new target of GPC3 for nephroblastoma, can detect the transcription expression and protein expression of GPC3, and can perform early diagnosis and prognosis evaluation on the nephroblastoma;

(3) the invention provides a new idea for diagnosing and treating the nephroblastoma as a new discovery of the nephroblastoma.

Drawings

FIG. 1 is a graph of the efficiency of lentivirus infection of T cells by GPC3-CAR of the present invention;

FIG. 2 is a graph showing that GPC3-CAR T cells detect in vitro immune killing effects on nephroblastoma cells by in vitro tumor cell killing assays in accordance with the present invention;

FIG. 3 is a graph showing the detection of IL-2 immunocytokine secretion by Eliza assay on CAR T cells, wherein Mock-CAR T is a blank control group and GPC3-CAR T is an experimental group;

FIG. 4 is a graph showing the detection of IFN- γ immunocytokine secretion from CAR T cells by Eliza assay, wherein Mock-CAR T is a blank control group and GPC3-CAR T is an experimental group;

FIG. 5 is a graph of the results of effective killing and inhibition of nephroblastoma in vivo by CAR GPC3T cells, wherein Mock-CAR T is a blank control group and GPC3-CAR T is an experimental group;

FIG. 6 is a graph showing the inhibition of wilms' tumors by GPC3 of the present invention, wherein V22 is a circle black dot, V209 is a square black box, V1608 is a regular triangle, and WT is a blank control inverted triangle;

FIG. 7 is an electrophoretogram of GPC3 detected by RT-PCR and compared in samples of nephroblastoma tissue and normal kidney tissue, wherein marker is 100-500bp, 1-nephroblastoma tissue 1, 2-nephroblastoma tissue 2, 3-nephroblastoma tissue 3, 4-nephroblastoma tissue 4, 5-nephroblastoma tissue 5, 6-normal kidney.

Detailed Description

To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Material

Invitrogen corporation, T4DNA ligase.

Example 1: construction of GPC3-CAR Lentiviral vectors

(1) GPC3-CD28-OX40 (abbreviation: G28O ζ, i.e., tandem human IgM signal peptide, anti-GPC 3 antibody extracellular domain, human CD28 transmembrane domain, human OX40 intracellular domain, and human CD3 ζ signaling domain), GPC3-CD28-ICOS (abbreviation: G28 28 ζ, i.e., tandem human IgM signal peptide, anti-GPC 28 antibody extracellular domain, human CD28 transmembrane domain, human ICOS intracellular domain, and human CD28 ζ signaling domain), GPC 28-CD 28-CD 28 (abbreviation: G2840 ζ, i.e., tandem human IgM signal peptide, anti-GPC 28 antibody extracellular domain, human CD28 transmembrane domain, human CD28 intracellular domain, and human CD28 ζ signaling domain), GPC 28-41 BB-CD 28 (abbreviation: GBB 28, i.e., tandem human IgM signal extracellular peptide, anti-GPC 28 antibody transmembrane domain, human CD28 intracellular domain, and human CD28 ζ signaling domain), GPC 28-CD 28-CD 28-CD 28 zeta signaling domain, total gene sequence was synthesized as a control nucleic acid sequence, the synthesized gene C end contains restriction enzyme Pme1 restriction enzyme cutting site and its protection base, and the N end contains restriction enzyme Spe1 restriction enzyme cutting site and its protection base;

(2) performing double enzyme digestion by restriction enzymes Pme1 and Spe1 respectively, and performing gel electrophoresis to recover and obtain synthetic DNA fragments G28O zeta, G28I zeta, G2840 zeta, GBB27 zeta and Mock containing cohesive ends and linearized DNA of a pWPXld-eGFP lentiviral vector containing cohesive ends;

(3) linearized pWPXLD-2A-EGFP was ligated to cohesive-ended synthetic DNA fragments G28O ζ, G28I ζ, G2840 ζ, GBB27 ζ, and Mock by T4DNA ligase (Invitrogen corporation) to obtain the series of GPC3CAR plasmid transformation vectors pWPXLD-G28O ζ -2A-EGFP, pWPXLD-G28I ζ -2A-EGFP, pWPXLD-G2840 ζ -2A-EGFP, pWPXLD-GBB27 ζ -2A-EGFP, and pWPXLD-Mock-2A-EGFP.

The amino acid sequence of IgM-GPC3-CD28-OX40-CD3 xi is shown as SEQ ID NO.1

The amino acid sequence of the IgM-GPC3-CD28-ICOS-CD3 ξ is shown as SEQ ID NO. 2;

the amino acid sequence of the IgM-GPC3-CD28-CD40-CD3 ξ is shown as SEQ ID NO. 3;

the amino acid sequence of the IgM-GPC3-41BB-CD27-CD3 ξ is shown as SEQ ID NO. 4.

Example 2 GPC3-CAR lentivirus infected T cells GPC3-CAR T cells

(1) 293T cells were cultured in 10cm dishes in the following media: DMEM high glucose medium + 10% FBS (fetal bovine serum) + 1% diabody (100 × penicillin-streptomycin mixed solution);

(2) when the density of 293T cells in a 150mm culture dish reaches 80-90%, the culture medium is replaced: DMEM high-glucose medium + 1% FBS + 1% double antibody;

(3) after culture for 2-6 hours by changing the culture medium, respectively co-transferring pWPXld-GPC3CAR-EGFP plasmids (pWPXld-G28O zeta-2A-EGFP, pWPXld-G28I zeta-2A-EGFP, pWPXld-G2840 zeta-2A-EGFP, pWPXld-GBB27 zeta-2A-EGFP and pWPXld-Mock-2A-EGFP) with lentivirus packaging helper plasmids pMD2.G and psX 2 into 293T cells by using PEI, wherein the reaction system is as follows:

reagent	Dosage form
		pWPXld-CAR-EGFP plasmid	9μg
pMD2.G helper plasmid	3μg
		psPAX2	12μg
PEI	72μg

(4) At 24, 48 and 72 hours post-transformation, respectively, medium supernatants were collected and fresh medium was added: DMEM high-glucose medium + 1% FBS + 1% double antibody;

(5) after collecting the culture medium supernatant, centrifuging 2500g of the supernatant for 0.5 hour;

(6) taking the centrifugal supernatant, filtering the centrifugal supernatant by using a 0.45um filter, and centrifuging the centrifugal supernatant for 1.5 hours by using a super high speed centrifuge at 28000 rpm;

(7) after ultra-high speed centrifugation, gently removing the supernatant, adding 200ul PBS, and dissolving at 4 ℃ for 12-16 hours to obtain CAR lentivirus;

(8) after the virus is dissolved, collecting virus solution, subpackaging in a PCR tube, and freezing and storing at-80 ℃ for later use.

The result of infecting the T cells with the GPC3-CAR lentivirus is shown in FIG. 1, and as can be seen from FIG. 1, the GPC3-CAR lentivirus infection efficiency can reach 56.3%, and the GPC3-CAR lentivirus can effectively infect the T cells.

Example 3 in vitro killing assay of Reniloblastoma by GPC3-CAR T cells

The CAR GPC3T cells and CAR Mock T cells prepared in example 2 were mixed with 1 × 10 cells at different ratios of 3:1, 1:3, 1:9, respectively⁴Mixing G401 cells (nephroblastoma cell line), adding into a 96-well U-shaped plate, setting 3 multiple wells for each group, setting a single tumor cell group as a positive control, centrifuging for 5min at 250G, and then placing in a 37-degree 5% CO2 incubator for CO-culture for 18 h;

luciferase (Luciferase) killing quantitative killing efficiency: after 18 hours after the CAR T cells were co-cultured with tumor cells or tumor cells were cultured alone, 100 μ l/well of luciferase substrate (1 ×) was added to a 96-well cell culture plate, the cells were resuspended and mixed well, rlu (relative light unit) was immediately measured by a multifunctional plate reader for 1 second;

the killing proportion calculation formula is as follows: 100% × (control well reading-experimental well reading)/control well reading (blank reading without cells negligible)

The results are shown in fig. 2, and indicate that the CAR GPC3T cell has strong cell killing capacity, which can reach more than 70%, compared with the control group, and thus, the CAR GPC3T cell can effectively kill the nephroblastoma cell.

Example 4 GPC3 antigen-specifically activated GPC3CAR T cells secrete cytokines

Cytokine IL-2 and IFN-gamma levels in the supernatant after 18h coculture of CAR GPC3T cells and CAR Mock T cells with nephroblastoma cells, respectively, were measured by ELISA and the results are shown in FIGS. 3-4.

As can be seen from the results in FIG. 3, the cytokine IL-2 level after stimulation can reach 1000pg/mL, and the cytokine IFN-gamma level after stimulation can reach 1400pg/mL, and it can be seen that nephroblastoma can stimulate CAR GPC3T cells to produce immune effects.

Example 5 GPC3-CAR T cells in vivo killing assay for Reniloblastoma

The specific method comprises the following steps:

(1) the number of cells was 1X 10⁵The nephroblastoma is transplanted into NOD/SCID IL2 rg-/-immunodeficient mice, a nephroblastoma xenograft mouse model is constructed, and the inhibition effect of CAR GPC3T cells on the growth of the nephroblastoma cells is evaluated in vivo;

(2) 7 and 14 days after tumor transplantation, the number of cells injected intravenously was 2X 10 in the wilms tumor xenograft mouse model⁶The GPC3CAR T cells and CAR Mock T cells of (a), for a total of two experimental groups, each set was set to 5 replicates;

(3) measuring the sizes of subcutaneous tumor masses of the mice of the two experimental groups by using vernier calipers at 7 days, 14 days, 21 days and 28 days after CAR T cell infusion, recording, and drawing a tumor growth curve graph, wherein the result is shown in FIG. 5;

from the results in FIG. 5, it was revealed that GPC3CAR T cells recognized and killed nephroblastoma cells in vivo, and that GPC3CAR T cells caused nephroblastoma to have a size of 10mm from the beginning by comparison with the control group³15mm after 14 days³And 8mm after 28 days³It can be seen that GPC3CAR T cells effectively inhibited wilms tumor growth.

Example 6 Effect of the monoclonal antibody GPC3 on nephroblastoma

(1) Constructing a vector pC-aGPChl for expressing the resistance of an anti-GPC 3 antibody and geneticin to obtain pC-aGPChl-anti GPC3, and transferring the pC-aGPChl-anti GPC3 into CHO cells in an electrotransformation mode; the CHO cells after electroporation were incubated at 37 ℃ with 8% CO₂Culturing in an incubator; after the transformed plasmid is stably expressed, the screening medium is replaced: CHO-S-SFMII Medium containing 400. mu.g/ml Geneticin, the selected cells were seeded at a density of 0.4 cells/100. mu.l/well into 96-well plates and CHO-S-SFMII Medium containing 400. mu.g/ml GeneticinObtaining monoclonal cells by limiting dilution;

the amino acid sequence of the heavy chain of the monoclonal antibody (SEQ ID NO.5) is as follows:

QVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHWVKQTPVHGLKWIGALDPKTGDTAYSQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRFYSYTYWGQGTLVTVSA；

the amino acid sequence of the light chain of the monoclonal antibody (SEQ ID NO.6) is as follows:

DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHVPPTFGSGTKLEIK；

(2) the culture supernatant of CHO cells expressing the antibody GPC3 was applied to an rProtein A Sepharose Fast Flow column equilibrated with 10mM citrate-phosphate buffer solution (pH7.5) containing 150mM NaCl, washed with 10mM citrate-phosphate buffer solution (pH7.5) containing 1M NaCl and then with 10mM citrate-phosphate buffer solution (pH7.5), and the protein adsorbed on the column was eluted with 20mM acetic acid. Adding 1M tris-HCl buffer (pH 8.5) to a 20mM acetic acid fraction containing an antibody to GPC3 to adjust the pH from 5 to 6, filtering with a 0.22 μ M filter, adding an equal amount of deionized water, loading onto a SP Sepharose Fast Flow column equilibrated with 20mM acetic acid buffer (pH6.0), washing the column with the same buffer, and then eluting the protein adsorbed on the column with 20mM acetic acid buffer (pH6.0) containing 20mM NaCl to obtain a purified GPC3 antibody;

(3) adding 50. mu.l of GPC3 antibody solutions prepared at different concentrations to the nephroblastoma cells, and reacting at room temperature for 15 minutes; next, 100. mu.l of effector cell suspension derived from human peripheral blood (5X 105 cells/well) was added, centrifuged, and cultured at 37 ℃ for 4 hours in a 5% CO2 incubator to examine the percentage of killing of wilms' tumor cells, and a data statistical chart was prepared, and the results are shown in FIG. 6.

From the results in fig. 6, it is shown that GPC3 antibody (V22, V209, and V1608 clones) is effective in promoting recognition killing and inhibition of nephroblastoma by immune effector cells.

Example 7 detection of the Effect of reverse transcription expression of GPC3 on nephroblastoma

(1) Treatment of peripheral blood leukocytes/ctcs (circulating tumor cells):

obtaining peripheral blood of a patient with nephroblastoma, adding erythrocyte lysate (eBioscience) to lyse erythrocytes for 5 minutes at room temperature, adding 5mL of PBS to neutralize the erythrocyte lysate, centrifuging for 5 minutes at 300g to obtain peripheral blood leukocytes, and preparing RNA for later use; or further obtaining CK18+ DAPI + cells (namely CTC cells) in peripheral blood leukocytes by a flow cytometry sorting technology (Beckman Coulter) to be ready for RNA extraction;

(2) treatment of kidney/nephroblastoma tissue samples:

solid kidney/nephroblastoma tissue was minced, washed with DMEM/F12(Lonza), and then incubated at 37 deg.C for 1 hour with Accumax 1(Innovative Cell Technologies). Filtering the digested tissue through a 70-m cell filter to obtain a single cell suspension, then gently placing the single cell suspension on a Histopaque-1077gradient (Sigma-Aldrich), centrifuging at the room temperature of 400Xg for 30 minutes, removing red blood cells, necrotic cells and debris at the bottom of a tube, collecting the viable nucleated cells, and preparing RNA for later use;

(3) RNA extraction:

freezing and storing the lysed cells, and standing for 5 minutes at room temperature to completely dissolve the cells;

② two-phase separation 0.2ml chloroform is added into each 1ml sample cracked by TRIZOL reagent, and the tube cover is tightly covered. After shaking the tube vigorously by hand for 15 seconds, incubating at 15-30 ℃ for 2-3 minutes, centrifuging at 12000rpm for 15 minutes at 4 ℃, separating the mixed liquid into a lower red phenol chloroform phase, an intermediate layer and a colorless aqueous phase, and distributing the RNA into the aqueous phase. The volume of the upper aqueous layer was approximately 60% of the TRIZOL reagent added during homogenization;

thirdly, transferring the upper layer of the water phase to a clean centrifugal tube without RNase by RNA precipitation, adding equal volume of isopropanol to mix so as to precipitate RNA in the water phase, incubating for 10 minutes at 15-30 ℃ after mixing uniformly, and centrifuging for 10 minutes at 12000rpm at 4 ℃, wherein the RNA precipitation which is not visible before centrifugation forms colloidal precipitation blocks on the bottom and the side wall of the tube;

fourthly, cleaning RNA, removing supernatant, adding at least 1ml of 75% ethanol (the 75% ethanol is prepared by DEPCH 2O) into each 1ml of sample cracked by the TRIZOL reagent, cleaning RNA sediment, mixing uniformly, and centrifuging at 7000rpm at 4 ℃ for 5 minutes;

fifthly, drying the RNA, carefully absorbing most of ethanol solution, and drying the RNA precipitate in air at room temperature for 5-10 minutes;

sixthly, when dissolving the RNA precipitate and dissolving the RNA, adding 40 mu l of RNase-free water and repeatedly blowing and beating the mixture by a gun for several times to completely dissolve the RNA precipitate, detecting the purity and the concentration of the RNA by a spectrophotometer, and storing the obtained RNA solution at-80 ℃ for later use;

(4) after DNA removal of the RNA sample by the TAKARA reverse transcription kit step, reverse transcription reaction is carried out to obtain sample cDNA.

Designing RT-PCR or qRT-PCR primers of GPC3, wherein the nucleotide sequence of the qRT-PCR primers is shown in SEQ ID NO. 17-22:

The expression of GPC3 in the sample was measured, and the results are shown in FIG. 7.

From the results in FIG. 7, it is shown that GPC3mRNA was significantly highly expressed in the patient sample of nephroblastoma, and therefore, the mRNA expression of GPC3 in the patient peripheral blood/kidney tissue sample was detected by RT-PCR/qRT-PCR method to diagnose nephroblastoma.

In summary, the pharmaceutical composition of the present invention can be a drug or a reagent constructed by methods such as lentivirus, qRT-PCR, Eliza, immunohistochemistry, flow cytometry, antibody construction, etc., which are novel targets of GPC3 of nephroblastoma, and can detect the transcription expression and protein expression of GPC3, and can perform early diagnosis and prognosis evaluation of nephroblastoma.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

<110> Shenzhen City internal biomedical science and technology Limited

<120> a pharmaceutical composition and application thereof

<130> 2017

<141> 2017-12-26

<160> 24

<170> SIPOSequenceListing 1.0

<210> 1

<211> 697

<212> PRT

<213> Artificial Synthesis sequence ()

<400> 1

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser

20 25 30

Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser

35 40 45

Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

50 55 60

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn

65 70 75 80

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

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

100 105 110

Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly

115 120 125

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

145 150 155 160

Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His

180 185 190

Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala

195 200 205

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

210 215 220

Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln

245 250 255

Gly Thr Leu Val Thr Val Ser Ala Ala Ala Ala Cys Val Gly Ala Arg

260 265 270

Arg Leu Gly Arg Gly Pro Cys Ala Ala Leu Leu Leu Leu Gly Leu Gly

275 280 285

Leu Ser Thr Val Thr Gly Leu His Cys Val Gly Asp Thr Tyr Pro Ser

290 295 300

Asn Asp Arg Cys Cys His Glu Cys Arg Pro Gly Asn Gly Met Val Ser

305 310 315 320

Arg Cys Ser Arg Ser Gln Asn Thr Val Cys Arg Pro Cys Gly Pro Gly

325 330 335

Phe Tyr Asn Asp Val Val Ser Ser Lys Pro Cys Lys Pro Cys Thr Trp

340 345 350

Cys Asn Leu Arg Ser Gly Ser Glu Arg Lys Gln Leu Cys Thr Ala Thr

355 360 365

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

370 375 380

Tyr Lys Pro Gly Val Asp Cys Ala Pro Cys Pro Pro Gly His Phe Ser

385 390 395 400

Pro Gly Asp Asn Gln Ala Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala

405 410 415

Gly Lys His Thr Leu Gln Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys

420 425 430

Glu Asp Arg Asp Pro Pro Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro

435 440 445

Pro Ala Arg Pro Ile Thr Val Gln Pro Thr Glu Ala Trp Pro Arg Thr

450 455 460

Ser Gln Gly Pro Ser Thr Arg Pro Val Glu Val Pro Gly Gly Arg Ala

465 470 475 480

Val Ala Ala Ile Leu Gly Leu Gly Leu Val Leu Gly Leu Leu Gly Pro

485 490 495

Leu Ala Ile Leu Leu Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu

500 505 510

Pro Pro Asp Ala His Lys Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro

515 520 525

Ile Gln Glu Glu Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile Lys

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Leu His Met Gln Ala Leu Pro Pro Arg

690 695

<210> 2

<211> 619

<212> PRT

<213> Artificial Synthesis sequence ()

<400> 2

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser

20 25 30

Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser

35 40 45

Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

50 55 60

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn

65 70 75 80

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

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

100 105 110

Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly

115 120 125

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

145 150 155 160

Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His

180 185 190

Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala

195 200 205

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

210 215 220

Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln

245 250 255

Gly Thr Leu Val Thr Val Ser Ala Ala Ala Ala Lys Ser Gly Leu Trp

260 265 270

Tyr Phe Phe Leu Phe Cys Leu Arg Ile Lys Val Leu Thr Gly Glu Ile

275 280 285

Asn Gly Ser Ala Asn Tyr Glu Met Phe Ile Phe His Asn Gly Gly Val

290 295 300

Gln Ile Leu Cys Lys Tyr Pro Asp Ile Val Gln Gln Phe Lys Met Gln

305 310 315 320

Leu Leu Lys Gly Gly Gln Ile Leu Cys Asp Leu Thr Lys Thr Lys Gly

325 330 335

Ser Gly Asn Thr Val Ser Ile Lys Ser Leu Lys Phe Cys His Ser Gln

340 345 350

Leu Ser Asn Asn Ser Val Ser Phe Phe Leu Tyr Asn Leu Asp His Ser

355 360 365

His Ala Asn Tyr Tyr Phe Cys Asn Leu Ser Ile Phe Asp Pro Pro Pro

370 375 380

Phe Lys Val Thr Leu Thr Gly Gly Tyr Leu His Ile Tyr Glu Ser Gln

385 390 395 400

Leu Cys Cys Gln Leu Lys Phe Trp Leu Pro Ile Gly Cys Ala Ala Phe

405 410 415

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

420 425 430

Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr Met Phe

435 440 445

Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

610 615

<210> 3

<211> 697

<212> PRT

<213> Artificial Synthesis sequence ()

<400> 3

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser

20 25 30

Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser

35 40 45

Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

50 55 60

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn

65 70 75 80

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

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

100 105 110

Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly

115 120 125

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

145 150 155 160

Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His

180 185 190

Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala

195 200 205

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

210 215 220

Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln

245 250 255

Gly Thr Leu Val Thr Val Ser Ala Ala Ala Ala Val Arg Leu Pro Leu

260 265 270

Gln Cys Val Leu Trp Gly Cys Leu Leu Thr Ala Val His Pro Glu Pro

275 280 285

Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln Cys Cys

290 295 300

Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr Glu Phe

305 310 315 320

Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr

325 330 335

Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp Pro Asn

340 345 350

Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile

355 360 365

Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser

370 375 380

Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile

385 390 395 400

Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe

405 410 415

Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser

420 425 430

Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr

435 440 445

Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg Ala Leu Val Val Ile

450 455 460

Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile Leu Leu Val Leu Val Phe

465 470 475 480

Ile Lys Lys Val Ala Lys Lys Pro Thr Asn Lys Ala Pro His Pro Lys

485 490 495

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

500 505 510

Thr Ala Ala Pro Val Gln Glu Thr Leu His Gly Cys Gln Pro Val Thr

515 520 525

Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser Val Gln Glu Arg Gln Lys

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Leu His Met Gln Ala Leu Pro Pro Arg

690 695

<210> 4

<211> 680

<212> PRT

<213> Artificial Synthesis sequence ()

<400> 4

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Val Val Met Thr Gln Thr Pro Leu Ser

20 25 30

Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser

35 40 45

Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu

50 55 60

Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn

65 70 75 80

Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr

85 90 95

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

100 105 110

Tyr Phe Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Ser Gly

115 120 125

Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

130 135 140

Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu

145 150 155 160

Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Asp Tyr Glu Met His Trp Val Lys Gln Thr Pro Val His

180 185 190

Gly Leu Lys Trp Ile Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala

195 200 205

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

210 215 220

Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Tyr Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln

245 250 255

Gly Thr Leu Val Thr Val Ser Ala Ala Ala Ala Ala Arg Pro His Pro

260 265 270

Trp Trp Leu Cys Val Leu Gly Thr Leu Val Gly Leu Ser Ala Thr Pro

275 280 285

Ala Pro Lys Ser Cys Pro Glu Arg His Tyr Trp Ala Gln Gly Lys Leu

290 295 300

Cys Cys Gln Met Cys Glu Pro Gly Thr Phe Leu Val Lys Asp Cys Asp

305 310 315 320

Gln His Arg Lys Ala Ala Gln Cys Asp Pro Cys Ile Pro Gly Val Ser

325 330 335

Phe Ser Pro Asp His His Thr Arg Pro His Cys Glu Ser Cys Arg His

340 345 350

Cys Asn Ser Gly Leu Leu Val Arg Asn Cys Thr Ile Thr Ala Asn Ala

355 360 365

Glu Cys Ala Cys Arg Asn Gly Trp Gln Cys Arg Asp Lys Glu Cys Thr

370 375 380

Glu Cys Asp Pro Leu Pro Asn Pro Ser Leu Thr Ala Arg Ser Ser Gln

385 390 395 400

Ala Leu Ser Pro His Pro Gln Pro Thr His Leu Pro Tyr Val Ser Glu

405 410 415

Met Leu Glu Ala Arg Thr Ala Gly His Met Gln Thr Leu Ala Asp Phe

420 425 430

Arg Gln Leu Pro Ala Arg Thr Leu Ser Thr His Trp Pro Pro Gln Arg

435 440 445

Ser Leu Cys Ser Ser Asp Phe Ile Arg Ile Leu Val Ile Phe Ser Gly

450 455 460

Met Phe Leu Val Phe Thr Leu Ala Gly Ala Leu Phe Leu His Gln Arg

465 470 475 480

Arg Lys Tyr Arg Ser Asn Lys Gly Glu Ser Pro Val Glu Pro Ala Glu

485 490 495

Pro Cys Arg Tyr Ser Cys Pro Arg Glu Glu Glu Gly Ser Thr Ile Pro

500 505 510

Ile Gln Glu Asp Tyr Arg Lys Pro Glu Pro Ala Cys Ser Pro Lys Arg

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

His Met Gln Ala Leu Pro Pro Arg

675 680

<210> 5

<211> 115

<212> PRT

<213> Artificial Synthesis sequence ()

<400> 5

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

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Lys Trp Ile

35 40 45

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

50 55 60

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

65 70 75 80

Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Val Ser Ala

115

<210> 6

<211> 112

<212> PRT

<213> Artificial Synthesis sequence ()

<400> 6

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Asn

85 90 95

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

100 105 110

<210> 7

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 7

cctgaaagta tttgggaat 19

<210> 8

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 8

ggctctgaat cttggaatt 19

<210> 9

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 9

gggactgatg atggttaaa 19

<210> 10

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 10

ccatgattct atccagtat 19

<210> 11

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 11

ccagtatgtc cagaagaat 19

<210> 12

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 12

ccattctcaa caacgccaa 19

<210> 13

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 13

gccaatatag atctgctta 19

<210> 14

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 14

ccgaagaagg gaactaatt 19

<210> 15

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 15

gccagtggtc agtcaaatt 19

<210> 16

<211> 19

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 16

ccttgcagaa ctggcctat 19

<210> 17

<211> 22

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 17

cctttgaaat tgttgttcgc ca 22

<210> 18

<211> 21

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 18

cctgggttca ttagctgggt a 21

<210> 19

<211> 21

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 19

cagtaaggac tgtggccgaa t 21

<210> 20

<211> 22

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 20

agcagtacgt tctccatgtc at 22

<210> 21

<211> 21

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 21

attggcaagt tatgtgccca t 21

<210> 22

<211> 22

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 22

ttcggctgga taaggtttct tc 22

<210> 23

<211> 28

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 23

ggtggtggcg atgctgctca gcttggac 28

<210> 24

<211> 26

<212> DNA

<213> Artificial Synthesis sequence ()

<400> 24

agctgggtat agatgactgg aaacag 26

Claims

1. A pharmaceutical composition comprising a drug that specifically targets renal tumor GPC3, wherein the drug that specifically targets renal tumor GPC3 comprises CAR-T cells that target GPC 3;

the GPC 3-targeted CAR-T cells are expressed by transfecting GPC 3-targeted recombinant virus into T cells; the recombinant virus targeting GPC3 is used for transfecting a chimeric antigen receptor targeting GPC3 into an immune effector cell;

the chimeric antigen receptor targeting GPC3 is IgM-GPC3-CD28-OX40-CD3 xi, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 1; and/or the presence of a gas in the gas,

the chimeric antigen receptor targeting GPC3 is IgM-GPC3-CD28-ICOS-CD3 xi, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 2; and/or the presence of a gas in the gas,

the chimeric antigen receptor targeting GPC3 is IgM-GPC3-CD28-CD40-CD3 xi, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 3; and/or the presence of a gas in the gas,

the chimeric antigen receptor targeting GPC3 is IgM-GPC3-41BB-CD27-CD3 xi, and the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 4.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is used in combination with any one or a combination of at least two of surgery, chemotherapy, or radiation therapy.

3. Use of a pharmaceutical composition according to any one of claims 1-2 for the preparation of a medicament for the treatment of renal tumor cells.

4. The use of claim 3, wherein the renal tumor cell is any one of or a combination of at least two of renal squamous cell carcinoma, renin tumor, renal angiomyolipoma, Belliny ductal carcinoma, renal clear cell sarcoma, mesoblastic nephroma, nephroblastoma, or mixed epithelial-mesenchymal tumor.

5. The use of claim 4, wherein the renal tumor cell is a nephroblastoma.

6. The use of claim 5, wherein the nephroblastoma is an adult nephroblastoma and/or a pediatric nephroblastoma.