CN109485734B

CN109485734B - Bispecific chimeric antigen receptor targeting BCMA and CD19 and application thereof

Info

Publication number: CN109485734B
Application number: CN201811645915.2A
Authority: CN
Inventors: 李光超; 郭***; 丁雯; 曾剑华; 罗敏; 莫文俊
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2018-12-30
Filing date: 2018-12-30
Publication date: 2020-05-12
Anticipated expiration: 2038-12-30
Also published as: CN109485734A

Abstract

The present invention relates to a bispecific chimeric antigen receptor targeting BCMA and CD19 comprising an extracellular domain capable of binding antigen, a transmembrane domain and at least one intracellular domain, wherein the extracellular domain comprises an antigen binding domain against BCMA and against CD19, and uses thereof. The chimeric antigen receptor has smaller clinical side effect and higher safety, can effectively reduce the solid tumor focus and effectively improve the treatment effect of the tumor.

Description

Bispecific chimeric antigen receptor targeting BCMA and CD19 and application thereof

Technical Field

The invention relates to the field of tumor cellular immunotherapy, in particular to a bispecific chimeric antigen receptor targeting BCMA and CD19 and application thereof, and specifically relates to a construction method of a chimeric antigen receptor T (CAR-T) cell technology based on specific target BCMA and CD19 and application thereof in antitumor therapy.

Background

Currently, Chimeric Antigen Receptor (CAR) modified T cells have achieved favorable results in clinical trials for the treatment of B cell malignancies, bringing eosin to CAR technology for the treatment of relapsed refractory Multiple Myeloma (MM). Multiple myeloma is a malignant disease that originates from the plasma cells in the bone marrow, which are the cells that develop B lymphocytes to a final functional stage.

BCMA (CD269) is expressed in mature B cells and plasma cells, and is also widely expressed in MM. ADC drugs against BCMA (GSK2857916) and dual targeting antibody drugs (BI836909) are now in the process of first clinical and preclinical development. Multiple clinical trial data for a new generation of CAR-T currently developed targeting B Cell Maturation Antigen (BCMA, CD269) show that BCMA-targeted CAR-T therapy has been highly successful in the treatment of multiple myeloma. However, similar to B-cell acute lymphocytic leukemia recurrence in the CD19CAR assay, MM recurrence, BCMA-CAR positive and BCMA negative malignant cell recurrence were observed with BCMA-CAR treatment. The former indicates lack of potency and persistence, the latter indicates selective pressure escape of antigen through.

Primary myeloma was originally thought to originate from malignant changes in plasma cells, and immunological and molecular biological studies suggest: multiple myeloma originates from a premalignant B cell malignancy, or from a malignancy of a hematopoietic precursor cell earlier than premalignant B. Therefore, the elimination of malignant B cells originated at an early stage will greatly improve the treatment effect of multiple myeloma and delay relapse. In addition, multiple myeloma does not typically express CD19 as a B cell line tumor, and thus CD19 is not a target for multiple myeloma immunotherapy. However, there have been reports of trace amounts of multiple myeloma clones with drug resistance, disease recurrence characteristics, with a B cell phenotype (i.e., CD19 positive). An article reported by U.S. Penn and Novartis in NEJM in the last year indicated that multiple myeloma tumor stem cells are CD19 positive in character. In one reported case of multiple myeloma, the patient still received complete cure after CAR-T treatment with CD19 alone, despite the lack of CD19 expression on 99.5% of the malignant proliferating plasma cells. Thus, CD19 is a potential therapeutic target for the clearance of relapsed, refractory and drug-resistant multiple myeloma cells.

The use of BCMA antigen as the extracellular domain for the treatment of B cell related diseases is disclosed, but if the antigen binding domain binds too strongly, CAR T cells induce massive cytokine release resulting in a potentially lethal immune response seen as a "cytokine storm", but if the antigen binding domain binds too weakly, CAR T cells do not exhibit sufficient therapeutic efficacy in eliminating cancer cells whereas in the prior art CN 106687483a discloses a humanized anti-BCMA chimeric antigen receptor for the treatment of cancer by administering T cells expressing genetic modifications of the CAR to achieve treatment of diseases associated with expression of B cell maturation antigen protein (BCMA) CN 104788573a discloses a chimeric antigen receptor hCD19scFv-CD8 α -CD28-CD3 ζ and its use, in which CD19 after a CAR-T cell transfusion, the expression of CD19 is reduced and readily escapes the immune mechanism.

Therefore, the preparation of a bispecific chimeric antigen receptor targeting BCMA and CD19 is effective in improving T cell killing and avoiding target escape, and must provide a more effective treatment option for multiple myeloma.

Disclosure of Invention

Aiming at the situations that the targeting in the current CAR-T technology for treating the tumor is not ideal and the tumor microenvironment influences the treatment effect of the CAR-T technology, the invention provides the bispecific chimeric antigen receptor targeting BCMA and CD19 and the application thereof.

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

in one aspect, the invention provides a bispecific chimeric antigen receptor targeting BCMA and CD19 comprising an extracellular domain capable of binding antigen, a transmembrane domain, and at least one intracellular domain, wherein the extracellular domain comprises an antigen binding domain of anti-BCMA and anti-CD 19.

According to the invention, the extracellular domain is a single-chain antibody against BCMA and CD19 or a single-chain antibody against BCMA and CD19, wherein the sequence of the antigen binding domain against BCMA and the antigen binding domain against CD19 does not affect the effect of the chimeric antigen, and the connection sequence can be, for example, the antigen binding domain against BCMA-linker-anti-CD 19 antigen binding domain, or the antigen binding domain against CD 19-linker-anti-BCMA antigen binding domain, and the adjustment of such sequence does not affect the effect of the chimeric antigen receptor.

In the invention, the inventor finds that the bispecific antibody obtained by connecting the anti-BCMA antibody and the anti-CD 19 antibody by a linker is prepared into a chimeric antigen receptor, so that not only can a T cell enhanced killing signal be effectively improved, but also a target escape phenomenon can be effectively avoided, and the inventor also finds that the connection sequence of the anti-BCMA antigen binding domain and the anti-CD 19 antigen binding domain does not influence the effect of the chimeric antigen receptor, wherein BCMA-linker-CD19 is named as B (G)19, and CD19-linker-BCMA is named as 19(G) B.

According to the invention, the antigen binding domain of anti-BCMA and the antigen binding domain of anti-CD 19 in the bispecific antibody of anti-BCMA and anti-CD 19 are connected by a linker, and the (G4S)3linker sequence is selected in the application, and the specific sequence is as follows:

(G4S) amino acid sequence of 3Linker (SEQ ID NO. 9): GGGGSGGGGSGGS;

(G4S) nucleotide sequence of 3Linker (SEQ ID NO. 10):

GGCGGCGGAGGATCTGGAGGAGGAGGAAGCGGAGGcGGAGGAAGC.

according to the invention, preferably, the amino acid sequences of the antigen binding domains of the anti-BCMA and anti-CD 19 are shown as SEQ ID NO.1-2, and the specific sequences are as follows:

BCMA-linker-CD19B(G)19(SEQ ID NO.1)：

DIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT；

CD19-linker-BCMA 19(G)B(SEQ ID NO.2)：

EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSS.

preferably, the nucleotide sequence of the bispecific antibody against BCMA and CD19 is shown in SEQ ID NO.3-4, and the specific sequence is as follows:

BCMA-linker-CD19B(G)19(SEQ ID NO.3)：

GACATCGTCCTGACCCAGTCACCTGCCAGCCTGGCCGTCAGCCTGGGCGAGAGAGCCACCATTAATTGCCGCGCTAGCGAATCAGTGTCAGTCATCGGCGCTCACCTGATCCACTGGTATCAGCAAAAGCCTGGGCAGCCACCAAAGCTGCTCATCTATCTCGCCTCCAACCTGGAGACAGGCGTGCCTGCTCGCTTTAGTGGTTCTGGTAGCGGCACCGATTTCACCCTGACTATTTCAAGCCTGCAGGCAGAGGACGCAGCCATATACTATTGCCTGCAGTCCCGGATTTTCCCTCGCACATTCGGCCAGGGCACAAAACTGGAAATCAAAGGATCTACCTCCGGCTCCGGGAAGCCCGGAAGCGGCGAAGGCTCAACCAAAGGCCAGGTTCAGCTCGTGCAGTCTGGTTCTGAACTGAAGAAACCCGGTGCATCTGTGAAAGTCTCCTGCAAGGCTTCCGGGTATACTTTCACAGACTACAGTATTAATTGGGTTCGCCAAGCTCCTGGACAGGGACTGGAGTGGATGGGATGGATAAACACAGAAACTAGGGAGCCTGCATACGCCTATGATTTCAGAGGTCGCTTCGTGTTCAGTCTGGATACAAGTGTTTCAACAGCCTATCTGCAAATTAGCTCCCTGAAAGCCGAGGACACCGCAGTTTACTACTGTGCACGGGATTATTCTTACGCTATGGACTATTGGGGGCAGGGCACACTCGTGACAGTGTCTAGCGGCGGCGGAGGATCTGGAGGAGGAGGAAGCGGcGGAGGAGGAAGCGAAGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCTCCTTCACAGTCTCTGAGCGTGACCTGTACCGTGTCCGGAGTGTCTCTGCCAGACTACGGAGTGTCTTGGATCAGACAGCCTCCTAGAAAGGGACTCGAGTGGCTCGGAGTGATTTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTATTGGGGACAGGGCACCAGCGTGACAGTGTCTAGCGGAGGAGGcGGcAGCGGAGGAGGAGGATCTGGAGGAGGCGGCAGCGATATTCAGATGACCCAGACCACCTCTTCTCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCTCTTGCAGGGCCAGCCAGGACATCAGCAAGTACCTGAATTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGGCTGCACAGCGGAGTGCCTAGCAGATTCAGCGGAAGCGGCAGCGGCACAGATTATAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTTTGCCAGCAGGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAGATCACA；

CD19-linker-BCMA 19(G)B(SEQ ID NO.4)：

GAAGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCTCCTTCACAGTCTCTGAGCGTGACCTGTACCGTGTCCGGAGTGTCTCTGCCAGACTACGGAGTGTCTTGGATCAGACAGCCTCCTAGAAAGGGACTCGAGTGGCTCGGAGTGATTTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTATTGGGGACAGGGCACCAGCGTGACAGTGTCTAGCGGcGGAGGcGGAAGCGGAGGAGGAGGATCTGGAGGAGGCGGCAGCGATATTCAGATGACCCAGACCACCTCTTCTCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCTCTTGCAGGGCCAGCCAGGACATCAGCAAGTACCTGAATTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGGCTGCACAGCGGAGTGCCTAGCAGATTCAGCGGAAGCGGCAGCGGCACAGATTATAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTTTGCCAGCAGGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAGATCACAGGCGGCGGAGGATCTGGAGGAGGAGGAAGCGGAGGcGGAGGAAGCGACATCGTCCTGACCCAGTCACCTGCCAGCCTGGCCGTCAGCCTGGGCGAGAGAGCCACCATTAATTGCCGCGCTAGCGAATCAGTGTCAGTCATCGGCGCTCACCTGATCCACTGGTATCAGCAAAAGCCTGGGCAGCCACCAAAGCTGCTCATCTATCTCGCCTCCAACCTGGAGACAGGCGTGCCTGCTCGCTTTAGTGGTTCTGGTAGCGGCACCGATTTCACCCTGACTATTTCAAGCCTGCAGGCAGAGGACGCAGCCATATACTATTGCCTGCAGTCCCGGATTTTCCCTCGCACATTCGGCCAGGGCACAAAACTGGAAATCAAAGGATCTACCTCCGGCTCCGGGAAGCCCGGAAGCGGCGAAGGCTCAACCAAAGGCCAGGTTCAGCTCGTGCAGTCTGGTTCTGAACTGAAGAAACCCGGTGCATCTGTGAAAGTCTCCTGCAAGGCTTCCGGGTATACTTTCACAGACTACAGTATTAATTGGGTTCGCCAAGCTCCTGGACAGGGACTGGAGTGGATGGGATGGATAAACACAGAAACTAGGGAGCCTGCATACGCCTATGATTTCAGAGGTCGCTTCGTGTTCAGTCTGGATACAAGTGTTTCAACAGCCTATCTGCAAATTAGCTCCCTGAAAGCCGAGGACACCGCAGTTTACTACTGTGCACGGGATTATTCTTACGCTATGGACTATTGGGGGCAGGGCACACTCGTGACAGTGTCTAGC。

according to the invention, the transmembrane domain is a CD28 transmembrane domain and/or a CD8 α transmembrane domain.

According to the invention, the intracellular domain further comprises an intracellular costimulatory signaling domain and/or a CD3 zeta signaling domain.

According to the present invention, the costimulatory signaling domain is any one of or a combination of at least two of the human 4-1BB intracellular domain, the human CD28 intracellular domain, the human CD27 intracellular domain, the human OX40 intracellular domain, the human CD30 intracellular domain, the human CD40 intracellular domain or the human OX40 intracellular domain, preferably the human 4-1BB intracellular domain.

According to the invention, the extracellular domain and the transmembrane domain are connected by a hinge region comprising an IgG1 hinge region and/or a CD8 α hinge region.

According to the present invention, the bispecific chimeric antigen receptor further comprises a signal peptide, wherein the signal peptide is a signal peptide capable of directing transmembrane transfer of the chimeric antigen receptor, and a person skilled in the art can select a signal peptide conventional in the art according to needs, and the chimeric antigen receptor further comprises a signal peptide, preferably a CD8 α signal peptide or a Secretory signal peptide.

The bispecific chimeric antigen receptor of the present invention further comprises a promoter, which can be EF α or any high expression promoter, and the skilled person can select the promoter according to the actual situation, and the promoter is not particularly limited herein, and the presence of the promoter does not affect the performance of the bispecific chimeric antigen receptor of the present invention.

According to the present invention, the bispecific chimeric antigen receptor comprises a signal peptide, an extracellular domain that binds BCMA and CD19 antigens, a hinge region, a transmembrane domain, a costimulatory signaling domain, and a CD3 zeta signaling domain in tandem.

According to the invention, the bispecific chimeric antigen receptor is a CD8 α signal peptide, and is formed by connecting an extracellular domain of BCMA and CD19 antigen, a CD8 α hinge region, a CD8 α transmembrane domain, a 4-1BB signaling domain and a CD3 zeta signaling domain in series.

According to the invention, the amino acid sequence of the bispecific chimeric antigen receptor comprises a sequence shown as SEQ ID NO.5-6, and the amino acid sequence shown as SEQ ID NO.5-6 is as follows:

B(G)19CAR(SEQ ID NO.5)：

MALPVTALLLPLALLLHAARPDIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；

19(G)B CAR(SEQ ID NO.6)：

MALPVTALLLPLALLLHAARPEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIKGSTSGSGKPGSGEGSTKGQVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

in a second aspect, a nucleic acid encoding the chimeric antigen receptor of the first aspect.

The nucleic acid is specifically shown as one of SEQ ID NO.7-8, and specifically comprises the following steps:

B(G)19CAR(SEQ ID NO.7)：

ATGGCACTGCCTGTGACTGCCCTGCTGCTCCCTCTCGCACTCCTGCTGCACGCAGCCCGCCCAGACATCGTCCTGACCCAGTCACCTGCCAGCCTGGCCGTCAGCCTGGGCGAGAGAGCCACCATTAATTGCCGCGCTAGCGAATCAGTGTCAGTCATCGGCGCTCACCTGATCCACTGGTATCAGCAAAAGCCTGGGCAGCCACCAAAGCTGCTCATCTATCTCGCCTCCAACCTGGAGACAGGCGTGCCTGCTCGCTTTAGTGGTTCTGGTAGCGGCACCGATTTCACCCTGACTATTTCAAGCCTGCAGGCAGAGGACGCAGCCATATACTATTGCCTGCAGTCCCGGATTTTCCCTCGCACATTCGGCCAGGGCACAAAACTGGAAATCAAAGGATCTACCTCCGGCTCCGGGAAGCCCGGAAGCGGCGAAGGCTCAACCAAAGGCCAGGTTCAGCTCGTGCAGTCTGGTTCTGAACTGAAGAAACCCGGTGCATCTGTGAAAGTCTCCTGCAAGGCTTCCGGGTATACTTTCACAGACTACAGTATTAATTGGGTTCGCCAAGCTCCTGGACAGGGACTGGAGTGGATGGGATGGATAAACACAGAAACTAGGGAGCCTGCATACGCCTATGATTTCAGAGGTCGCTTCGTGTTCAGTCTGGATACAAGTGTTTCAACAGCCTATCTGCAAATTAGCTCCCTGAAAGCCGAGGACACCGCAGTTTACTACTGTGCACGGGATTATTCTTACGCTATGGACTATTGGGGGCAGGGCACACTCGTGACAGTGTCTAGCGGCGGCGGAGGATCTGGAGGAGGAGGAAGCGGcGGAGGAGGAAGCGAAGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCTCCTTCACAGTCTCTGAGCGTGACCTGTACCGTGTCCGGAGTGTCTCTGCCAGACTACGGAGTGTCTTGGATCAGACAGCCTCCTAGAAAGGGACTCGAGTGGCTCGGAGTGATTTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTATTGGGGACAGGGCACCAGCGTGACAGTGTCTAGCGGAGGAGGcGGcAGCGGAGGAGGAGGATCTGGAGGAGGCGGCAGCGATATTCAGATGACCCAGACCACCTCTTCTCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCTCTTGCAGGGCCAGCCAGGACATCAGCAAGTACCTGAATTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGGCTGCACAGCGGAGTGCCTAGCAGATTCAGCGGAAGCGGCAGCGGCACAGATTATAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTTTGCCAGCAGGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAGATCACAACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAGCGCGGGAGGAAGAAGCTGCTGTATATCTTCAAACAGCCCTTTATGCGGCCAGTTCAGACCACACAAGAGGAAGATGGCTGCAGCTGTAGATTTCCAGAAGAGGAGGAAGGAGGATGTGAACTGAGGGTCAAATTTTCACGCTCAGCAGACGCTCCTGCTTACCAACAAGGCCAAAATCAGCTGTACAACGAGCTGAATCTGGGTCGCCGGGAGGAATACGACGTTCTGGATAAAAGGCGCGGGAGGGACCCAGAGATGGGTGGCAAGCCCAGGCGCAAGAACCCTCAAGAAGGCCTGTATAACGAGCTGCAGAAAGATAAGATGGCAGAAGCCTACTCTGAAATAGGAATGAAGGGCGAGAGACGGCGCGGAAAGGGCCATGATGGCCTCTACCAGGGACTGTCCACCGCCACAAAAGATACCTACGACGCACTCCATATGCAGGCACTGCCTCCTCGGTAA；

19(G)B CAR(SEQ ID NO.8)：

ATGGCACTGCCTGTGACTGCCCTGCTGCTCCCTCTCGCACTCCTGCTGCACGCAGCCCGCCCAGAAGTGAAGCTGCAGGAGAGCGGACCAGGACTGGTGGCTCCTTCACAGTCTCTGAGCGTGACCTGTACCGTGTCCGGAGTGTCTCTGCCAGACTACGGAGTGTCTTGGATCAGACAGCCTCCTAGAAAGGGACTCGAGTGGCTCGGAGTGATTTGGGGCAGCGAGACCACCTACTACAACAGCGCCCTGAAGAGCAGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGACTATTGGGGACAGGGCACCAGCGTGACAGTGTCTAGCGGcGGAGGcGGAAGCGGAGGAGGAGGATCTGGAGGAGGCGGCAGCGATATTCAGATGACCCAGACCACCTCTTCTCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCTCTTGCAGGGCCAGCCAGGACATCAGCAAGTACCTGAATTGGTACCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAGCAGGCTGCACAGCGGAGTGCCTAGCAGATTCAGCGGAAGCGGCAGCGGCACAGATTATAGCCTGACCATCAGCAACCTGGAGCAGGAGGACATCGCCACCTACTTTTGCCAGCAGGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACCAAGCTGGAGATCACAGGCGGCGGAGGATCTGGAGGAGGAGGAAGCGGAGGcGGAGGAAGCGACATCGTCCTGACCCAGTCACCTGCCAGCCTGGCCGTCAGCCTGGGCGAGAGAGCCACCATTAATTGCCGCGCTAGCGAATCAGTGTCAGTCATCGGCGCTCACCTGATCCACTGGTATCAGCAAAAGCCTGGGCAGCCACCAAAGCTGCTCATCTATCTCGCCTCCAACCTGGAGACAGGCGTGCCTGCTCGCTTTAGTGGTTCTGGTAGCGGCACCGATTTCACCCTGACTATTTCAAGCCTGCAGGCAGAGGACGCAGCCATATACTATTGCCTGCAGTCCCGGATTTTCCCTCGCACATTCGGCCAGGGCACAAAACTGGAAATCAAAGGATCTACCTCCGGCTCCGGGAAGCCCGGAAGCGGCGAAGGCTCAACCAAAGGCCAGGTTCAGCTCGTGCAGTCTGGTTCTGAACTGAAGAAACCCGGTGCATCTGTGAAAGTCTCCTGCAAGGCTTCCGGGTATACTTTCACAGACTACAGTATTAATTGGGTTCGCCAAGCTCCTGGACAGGGACTGGAGTGGATGGGATGGATAAACACAGAAACTAGGGAGCCTGCATACGCCTATGATTTCAGAGGTCGCTTCGTGTTCAGTCTGGATACAAGTGTTTCAACAGCCTATCTGCAAATTAGCTCCCTGAAAGCCGAGGACACCGCAGTTTACTACTGTGCACGGGATTATTCTTACGCTATGGACTATTGGGGGCAGGGCACACTCGTGACAGTGTCTAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAGCGCGGGAGGAAGAAGCTGCTGTATATCTTCAAACAGCCCTTTATGCGGCCAGTTCAGACCACACAAGAGGAAGATGGCTGCAGCTGTAGATTTCCAGAAGAGGAGGAAGGAGGATGTGAACTGAGGGTCAAATTTTCACGCTCAGCAGACGCTCCTGCTTACCAACAAGGCCAAAATCAGCTGTACAACGAGCTGAATCTGGGTCGCCGGGAGGAATACGACGTTCTGGATAAAAGGCGCGGGAGGGACCCAGAGATGGGTGGCAAGCCCAGGCGCAAGAACCCTCAAGAAGGCCTGTATAACGAGCTGCAGAAAGATAAGATGGCAGAAGCCTACTCTGAAATAGGAATGAAGGGCGAGAGACGGCGCGGAAAGGGCCATGATGGCCTCTACCAGGGACTGTCCACCGCCACAAAAGATACCTACGACGCACTCCATATGCAGGCACTGCCTCCTCGGTAA.

in a third aspect, the present invention provides a viral vector comprising the nucleic acid of the second aspect.

According to the invention, the viral vector is a lentiviral vector and/or a retroviral vector, preferably a lentiviral vector.

In a fourth aspect, the invention provides a T cell for expression by transfection of a nucleic acid sequence encoding the chimeric antigen receptor according to the first aspect into the T cell.

According to the invention, the transfection is carried out by transfection of T cells with viral vectors and/or eukaryotic expression plasmids, preferably with viral vectors.

In the invention, the T cell has good target killing effect, can release low-dose immune factors, and has the properties of low toxicity and high immune killing reaction.

In a fifth aspect, the present invention provides a recombinant lentivirus comprising a mammalian cell co-transfected with a viral vector as described in the third aspect and packaging helper plasmids gag/pol, Rev and VSV-G.

According to the present invention, the mammalian cell is any one of 293 cells, 293T cells or 293F cells or a combination of at least two thereof.

In a sixth aspect, the present invention provides a composition comprising a chimeric antigen receptor according to the first aspect and/or a recombinant lentivirus according to the fifth aspect.

In a seventh aspect, the present invention provides a use of the chimeric antigen receptor of the first aspect, the nucleic acid of the second aspect, the viral vector of the third aspect, the T cell of the fourth aspect, the recombinant lentivirus of the fifth aspect or the composition of the sixth aspect for the preparation of a chimeric antigen receptor T cell, an immune cell or in a medicament for the treatment of a tumor.

According to the invention, the tumor is a B cell tumor, preferably multiple myeloma, B cell leukemia and B cell lymphoma.

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

(1) according to the invention, the extracellular domain in the chimeric antigen receptor is set as the antigen binding domain of anti-BCMA and anti-CD 19, so that not only can the enhanced killing signal of T cells be effectively improved, but also the target escape phenomenon can be effectively avoided, and the tumor recurrence can be delayed;

(2) the bispecific chimeric antigen receptor of the invention has expression positive rates of 26.98% and 17.99%, effectively kills cells expressing CD19 and/or BCMA, has no killing effect on double negative cells K562, and has high specificity and good treatment effect.

Drawings

FIG. 1 is a schematic sequence diagram of a chimeric antigen receptor of the present invention;

FIG. 2(a) is a schematic representation of the elements of the lentiviral expression vector pLVX-BCMA CAR, and FIG. 2(b) is a plasmid map of the lentiviral expression vector pLVX-BCMA CAR;

FIG. 3(a) is a schematic diagram of the elements of the lentiviral expression vector pLVX-CD19CAR, and FIG. 3(b) is a plasmid map of the lentiviral expression vector pLVX-CD19 CAR;

FIG. 4(a) is a schematic representation of the lentiviral expression vector pLVX-B (G)19CAR element, and FIG. 4(b) is a plasmid map of the lentiviral expression vector pLVX-B (G)19 CAR;

FIG. 5(a) is a schematic representation of the lentiviral expression vector pLVX-19(G) B CAR element and FIG. 5(B) is a plasmid map of the lentiviral expression vector pLVX-19(G) B CAR;

FIG. 6 is a graph showing the results of flow assay of CAR-T expression in K562 cells, wherein FIG. 6(a) shows the results of BCMA-CAR expression in K562 cells, and FIG. 6(b) shows the results of CD19-CAR expression in K562 cells;

FIG. 7 is a graph showing the results of the expression of CAR-T by Daudi and Jurkat cells wherein FIG. 7(a) is the result of the expression of BCMA-CAR by Daudi cells, FIG. 7(b) is the result of the expression of CD19-CAR by Daudi cells, FIG. 7(c) is the result of the expression of BCMA-CAR by Jurkat cells, and FIG. 7(d) is the result of the expression of CD19-CAR by Jurkat cells;

figure 8 is a flow chart of expression of BCMA CAR with FITC labeled human BCMA protein on the ordinate;

figure 9 is a flow chart detecting expression of CD19CAR with FITC labeled human CD19 protein on the ordinate;

FIG. 10 is a graph showing the results of IFN-gama secretion in the supernatant after co-culture of CART and test cells.

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.

Example 1: design of chimeric antigen receptors

In this example, bispecific chimeric antigen receptors against BCMA and CD19 (B (G)19 and 19(G) B), chimeric antigen receptor against BCMA and chimeric antigen receptor against CD19 were constructed, the sequence schematic is shown in fig. 1, and the chimeric antigen receptors include a signal peptide sequence (Leader) of CD8 α, a bispecific antibody sequence specifically binding to BCMA and CD19 antigen, a single domain antibody against BCMA and a single domain antibody against CD19, a Hinge region (hinde) and Transmembrane region sequence (Transmembrane) of CD8 α, a 4-1BB co-stimulatory domain sequence and a CD3 zeta signaling domain sequence, specifically as follows:

the amino acid sequence (SEQ ID NO.11) of the CD8 α signal peptide (leader) is MALPVTALLLPLALLLHAARP;

nucleotide sequence of CD8 α signal peptide (leader) (SEQ ID NO. 12):

ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCT；

amino acid sequence of the hinge region (hinge) of CD8 α (SEQ ID NO. 13):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD；

nucleotide sequence of CD8 α hinge region (hinge) (SEQ ID NO. 14):

ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT；

the amino acid sequence (SEQ ID NO.15) of the CD8 α transmembrane region (TM) is IYIWAPLAGTCGVLLLSLVITLYC;

nucleotide sequence of CD8 α transmembrane region (TM) (SEQ ID NO. 16):

ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC；

4-1BB intracellular co-stimulatory domain (ICD) (SEQ ID NO. 17):

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；

4-1BB Intracellular Costimulatory Domain (ICD) (SEQ ID NO. 18):

AAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTG；

amino acid sequence of the zeta-signaling domain of CD3 (SEQ ID NO. 19):

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；

nucleotide sequence of the CD3 zeta signaling domain (SEQ ID No. 20):

AGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG.

example 2: construction of chimeric antigen receptor expression vectors against BCMA

(1) The B (G)19CAR, 19(G) B CAR, BCMA CAR and CD19CAR sequences are synthesized by whole gene, the synthesized CAR and an empty vector are subjected to double enzyme digestion by EcoRI and BamHI, after the enzyme digestion is carried out for 30min in water bath at 37 ℃, DNA electrophoresis is carried out by using 1.5% agarose gel, and then purification and recovery treatment are carried out by using a Tiangen agarose gel kit;

(2) ligation of pLVX-EF1-MCS vector to CAR gene fragment:

the linking system is as follows:

ligation was performed at 22 ℃ for 1h, the ligation products were directly transformed into Stbl3 E.coli competent cells, 200. mu.l of the transformation products were spread on ampicillin-resistant LB plates, and the LB plates were cultured in an incubator at 37 ℃ overnight in an inverted manner. Randomly selecting 3 monoclonals the next morning for colony PCR identification, and sending positive clones to sequence.

The elements of the anti-BCMA chimeric antigen receptor lentiviral expression vector pLVX-BCMA CAR are shown in fig. 2(a), the vector map is shown in fig. 2 (b); the elements of the anti-CD 19 chimeric antigen receptor lentiviral expression vector pLVX-CD19CAR are shown in fig. 3(a), the vector map is shown in fig. 3 (b); the elements of the anti-BCMA and anti-CD 19 bispecific chimeric antigen receptor lentiviral expression vector pLVX-b (g)19CAR are shown in fig. 4(a), and the vector map is shown in fig. 4 (b); the elements of the anti-CD 19 and anti-BCMA bispecific chimeric antigen receptor lentiviral expression vector pLVX-19(G) B CAR are shown in fig. 5(a), and the vector map is shown in fig. 5 (B).

Example 3: lentiviral packaging

The lentiviral expression vectors in the examples were each subjected to lentiviral packaging using a four plasmid system, the specific steps of which were as follows:

(1) four plasmid systems respectively express gag/pol, Rev, VSV-G and artificial chimeric antigen receptor composed of the engineering stable single chain antibody of the invention, which are required by slow virus vector packaging: transient transfection is carried out on the four plasmids to 293T cells, and the total mass is 10 mug;

(2) adding the plasmid into serum-free DMEM with a certain volume, uniformly mixing, standing for 15 minutes, and mixingAdding the above mixture into 293T cell-spread T75 culture bottle, mixing, and adding 5% CO at 37 deg.C₂Culturing for 6h in a cell culture box;

(3) after 6h, the culture medium was replaced with fresh medium and the culture was continued, and 10mM sodium butyrate solution was added, and culture supernatant of lentivirus was collected 72 hours later for purification assay.

Example 4: expansion of CAR-T cells

Collecting 30ml of whole blood from each volunteer, diluting peripheral blood with physiological saline 1:1, adding Ficoll into a centrifugal tube, slowly adding diluted peripheral blood, centrifuging at 1500rpm for 30min, gently sucking PBMC layer, and transferring into another centrifugal tube;

PBMC are washed with physiological saline for multiple times, transferred into X-VIVO culture medium (containing 50ng/mL OKT3, 300IU/mL IL-2) for culture, after PBMC is separated, X-VIVO containing 50ng/mL OKT3, 300IU/mL IL-2 is needed for activation, after 2 days, the culture medium is changed into X-VIVO containing 300IU/mL for amplification culture, then counting and changing the X-VIVO containing 300IU/mL are carried out every two days, and the cell concentration is maintained at 0.5X 10⁶-1×10⁶mL, for 10 consecutive days.

The expression of CAR in K562 cells was detected by flow cytometry, and the results are shown in fig. 6, and as can be seen from fig. 6(a) and 6(b), K562 cells are BCMA and CD19 double negative cells; the BCMA expression percentage of the K562-BCMA stable cell line is 99.2 percent; the percentage expression of CD19 by the K562-CD19 stable cell line was 100%.

The expression of CAR in Daudi and Jurkat cells was examined by flow cytometry, and the results are shown in fig. 7, and as can be seen from fig. 7(a) and 7(b), Daudi cells simultaneously expressed CD19 and BCMA, expressed 99.7% BCMA and 100% CD19, and as can be seen from fig. 7(c) and 7(d), Jurkat cells simultaneously expressed CD19 and BCMA, expressed 99.5% BCMA and 100% CD 19.

Example 5: lentiviral infection of T cells

The infection efficiency of lentivirus to T cells is improved by using retroNectin, and 30 mu g of retroNectin is coated in a 6-hole plate and placed in a cell culture box at 37 ℃ for 2 h; extracting RetroNectin, blocking the coated with Hank's solution containing 2.5% BSAPlacing the 6-hole plate in a cell culture box at 37 ℃ for 0.5 h; sucking the confining liquid, washing a 6-well plate by using a Hank's solution containing 2% Hepes, adding an X-VIVO culture medium, adding a proper amount of a lentivirus solution, adding 2000g of the lentivirus solution, and centrifuging for 2 hours; discard the supernatant and add 1X 10⁶T cells (CD3 positive)>90%), 1000g, centrifugation for 10min at 37 deg.C and 5% CO₂And culturing in a cell culture box with certain humidity, and repeating the process the next day.

Expression of CAR was measured 5 days after infection and measured by flow cytometry using FITC-Labeled Human BCMA (cat No. BCA-HF2H1, Acrobiosystems) bound to anti-BCMA antibody scFv (α BCMA), as shown in fig. 8, from which the positive expression rate of BCMA CAR was 8.93%, the positive expression rate of 19(G) B CAR was 31.88%, the positive expression rate of B (G)19CAR was 35.53%, T mock cells (untransfected T cells) and CD19CAR-T cells did not express BCMA CAR.

The expression of CAR was also detected by flow cytometry using FITC-Labeled Human CD19(20-291) Protein, Fc Tag (cat # CD9-HF251, Acrobiosystems) in combination with anti-CD 19 antibody scFv (derived from FMC63 clone, α CD19), as shown in FIG. 9. from the results in FIG. 9, the expression positivity for CD19CAR was 65.15%, for 19(G) BCAR was 26.98%, for B (G)19CAR was 17.99%, and for T mock cells (untransfected T cells) and BCMA cells did not express CD19 CAR.

Example 6: ELISA detection of IFN-gamma levels in cell lines and CAR-T cell coculture supernatants

K562, K562-CD19 (stably expressing CD 19K 562 cells), K562-BCMA (stably expressing BCMA K562 cells) and RPMI 8226 cells were seeded into 24-well plates at 5X 105 cells/well, respectively. Adding CAR-T and untransfected T cell (T mock) cells into 5X 105 cells per well, supplementing the culture solution to 1.5mL, and co-culturing in an incubator for 12 hours; the co-culture supernatant was assayed using human IL-2 and IFN- γ ELISA assay kit (Xinbo Sheng Biopsis) (see ELISA assay kit for details), and the results are shown in FIG. 10.

As can be seen from FIG. 10, the IFN- γ cytokine levels in the coculture supernatants of BCMA CAR-T cells and BCMA positive cells (K562-BCMA, Daudi and Jurkat) were significantly higher than those in the BCMA negative cell (K562 and K562-CD19) group; the IFN-gamma cytokine level in the co-culture supernatant of the CD19CAR-T cells and CD19 positive cells (K562-CD19, Daudi and Jurkat) is obviously higher than that of the CD19 negative cells (K562 and K562-BCMA), and the IFN-gamma cytokine level in the co-culture supernatant of the bispecific CAR-T cells B (G)19 and 19(B) and CD19 and BCMA single-positive or double-positive target cells is obviously higher than that of the double-negative cell K562.

Taken together, BCMA CAR-T cells were able to specifically kill BCMA positive cells (K562-BCMA, Daudi and Jurkat), while there was little killing ability on BCMA negative cells (K562 and K562-CD 19). Furthermore, CD19CAR-T cells were able to specifically kill CD19 positive cells (K562-CD19, Daudi and Jurkat) with little killing ability on CD19 negative cells (K562 and K562-BCMA), whereas bispecific CAR-T cells B (g)19 and 19(B) of the present application were able to effectively kill cells expressing CD19 and/or BCMA without killing effect on double negative cells K562.

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> Guangzhou Bai-and-Gen-Tech Co Ltd

<120> bispecific chimeric antigen receptor targeting BCMA and CD19 and application thereof

<130>2018

<160>20

<170>PatentIn version 3.3

<210>1

<211>503

<212>PRT

<213> artificially synthesized sequence

<400>1

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Glu Ser Val Ser Val Ile

20 25 30

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

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro Ala

5055 60

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

65 70 75 80

Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

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

100 105 110

Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

115 120 125

Gly Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly

130 135 140

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

145 150 155 160

Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp

180 185 190

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

195 200 205

Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr

210 215220

Cys Ala Arg Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

245 250 255

Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu

260 265 270

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

275 280 285

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys

290 295 300

Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

305 310 315 320

Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys

325 330 335

Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala

340 345 350

Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

355 360 365

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

370 375380

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

385 390 395 400

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

405 410 415

Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

420 425 430

Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser

435 440 445

Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

450 455 460

Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala

465 470 475 480

Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly

485 490 495

Gly Thr Lys Leu Glu Ile Thr

500

<210>2

<211>503

<212>PRT

<213> artificially synthesized sequence

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

1 510 15

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

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Thr Thr Ser

130 135 140

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

145 150 155 160

Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp

165170 175

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

180 185 190

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

195 200 205

Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln

210 215 220

Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu

225 230 235 240

Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

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

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

275 280 285

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

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Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro

305 310 315 320

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

325 330335

Ser Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr Tyr Cys Leu Gln Ser

340 345 350

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

355 360 365

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr

370 375 380

Lys Gly Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro

385 390 395 400

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

405 410 415

Asp Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

420 425 430

Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr

435 440 445

Asp Phe Arg Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr

450 455 460

Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr

465 470 475 480

Tyr Cys Ala Arg Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly

485 490495

Thr Leu Val Thr Val Ser Ser

500

<210>3

<211>1509

<212>DNA

<213> artificially synthesized sequence

<400>3

gacatcgtcc tgacccagtc acctgccagc ctggccgtca gcctgggcga gagagccacc 60

attaattgcc gcgctagcga atcagtgtca gtcatcggcg ctcacctgat ccactggtat 120

cagcaaaagc ctgggcagcc accaaagctg ctcatctatc tcgcctccaa cctggagaca 180

ggcgtgcctg ctcgctttag tggttctggt agcggcaccg atttcaccct gactatttca 240

agcctgcagg cagaggacgc agccatatac tattgcctgc agtcccggat tttccctcgc 300

acattcggcc agggcacaaa actggaaatc aaaggatcta cctccggctc cgggaagccc 360

ggaagcggcg aaggctcaac caaaggccag gttcagctcg tgcagtctgg ttctgaactg 420

aagaaacccg gtgcatctgt gaaagtctcc tgcaaggctt ccgggtatac tttcacagac 480

tacagtatta attgggttcg ccaagctcct ggacagggac tggagtggat gggatggata 540

aacacagaaa ctagggagcc tgcatacgcc tatgatttca gaggtcgctt cgtgttcagt 600

ctggatacaa gtgtttcaac agcctatctg caaattagct ccctgaaagc cgaggacacc 660

gcagtttact actgtgcacg ggattattct tacgctatgg actattgggg gcagggcaca 720

ctcgtgacag tgtctagcgg cggcggagga tctggaggag gaggaagcgg cggaggagga 780

agcgaagtga agctgcagga gagcggacca ggactggtgg ctccttcaca gtctctgagc 840

gtgacctgta ccgtgtccgg agtgtctctg ccagactacg gagtgtcttg gatcagacag 900

cctcctagaa agggactcga gtggctcgga gtgatttggg gcagcgagac cacctactac 960

aacagcgccc tgaagagcag gctgaccatc atcaaggaca acagcaagag ccaggtgttc 1020

ctgaagatga acagcctgca gaccgacgac accgccatct actactgcgc caagcactac 1080

tactacggcg gcagctacgc catggactat tggggacagg gcaccagcgt gacagtgtct 1140

agcggaggag gcggcagcgg aggaggagga tctggaggag gcggcagcga tattcagatg 1200

acccagacca cctcttctct gagcgccagc ctgggcgaca gagtgaccat ctcttgcagg 1260

gccagccagg acatcagcaa gtacctgaat tggtaccagc agaagcccga cggcaccgtg 1320

aagctgctga tctaccacac cagcaggctg cacagcggag tgcctagcag attcagcgga 1380

agcggcagcg gcacagatta tagcctgacc atcagcaacc tggagcagga ggacatcgcc 1440

acctactttt gccagcaggg caacaccctg ccttacacct ttggcggagg caccaagctg 1500

gagatcaca 1509

<210>4

<211>1509

<212>DNA

<213> artificially synthesized sequence

<400>4

gaagtgaagc tgcaggagag cggaccagga ctggtggctc cttcacagtc tctgagcgtg 60

acctgtaccg tgtccggagt gtctctgcca gactacggag tgtcttggat cagacagcct 120

cctagaaagg gactcgagtg gctcggagtg atttggggca gcgagaccac ctactacaac 180

agcgccctga agagcaggct gaccatcatc aaggacaaca gcaagagcca ggtgttcctg 240

aagatgaaca gcctgcagac cgacgacacc gccatctact actgcgccaa gcactactac 300

tacggcggca gctacgccat ggactattgg ggacagggca ccagcgtgac agtgtctagc 360

ggcggaggcg gaagcggagg aggaggatct ggaggaggcg gcagcgatat tcagatgacc 420

cagaccacct cttctctgag cgccagcctg ggcgacagag tgaccatctc ttgcagggcc 480

agccaggaca tcagcaagta cctgaattgg taccagcaga agcccgacgg caccgtgaag 540

ctgctgatct accacaccag caggctgcac agcggagtgc ctagcagatt cagcggaagc 600

ggcagcggca cagattatag cctgaccatc agcaacctgg agcaggagga catcgccacc 660

tacttttgcc agcagggcaa caccctgcct tacacctttg gcggaggcac caagctggag 720

atcacaggcg gcggaggatc tggaggagga ggaagcggag gcggaggaag cgacatcgtc 780

ctgacccagt cacctgccag cctggccgtc agcctgggcg agagagccac cattaattgc 840

cgcgctagcg aatcagtgtc agtcatcggc gctcacctga tccactggta tcagcaaaag 900

cctgggcagc caccaaagct gctcatctat ctcgcctcca acctggagac aggcgtgcct 960

gctcgcttta gtggttctgg tagcggcacc gatttcaccc tgactatttc aagcctgcag 1020

gcagaggacg cagccatata ctattgcctg cagtcccgga ttttccctcg cacattcggc 1080

cagggcacaa aactggaaat caaaggatct acctccggct ccgggaagcc cggaagcggc 1140

gaaggctcaa ccaaaggcca ggttcagctc gtgcagtctg gttctgaact gaagaaaccc 1200

ggtgcatctg tgaaagtctc ctgcaaggct tccgggtata ctttcacaga ctacagtatt 1260

aattgggttc gccaagctcc tggacaggga ctggagtgga tgggatggat aaacacagaa 1320

actagggagc ctgcatacgc ctatgatttc agaggtcgct tcgtgttcag tctggataca 1380

agtgtttcaa cagcctatct gcaaattagc tccctgaaag ccgaggacac cgcagtttac 1440

tactgtgcac gggattattc ttacgctatg gactattggg ggcagggcac actcgtgaca 1500

gtgtctagc 1509

<210>5

<211>747

<212>PRT

<213> artificially synthesized sequence

<400>5

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 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu

20 25 30

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

35 40 45

Ser Val Ser Val Ile Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu

65 70 75 80

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

85 90 95

Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr Tyr

100 105110

Cys Leu Gln Ser Arg Ile Phe Pro Arg Thr Phe Gly Gln Gly Thr Lys

115 120 125

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

130 135 140

Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln Ser Gly Ser Glu

145 150 155 160

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

165 170 175

Tyr Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly

180 185 190

Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro

195 200 205

Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Val Phe Ser Leu Asp Thr

210 215 220

Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp

225 230 235 240

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

245 250 255

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

260 265 270

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu

275 280 285

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

290 295 300

Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg

305 310 315 320

Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser

325 330 335

Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile

340 345 350

Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln

355 360 365

Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly

370 375 380

Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val

385 390 395 400

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

405 410 415

Ser Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu

420 425 430

Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys

435 440 445

Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu

450 455 460

Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser

465 470 475 480

Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu

485 490 495

Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro

500 505 510

Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Thr Thr Thr Pro

515 520 525

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

530 535 540

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

545 550 555 560

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

565 570 575

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

740 745

<210>6

<211>747

<212>PRT

<213> artificially synthesized sequence

<400>6

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 Lys Leu Gln Glu Ser Gly Pro Gly Leu

20 25 30

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

35 40 45

Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys

50 55 60

Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr

65 70 75 80

Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys

85 90 95

Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala

100 105 110

Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met

115 120 125

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

130 135140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met

145 150 155 160

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

165 170 175

Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr

180 185 190

Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr His Thr Ser

195 200 205

Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala

225 230 235 240

Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly

245 250 255

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

260 265 270

Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ala Ser

275 280 285

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

290 295 300

Glu Ser Val Ser Val Ile Gly Ala His Leu Ile His Trp Tyr Gln Gln

305 310 315 320

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

325 330 335

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

340 345 350

Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Ala Ala Ile Tyr

355 360 365

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

370 375 380

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

385 390 395 400

Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Val Gln Ser Gly Ser

405 410 415

Glu Leu Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser

420 425 430

Gly Tyr Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro

435 440 445

Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu

450 455 460

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

465 470 475 480

Thr Ser Val Ser Thr Ala Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu

485 490 495

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

500 505 510

Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro

515 520 525

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

530 535 540

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

545 550 555 560

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

565 570 575

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

740 745

<210>7

<211>2244

<212>DNA

<213> artificially synthesized sequence

<400>7

atggcactgc ctgtgactgc cctgctgctc cctctcgcac tcctgctgca cgcagcccgc 60

ccagacatcg tcctgaccca gtcacctgcc agcctggccg tcagcctggg cgagagagcc 120

accattaatt gccgcgctag cgaatcagtg tcagtcatcg gcgctcacct gatccactgg 180

tatcagcaaa agcctgggca gccaccaaag ctgctcatct atctcgcctc caacctggag 240

acaggcgtgc ctgctcgctt tagtggttct ggtagcggca ccgatttcac cctgactatt 300

tcaagcctgc aggcagagga cgcagccata tactattgcc tgcagtcccg gattttccct 360

cgcacattcg gccagggcac aaaactggaa atcaaaggat ctacctccgg ctccgggaag 420

cccggaagcg gcgaaggctc aaccaaaggc caggttcagc tcgtgcagtc tggttctgaa 480

ctgaagaaac ccggtgcatc tgtgaaagtc tcctgcaagg cttccgggta tactttcaca 540

gactacagta ttaattgggt tcgccaagct cctggacagg gactggagtg gatgggatgg 600

ataaacacag aaactaggga gcctgcatac gcctatgatt tcagaggtcg cttcgtgttc 660

agtctggata caagtgtttc aacagcctat ctgcaaatta gctccctgaa agccgaggac 720

accgcagttt actactgtgc acgggattat tcttacgcta tggactattg ggggcagggc 780

acactcgtga cagtgtctag cggcggcgga ggatctggag gaggaggaag cggcggagga 840

ggaagcgaag tgaagctgca ggagagcgga ccaggactgg tggctccttc acagtctctg 900

agcgtgacct gtaccgtgtc cggagtgtct ctgccagact acggagtgtc ttggatcaga 960

cagcctccta gaaagggact cgagtggctc ggagtgattt ggggcagcga gaccacctac 1020

tacaacagcg ccctgaagag caggctgacc atcatcaagg acaacagcaa gagccaggtg 1080

ttcctgaaga tgaacagcct gcagaccgac gacaccgcca tctactactg cgccaagcac 1140

tactactacg gcggcagcta cgccatggac tattggggac agggcaccag cgtgacagtg 1200

tctagcggag gaggcggcag cggaggagga ggatctggag gaggcggcag cgatattcag 1260

atgacccaga ccacctcttc tctgagcgcc agcctgggcg acagagtgac catctcttgc 1320

agggccagcc aggacatcag caagtacctg aattggtacc agcagaagcc cgacggcacc 1380

gtgaagctgc tgatctacca caccagcagg ctgcacagcg gagtgcctag cagattcagc 1440

ggaagcggca gcggcacaga ttatagcctg accatcagca acctggagca ggaggacatc 1500

gccacctact tttgccagca gggcaacacc ctgccttaca cctttggcgg aggcaccaag 1560

ctggagatca caaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 1620

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 1680

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 1740

ggggtccttc tcctgtcact ggttatcacc ctttactgca agcgcgggag gaagaagctg 1800

ctgtatatct tcaaacagcc ctttatgcgg ccagttcaga ccacacaaga ggaagatggc 1860

tgcagctgta gatttccaga agaggaggaa ggaggatgtg aactgagggt caaattttca 1920

cgctcagcag acgctcctgc ttaccaacaa ggccaaaatc agctgtacaa cgagctgaat 1980

ctgggtcgcc gggaggaata cgacgttctg gataaaaggc gcgggaggga cccagagatg 2040

ggtggcaagc ccaggcgcaa gaaccctcaa gaaggcctgt ataacgagct gcagaaagat 2100

aagatggcag aagcctactc tgaaatagga atgaagggcg agagacggcg cggaaagggc 2160

catgatggcc tctaccaggg actgtccacc gccacaaaag atacctacga cgcactccat 2220

atgcaggcac tgcctcctcg gtaa 2244

<210>8

<211>2244

<212>DNA

<213> artificially synthesized sequence

<400>8

atggcactgc ctgtgactgc cctgctgctc cctctcgcac tcctgctgca cgcagcccgc 60

ccagaagtga agctgcagga gagcggacca ggactggtgg ctccttcaca gtctctgagc 120

gtgacctgta ccgtgtccgg agtgtctctg ccagactacg gagtgtcttg gatcagacag 180

cctcctagaa agggactcga gtggctcgga gtgatttggg gcagcgagac cacctactac 240

aacagcgccc tgaagagcag gctgaccatc atcaaggaca acagcaagag ccaggtgttc 300

ctgaagatga acagcctgca gaccgacgac accgccatct actactgcgc caagcactac 360

tactacggcg gcagctacgc catggactat tggggacagg gcaccagcgt gacagtgtct 420

agcggcggag gcggaagcgg aggaggagga tctggaggag gcggcagcga tattcagatg 480

acccagacca cctcttctct gagcgccagc ctgggcgaca gagtgaccat ctcttgcagg 540

gccagccagg acatcagcaa gtacctgaat tggtaccagc agaagcccga cggcaccgtg 600

aagctgctga tctaccacac cagcaggctg cacagcggag tgcctagcag attcagcgga 660

agcggcagcg gcacagatta tagcctgacc atcagcaacc tggagcagga ggacatcgcc 720

acctactttt gccagcaggg caacaccctg ccttacacct ttggcggagg caccaagctg 780

gagatcacag gcggcggagg atctggagga ggaggaagcg gaggcggagg aagcgacatc 840

gtcctgaccc agtcacctgc cagcctggcc gtcagcctgg gcgagagagc caccattaat 900

tgccgcgcta gcgaatcagt gtcagtcatc ggcgctcacc tgatccactg gtatcagcaa 960

aagcctgggc agccaccaaa gctgctcatc tatctcgcct ccaacctgga gacaggcgtg 1020

cctgctcgct ttagtggttc tggtagcggc accgatttca ccctgactat ttcaagcctg 1080

caggcagagg acgcagccat atactattgc ctgcagtccc ggattttccc tcgcacattc 1140

ggccagggca caaaactgga aatcaaagga tctacctccg gctccgggaa gcccggaagc 1200

ggcgaaggct caaccaaagg ccaggttcag ctcgtgcagt ctggttctga actgaagaaa 1260

cccggtgcat ctgtgaaagt ctcctgcaag gcttccgggt atactttcac agactacagt 1320

attaattggg ttcgccaagc tcctggacag ggactggagt ggatgggatg gataaacaca 1380

gaaactaggg agcctgcata cgcctatgat ttcagaggtc gcttcgtgtt cagtctggat 1440

acaagtgttt caacagccta tctgcaaatt agctccctga aagccgagga caccgcagtt 1500

tactactgtg cacgggatta ttcttacgct atggactatt gggggcaggg cacactcgtg 1560

acagtgtcta gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 1620

tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 1680

acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 1740

ggggtccttc tcctgtcact ggttatcacc ctttactgca agcgcgggag gaagaagctg 1800

ctgtatatct tcaaacagcc ctttatgcgg ccagttcaga ccacacaaga ggaagatggc 1860

tgcagctgta gatttccaga agaggaggaa ggaggatgtg aactgagggt caaattttca 1920

cgctcagcag acgctcctgc ttaccaacaa ggccaaaatc agctgtacaa cgagctgaat 1980

ctgggtcgcc gggaggaata cgacgttctg gataaaaggc gcgggaggga cccagagatg 2040

ggtggcaagc ccaggcgcaa gaaccctcaa gaaggcctgt ataacgagct gcagaaagat 2100

aagatggcag aagcctactc tgaaatagga atgaagggcg agagacggcg cggaaagggc 2160

catgatggcc tctaccaggg actgtccacc gccacaaaag atacctacga cgcactccat 2220

atgcaggcac tgcctcctcg gtaa 2244

<210>9

<211>15

<212>PRT

<213> artificially synthesized sequence

<400>9

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210>10

<211>45

<212>DNA

<213> artificially synthesized sequence

<400>10

ggcggcggag gatctggagg aggaggaagc ggaggcggag gaagc 45

<210>11

<211>21

<212>PRT

<213> artificially synthesized sequence

<400>11

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

<210>12

<211>63

<212>DNA

<213> artificially synthesized sequence

<400>12

atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60

cct 63

<210>13

<211>45

<212>PRT

<213> artificially synthesized sequence

<400>13

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>14

<211>135

<212>DNA

<213> artificially synthesized sequence

<400>14

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210>15

<211>24

<212>PRT

<213> artificially synthesized sequence

<400>15

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>16

<211>72

<212>DNA

<213> artificially synthesized sequence

<400>16

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210>17

<211>42

<212>PRT

<213> artificially synthesized sequence

<400>17

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>18

<211>126

<212>DNA

<213> artificially synthesized sequence

<400>18

aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60

acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120

gagctg 126

<210>19

<211>112

<212>PRT

<213> artificially synthesized sequence

<400>19

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

1 5 10 15

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

20 25 30

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

35 40 45

Pro 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 105110

<210>20

<211>336

<212>DNA

<213> artificially synthesized sequence

<400>20

agggtgaagt tttctcggag cgccgatgca ccagcatatc agcagggaca gaatcagctg 60

tacaacgagc tgaatctggg caggcgcgag gagtacgacg tgctggataa gcggagaggc 120

agagatcccg agatgggagg caagccaagg aggaagaacc ctcaggaggg cctgtataat 180

gagctgcaga aggacaagat ggccgaggcc tactctgaga tcggcatgaa gggagagcgg 240

agaaggggca agggacacga tggcctgtat cagggcctga gcacagccac caaggacacc 300

tacgatgcac tgcacatgca ggccctgcca cctagg 336

Claims

1. A bispecific chimeric antigen receptor targeting BCMA and CD19, wherein said bispecific chimeric antigen receptor is a CD8 α signal peptide, which binds to the extracellular domain of BCMA and CD19 antigens, a CD8 α hinge region, a CD8 α transmembrane domain, a 4-1BB signaling domain and a CD3 zeta signaling domain in tandem;

the amino acid sequence of the bispecific chimeric antigen receptor is shown in one of SEQ ID NO. 5-6.

2. The bispecific chimeric antigen receptor according to claim 1, wherein the nucleotide sequences of the extracellular domains that bind to BCMA and CD19 antigens are shown as SEQ ID No.3 and SEQ ID No.4, respectively.

3. A nucleic acid encoding the bispecific chimeric antigen receptor of claim 1 or 2.

4. A viral vector comprising the nucleic acid of claim 3.

5. The viral vector according to claim 4, wherein the viral vector is a lentiviral vector and/or a retroviral vector.

6. The viral vector according to claim 5, wherein the viral vector is a lentiviral vector.

7. A T cell transfected with a nucleic acid sequence encoding the bispecific chimeric antigen receptor of claim 1 or 2.

8. The T-cell according to claim 7, wherein the transfection is by transfection into the T-cell with a viral vector and/or a eukaryotic expression plasmid.

9. The T-cell of claim 8, wherein the transfection is by transfection into the T-cell with a viral vector.

10. A recombinant lentivirus comprising a mammalian cell co-transfected with the viral vector of any one of claims 4-6 and a packaging helper plasmid.

11. The recombinant lentivirus of claim 10, wherein the mammalian cell is any one of 293 cells, 293T cells or 293F cells or a combination of at least two thereof.

12. A composition comprising the bispecific chimeric antigen receptor of claim 1 or 2 and/or the recombinant lentivirus of claim 10 or 11.

13. Use of a bispecific chimeric antigen receptor according to claim 1 or 2, a recombinant lentivirus according to claim 10 or 11 or a composition according to claim 12 for the preparation of chimeric antigen receptor T cells or for the preparation of a medicament for the treatment of B-cell tumors.

14. The use of claim 13, wherein the tumor is multiple myeloma, B-cell leukemia, and B-cell lymphoma.