CN111499723B

CN111499723B - Chimeric antigen receptor and application thereof

Info

Publication number: CN111499723B
Application number: CN202010468343.6A
Authority: CN
Inventors: 李光超; 罗敏; 周兆; 丁雯
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2020-05-28
Filing date: 2020-05-28
Publication date: 2021-04-09
Anticipated expiration: 2040-05-28
Also published as: CN111499723A

Abstract

The invention provides a chimeric antigen receptor and application thereof, wherein the chimeric antigen receptor adopts modified BAFF as an antigen binding domain, the modified BAFF retains the binding capacity of BAFF-R, but does not cause malignant B cell proliferation caused by BAFF activation, and the constructed CAR molecule can identify and bind the BAFF-R, so that the prepared CAR-T cell has specific targeted killing effect on B tumor cells positive to BAFF-R and negative to CD19, and is expected to become a new means for treating tumor recurrence caused by CD19 antigen deletion.

Description

Chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the technical field of biology, belongs to the technical field of cellular immunotherapy of diseases, relates to a chimeric antigen receptor and application thereof, and particularly relates to a chimeric antigen receptor targeting a BAFF-R positive tumor cell and application thereof in preparation of a medicine for treating tumor recurrence caused by CD19 antigen deletion.

Background

Chimeric Antigen Receptor (CAR) T cells are widely applied to treatment of B cell malignant tumors, CD 19-targeted CAR-T cells are precursors for treatment of B cell malignant tumors through CAR-T therapy, and an effective scheme is provided for treatment of B cell malignant tumors. However, tumor recurrence caused by the deletion of CD19 antigen greatly attenuated the therapeutic effect. CD 19-negative relapse has become an important problem, with CD 19-negative relapse occurring in approximately 30% of patients. Relevant targets for B cells also include CD20 and CD22, etc., but their expression levels in B cell leukemias and lymphomas are much lower than CD 19. Studies have shown that CAR-T therapy targeting CD19 is more effective than CAR-T targeting CD20 and CD22, suggesting that high density CAR target expression has better therapeutic effect. Therefore, the search for new antigen targets highly expressed on the surface of B cells is an important research direction for solving the problem of escape of CD19 antigen loss.

The B cell activating factor (BAFF) of TNF family is an important member of Tumor Necrosis Factor (TNF) superfamily, has two forms of membrane-bound form and soluble form, and the biological activities of the two forms are basically consistent, so that the B cell activating factor (BAFF) not only is widely involved in regulating B cell development, differentiation and antibody production, but also is widely involved in regulating T cell activation and response processes. BAFF binds to three different receptors, and thus has the functions of promoting the differentiation and maturation of B cells, class switching, promoting humoral immune response and T cell activation, and the like, and also can cause the degeneration of B cells and induce the generation of lymphoma (Novak A, Darce J, Arendt B, et al. expression of BCMA, TACI, BAFF-R in multiple myeloma: a mechanism for growth and survival. blood,2004,103(2): 689-694.; Klein B, Tarte K, Jourdan M, et al. survival and promotion factor of normal and monoclonal cells. int-J-Hematol,2003,78(2): 106-113.). BAFF has high homology with proliferation inducing ligand (APRIL). As shown in fig. 1, BAFF can bind to 3 receptors of B cell activating factor receptor (BAFF-R), B Cell Maturation Antigen (BCMA), and transmembrane activator and cyclophilin ligand activator (TACI), while APRIL binds to BCMA and TACI. It has been reported in the literature that BCMA has significantly higher affinity for APRIL than for BAFF, and this binding selectivity is probably due to sequence variability within the cysteine-rich domains (CRDs) of the receptor outside the Dx L motif.

BAFF-R is a specific receptor of BAFF and has important influence on BAFF-mediated B cell survival and maturation. BAFF-R is widely expressed in B cell subsets including immature B, transitional B, mature B, memory B, germinal center B, and plasma cells. The inventor previously found that BAFF-R and CD19 are co-expressed on various B-lymphoid tumor cell lines, and the BAFF-R is a potential target for treating CD19 negative relapse. CN109311991A discloses the construction of CAR-T with anti-BAFF-R antibodies, which were found to be cytotoxic to human lymphoma and Acute Lymphoblastic Leukemia (ALL) line cells. BAFF-R-CAR T cells can eliminate CD19 deficient tumor cells. (Hong Qin et al, CAR T cells targeting BAFF-R can overview CD19 anti loss in B cells malignnances science relative Medicine 2019: Vol.11, Issue 511, eaaw9414, DOI: 10.1126/sciitranslmed. aaw 9414).

Disclosure of Invention

The invention aims to provide a novel chimeric antigen receptor targeting BAFF-R and construct a CAR-T cell targeting BAFF-R, wherein the chimeric antigen receptor adopts BAFF monomer or trimer (TriBAFF) as an antigen binding domain of a CAR molecule, and the prepared CAR-T cell targets BAFF-R on the surface of a B cell, is used for treating B cell related diseases positively expressed by BAFF-R, and provides an effective scheme for solving negative relapse of CD19 CAR-T cell therapy.

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

in a first aspect, the present invention provides an engineered BAFF, which is a peptide fragment obtained by deletion mutation of an activated amino acid of soluble BAFF.

In the invention, the amino acid VHVFGDEL which is necessary for the 217-224 th activation of soluble BAFF (soluble form: 134-285) is replaced by GG to obtain the modified BAFF, the modified BAFF lacks the BAFF activation capability but retains the BAFF-R binding capability, and the CAR-T cell constructed as the antigen binding domain can inhibit the malignant B cell proliferation caused by the BAFF activation and retains the B cell recognition capability.

Preferably, the modified BAFF comprises an amino acid sequence shown in SEQ ID NO. 1;

SEQ ID NO:1：

AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGY FFIYGQVLYTDKTYAMGHLIQRKKGGSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDEL QLAIPRENAQISLDGDVTFFGALKLL。

in a second aspect, the present invention provides a chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signaling domain;

the antigen binding domain comprises an engineered BAFF monomer and/or multimer according to the first aspect.

In the invention, the modified BAFF shown in SEQ ID NO. 1 is used as an antigen binding domain, the CAR molecule is constructed, the binding with BAFF-R can be identified, but malignant B cell proliferation caused by BAFF activation can not be caused, and the prepared CAR-T cell has a specific targeted killing effect on B tumor cells with CD19 antigen deletion, and is expected to become a new means for treating tumor recurrence caused by CD19 antigen deletion.

Preferably, the antigen binding domain comprises an engineered BAFF monomer according to the first aspect.

Preferably, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO. 1.

Preferably, the antigen binding domain comprises an engineered BAFF trimer as described in the first aspect.

Preferably, the modified BAFF trimers are linked using GGGGS linkers.

Preferably, the antigen binding domain comprises the amino acid sequence shown in SEQ ID NO. 2;

SEQ ID NO:2：

AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKGGSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLLGGGGSAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKGGSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLLGGGGSAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKGGSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL。

in the invention, the modified BAFF tripolymer is used as an antigen binding structural domain, and the killing capacity of the prepared CAR-T cell to BAFF-R positive tumor cells is obviously enhanced, because the binding capacity of the BAFF tripolymer and a receptor BAFF-R is obviously improved compared with that of a BAFF monomer or wild type BAFF.

Preferably, the signal peptide comprises a CD8a signal peptide.

Preferably, the CD8 alpha signal peptide comprises the amino acid sequence shown in SEQ ID NO. 3;

SEQ ID NO:3：

MALPVTALLLPLALLLHAARP。

preferably, the hinge region comprises a CD8a hinge region.

Preferably, the CD8a hinge region comprises the amino acid sequence set forth in SEQ ID NO 4;

SEQ ID NO:4：

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD。

preferably, the transmembrane domain comprises a CD8a transmembrane domain.

Preferably, the CD8a transmembrane domain comprises the amino acid sequence shown in SEQ ID NO. 5;

SEQ ID NO:5：

IYIWAPLAGTCGVLLLSLVITLYC。

preferably, the co-stimulatory domain comprises 4-1 BB.

Preferably, the 4-1BB comprises the amino acid sequence shown in SEQ ID NO 6;

SEQ ID NO:6：

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL。

preferably, the signaling domain comprises CD3 ζ.

Preferably, the CD3 ζ comprises the amino acid sequence set forth in SEQ ID NO 7;

SEQ ID NO:7：

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

preferably, the chimeric antigen receptor consists of a CD8a signal peptide, an engineered BAFF monomer, a CD8a hinge region, a CD8a transmembrane domain, 4-1BB, and CD3 ζ in tandem.

Preferably, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 8;

SEQ ID NO:8：

MALPVTALLLPLALLLHAARPAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKGGSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLLSGGGSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

in the invention, a chimeric antigen receptor BAFF CAR shown in SEQ ID NO. 8 is constructed by taking a modified BAFF monomer as an antigen binding domain, and represents amino acid corresponding to a stop codon.

Preferably, the chimeric antigen receptor consists of a CD8a signal peptide, an engineered BAFF trimer, a CD8a hinge region, a CD8a transmembrane domain, 4-1BB and CD3 ζ in tandem.

Preferably, the chimeric antigen receptor comprises an amino acid sequence shown as SEQ ID NO. 9;

SEQ ID NO:9：

MALPVTALLLPLALLLHAARPAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKGGSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLLGGGGSAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKGGSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLLGGGGSAVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKETGYFFIYGQVLYTDKTYAMGHLIQRKKGGSLVTLFRCIQNMPETLPNNSCYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLLSGGGSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

in the invention, the chimeric antigen receptor TriBAFF CAR shown in SEQ ID NO. 9 is constructed by taking the modified BAFF trimer as an antigen binding domain.

In a third aspect, the present invention provides a gene encoding the chimeric antigen receptor of the second aspect.

In a fourth aspect, the present invention provides a lentiviral vector comprising the coding gene of the third aspect.

In a fifth aspect, the present invention provides a recombinant lentivirus which is a mammalian cell transfected with the lentiviral vector of the fourth aspect and a helper plasmid.

Preferably, the mammalian cells comprise any one or a combination of at least two of 293 cells, 293T cells or 293F cells, preferably 293T cells.

In a sixth aspect, the invention provides a CAR-T cell expressing an engineered BAFF of the first aspect and/or a chimeric antigen receptor of the second aspect.

In the invention, the modified BAFF is used as an antigen binding domain, the CAR molecule is constructed to recognize BAFF-R bound on the surface of a tumor cell, so that the prepared CAR-T cell has a specific targeted killing effect on BAFF-R positive cells and even CD19 antigen-deleted B tumor cells, and a new means is provided for treating tumor recurrence caused by CD19 antigen deletion.

Preferably, the CAR-T cell has integrated into its genome the gene encoding the gene of the third aspect.

Preferably, the CAR-T cell comprises the lentiviral vector of the fourth aspect and/or the recombinant lentivirus of the fifth aspect.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising any one of the engineered BAFF of the first aspect, the chimeric antigen receptor of the second aspect, the coding gene of the third aspect, the lentiviral vector of the fourth aspect, the recombinant lentivirus of the fifth aspect or the CAR-T cell of the sixth aspect, or a combination of at least two thereof.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In an eighth aspect, the invention provides a use of the modified BAFF of the first aspect, the chimeric antigen receptor of the second aspect, the coding gene of the third aspect, the lentiviral vector of the fourth aspect, the recombinant lentivirus of the fifth aspect, the CAR-T cell of the sixth aspect or the pharmaceutical composition of the seventh aspect in the manufacture of a medicament for the treatment of a disease.

Preferably, the disease comprises a B cell malignancy.

Preferably, the disease comprises recurrence of a B cell malignancy resulting from loss of CD19 antigen.

According to the invention, the treatment effect of CD19 CAR-T therapy is greatly weakened by tumor recurrence caused by CD19 antigen deletion, CD19 negative recurrence is an important problem, soluble BAFF is modified, the binding capacity of BAFF and BAFF-R is retained, the proliferation capacity of B tumor cells caused by BAFF activation is deleted, the obtained modified BAFF is used as an antigen binding domain to prepare CAR molecules, and the constructed CAR-T cells have a remarkable targeted killing effect on BAFF-R positive tumor cells, especially CD19 antigen deleted tumor cells, so that an effective means is provided for treating B cell malignant tumor recurrence caused by CD19 antigen deletion.

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

(1) the invention modifies the soluble BAFF, replaces the amino acid VHVFGDEL necessary for activation with GG, the modified BAFF lacks the BAFF activation capability, but retains the BAFF-R binding capability, and the CAR-T cell constructed as an antigen binding domain can inhibit malignant B cell proliferation caused by BAFF activation and retains the B cell recognition capability;

(2) the modified BAFF monomer or trimer is used as an antigen binding domain to construct the CAR molecule, and can be identified and combined with BAFF-R, so that the prepared CAR-T cell has a specific targeted killing effect on BAFF-R positive cells, wherein the TriBAFF CAR-T cell has a strong killing effect on BAFF-R positive tumor cells;

(3) the CAR-T cell constructed by taking the modified BAFF as the antigen binding domain has a specific targeted killing effect on the B tumor cell with the CD19 antigen deletion, and an effective means is provided for treating tumor recurrence caused by the CD19 antigen deletion.

Drawings

FIG. 1 is a schematic representation of the binding of BAFF and APRIL to receptors;

FIG. 2 is a graph of the effect of engineered BAFF on Raji cell proliferation;

FIG. 3(A) is the BAFF CAR and TriBAFF CAR structure with recognition site BAFF-R, FIG. 3(B) is the CD19 CAR structure with recognition site CD 19;

FIG. 4(A) is a BAFF CAR lentiviral vector structure, FIG. 4(B) is a TriBAFF CAR lentiviral vector structure, and FIG. 4(C) is a CD19 CAR lentiviral vector structure;

FIG. 5 is the results of flow assays of expression of T cell surface CAR molecules;

FIG. 6 shows that malignant B cell lines co-express CD19 and BAFF-R;

FIG. 7 is the killing effect of CAR-T cells on K562 cell line;

FIG. 8 shows the killing effect of CAR-T cells on CD19 and BAFF-R double positive tumor cells;

FIG. 9 shows the expression of Raji cells after knocking out CD 19;

FIG. 10 is a graph of the killing effect of CAR-T cells on Raji-CD 19-KO.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

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 and function of BAFF antigen binding Domain

In order to use BAFF as the antigen binding domain of CAR molecule, this example modifies soluble BAFF by replacing the amino acid VHVFGDEL essential for the 217-224 position activation of soluble BAFF (soluble form: 134-285) with GG to obtain modified BAFF shown in SEQ ID NO: 1.

(1) Modified binding ability of BAFF to BAFF-R

As shown in Table 1, the modified BAFF (mBAFF) lacks BAFF activation ability, but retains BAFF-R binding ability.

TABLE 1 analysis of binding of recombinant BAFF and post-engineering proteins to K562-BAFFR cells

Target protein	Mean Fluorescence Intensity (MFI)	Binding Positive Rate
			Wild soluble type BAFF	856	59.35％
Modification of soluble BAFF	969	62.56％
			Wild soluble BAFF trimer	2968	97.45％
Engineering soluble BAFF trimers	2565	95.67％
			PBS	25	0.12％

(2) Modified BAFF (B-factor-associated factor) on Raji cell proliferation

1×10⁴Each Raji-luc (luciferase-expressing Raji) cell was seeded in a black 96-well plate (Shanghai Jingan, cat # J09602) and recombinant BAFF protein (5. mu.g/mL) was added daily. After further culturing for 5 days, 100. mu.L of Reconstituted reagent (Bright-Glo Luciferase Assay System, cat. No. E2620) was added, and after incubation for 2min, fluorescence detection was carried out using a FLUOstar OMEGA instrument. The fluorescence value of the control group (with PBS added) was set to 100%, and proliferation of Raji cells in each group was evaluated.

The results are shown in FIG. 2: both wild soluble BAFF and wild soluble BAFF trimer significantly promote the proliferation of Raji cells, while both engineered soluble BAFF and engineered soluble BAFF trimer lose the ability to promote Raji cell proliferation.

Example 2 design of CAR molecules

In the embodiment, a modified BAFF shown as SEQ ID NO. 1 is used as an antigen binding domain to construct a monomer BAFF-based chimeric antigen receptor (BAFF CAR) and a trimer BAFF-based chimeric antigen receptor (TriBAFF CAR) shown as a figure 3(A), wherein three BAFFs in TriBAFF CAR molecules are connected through GGGGS connectors to recognize and bind to BAFF-R; a CD19 CAR based on anti-CD 19 antibody (FMC63) scFv, as shown in fig. 3(B), was also designed to specifically recognize CD19 antigen.

In addition to the antigen binding domain, the CAR molecule described above includes a CD8 α signal peptide sequence (Leader), a CD8 α Hinge region (Hinge) and Transmembrane region (Transmembrane) sequence, a 4-1BB co-stimulatory domain sequence, and a CD3 zeta signaling domain sequence.

EXAMPLE 3 construction of Lentiviral vectors

(1) The nucleic acid molecules of the following CAR molecules were synthesized in whole gene:

BAFF CAR: the polypeptide consists of a CD8a signal peptide sequence, an engineered BAFF sequence, a CD8a hinge region and transmembrane region sequence, 4-1BB and CD3 zeta in tandem (an amino acid sequence is shown as SEQ ID NO: 8);

TriBAFF CAR: the polypeptide consists of a CD8a signal peptide sequence, a three-segment repeated modified BAFF sequence (three BAFFs are connected by a GGGGS connector), a CD8a hinge region and transmembrane region sequence, and a 4-1BB and CD3 zeta tandem (an amino acid sequence is shown as SEQ ID NO: 9);

CD19 CAR: consists of a CD8a signal peptide sequence, a CD19 scFv, a CD8a hinge region and transmembrane region sequence, 4-1BB and CD3 zeta in series (the amino acid sequence is shown as SEQ ID NO: 10);

SEQ ID NO:10：

amino acid sequence of CD19 CAR:

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*。

and carrying out double enzyme digestion on the nucleic acid molecules of the CAR molecules by using EcoRI and BamHI, incubating in water bath at 37 ℃ for 30min, carrying out DNA electrophoresis on the digestion products in 1.5% agarose gel, and purifying and recovering the digestion fragments by using an agarose gel kit of Tiangen.

(2) Linearized pCDH-EF1-MCS vectors were ligated to CAR nucleic acid fragments

Preparing a connecting system shown in the table 2, and connecting for 1h at 22 ℃; the ligation products were transformed into Stbl3 E.coli competent cells, 200. mu.L of the transformation products were spread on an ampicillin-resistant LB plate and cultured in an inverted state in an incubator at 37 ℃ overnight; randomly selecting 3 monoclonals the next morning for colony PCR identification, and sending positive clones to sequence.

TABLE 2 connection System

Composition (I)	Dosage of
		Linearized pCDH-EF1-MCS vector	2μL(50ng)
CAR gene	10μL(150ng)
		T4 DNA ligase buffer	2μL
T4 DNA ligase (NEB)	1μL
		ddH₂O	5μL

Schematic diagrams of successfully constructed lentiviral vector elements BAFF CAR, TriBAFF CAR and CD19 CAR are shown in fig. 4(a), 4(B) and 4(C), respectively.

Example 4 Lentiviral packaging

In the embodiment, a four-plasmid system is adopted to carry out lentivirus packaging on the three lentivirus vectors constructed in the embodiment 3, auxiliary plasmids gag/pol, Rev and VSV-G are mixed with one of the three lentivirus vectors according to a proportion, added into serum-free DMEM with a certain volume, mixed uniformly and placed for 15 min; adding the above mixture into 293T cell culture bottle, mixing, and adding 5% CO at 37 deg.C₂Culturing for 6h in a cell culture box; after 6h, replacing a fresh culture medium, continuing to culture, and adding 10mM sodium butyrate solution; after 72h, collecting lentivirus culture supernatant for purification detection.

Example 5 isolation and expansion of T cells

Collecting 30mL of whole blood, and diluting peripheral blood by using physiological saline according to a ratio of 1: 1; adding a Ficoll lymphocyte separation solution into a centrifuge tube, slowly adding diluted peripheral blood, performing density gradient centrifugation, and gently sucking a PBMC layer to transfer into another centrifuge tube; washing PBMC layer with physiological saline, transferring into X-VIVO culture medium (containing 50ng/mL OKT3 and 300IU/mL IL-2) for activation, and changing the culture medium into X-VIVO containing 300IU/mL for expanded culture 2 days later; then counting every two days, replacing fresh X-VIVO culture medium containing 300IU/mL, and maintaining the cell concentration at (0.5-1) X10⁶one/mL.

Example 6 Lentiviral infection of T cells

This example utilizes RetroNectin to increase the efficiency of T cell infection by lentiviruses, as follows:

coating 30 mu g of RetroNectin in a 6-well plate, and placing the plate in a cell culture box at 37 ℃ for 2 hours; extracting RetroNectin, sealing the coated 6-well plate by using Hank's solution containing 2.5% BSA, and placing the plate in a 37 ℃ cell culture box for keeping 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, centrifuging for 2 hours at 2000g, and removing the supernatant; adding 1X 10⁶T cells (CD3 positive)>90%), 1000g, centrifuging for 10min, placing at 37 deg.C and 5% CO₂And culturing in a cell culture box with certain humidity.

Expression of CAR molecules on the T cell surface was detected 5 days after infection using flow cytometry, CD19 CAR expression using FITC-Labeled Human CD19 protein (cat No. CD9-HF251, Acrobiosystems), BAFF CAR and TriBAFF CAR expression using anti-BAFF antibodies (anti-BAFF mAb).

Results as shown in figure 5, the CAR T cell surface constructed successfully expressed CD19, BAFF or TriBAFF.

Example 7 flow assay of the Positive proportion of CD19 and BAFF-R expression in individual cells

This example uses FITC labeled CD19 antibody and BAFF-R antibody to detect CD19 and BAFF-R expression in a variety of tumor cell lines, including the human myeloma cell line NCI-H929, the human multiple myeloma cell line RPMI8226, the human Burkitt's lymphoma cell line Daudi, the human mantle cell lymphoma cell Jeko-1, the human T lymphoblastoid cell line Jurkat, the human Burkitt's lymphoma cell Raji, the human B lymphoblastic leukemia cell NALM-6, the human B lymphoblastic acute leukemia cell BALL-1, the histiocytic lymphoma cell U937, the human chronic myelogenous leukemia cell K562, the CD19 overexpressing K562 cell K562-CD19, and the BAFF-R overexpressing K562 cell K562-BAFF-R. The expression positive rate is shown in Table 3.

TABLE 3 Positive ratio of CD19 and BAFF-R expression by individual cells

Cell type	Name (R)	CD19(％)	BAFF-R(％)
				Human myeloma cell line	NCI-H929	0	1.5
Human multiple myeloma cell line	RPMI8226		0					1.55
				Human Burkitt's lymphoma cell line	Daudi	96.44	94.42
Human mantle cell lymphoma cell	Jeko-1	95.22	84.25
				Human T lymphoblast cell line	Jurkat	1.23	0.45
Human Burkitt's lymphoma cells	Raji	97.83	94.53
				Human B lymphoid leukemia cell	NALM-6	96.03	91.32
Human B lymphocyte acute leukemia cell	BALL-1	92.13	97.45
				Histiocytic lymphoma cells	U937	1.32	2.31
Human chronic myelogenous leukemia cell	K562	2.14	4.58
				Human chronic myelogenous leukemia cell	K562-CD19	98.24	2.16
Human chronic myelogenous leukemia cell	K562-BAFF-R	3.63	97.26

It can be seen that CD19 and BAFF-R exhibited co-expression in the malignant B cell lines Daudi, Jeko-1, Raji, NALM-6 and BALL-1, with the corresponding results shown in FIG. 6; while neither the myeloma cell lines NCI-H929 and RPMI8226, the T cell line Jurkat, nor the myeloid cells U937 and K562 expressed CD19 and BAFF-R.

Example 8CAR-T cells kill tumor cells

To verify the specific killing effect of the CAR-T cells prepared in example 6 on target cells, the inventors constructed K562-CD19 cells overexpressing CD19 protein and K562-BAFF-R cells overexpressing BAFF-R protein on the double negative cell line K562 with positive rates of 98.24% and 97.26%, respectively, see table 3; meanwhile, the double-positive Raji, Jeko-1 and BALL-1 cell lines also serve as tumor target cells.

Cytotoxicity assay experiments were performed with CD19-CAR-T, BAFF-CAR-T and TriBAFF-CAR-T, and LDH was used to assay the cytotoxicity of CAR-T cells on K562, K562-CD19, K562-BAFF-R, Raji, Jeko-1 and BALL-1, using untransfected T cells (Mock T) as a blank, as follows:

after various tumor target cells are centrifuged, the cells are washed with waterCounting after washing the serum phenol red-free RPMI1640 culture medium for multiple times; 50 μ L of 1X 10 was taken⁶K562, K562-CD19, K562-BAFF-R, Raji, Jeko-1 and BALL-1 cells were plated in a 96-well plate as target cells; adding untransfected T cells and three CAR-T cells according to different effective target ratios E: T (target cells: effector cells), respectively, and performing 5% CO treatment at 37 deg.C₂Culturing in a cell culture box with certain humidity for 12 h; adding lysis solution as positive control; centrifuging for 5min at 250g, taking 100 mu L of culture supernatant per well, adding the culture supernatant into a new 96-well plate, adding 20 mu L of reaction liquid, placing the plate in a dark room for reaction for 20-30 min, and measuring the result at 590nm by using an enzyme-labeling instrument; specific cleavage rate (Specific lysine) was calculated according to the following formula:

cytotoxicity (%) - [ (experimental well-medium background well) - (effector cell spontaneous LDH release well-medium background well) - (target cell spontaneous LDH release well-medium background well) ]/[ (target cell maximum LDH release well-volume corrected well) - (target cell spontaneous LDH release well-medium background well) ] × 100%;

the specific steps refer to the patent with the application number of 201510362935.5, CD33 specific chimeric antigen receptor and the application thereof.

Figure 7 shows that CD19-CAR-T cells can specifically kill only K562-CD19 cells overexpressing CD 19; BAFF-CAR-T and TriBAFF-CAR-T cells can only specifically kill K562-BAFF-R cells over-expressing BAFF-R, and the killing effect of the TriBAFF-CAR-T cells is better than that of the BAFF-CAR-T cells.

Figure 8 shows that both CD19-CAR-T, BAFF-CAR-T and TriBAFF-CAR-T kill tumor target cells that are double positive for CD19 and BAFF-R, with comparable killing of CD19-CAR-T cells and TriBAFF-CAR-T cells, but slightly weaker killing of BAFF-CAR-T cells.

EXAMPLE 9 construction of CD19 knock-out stable transgenic cell line Raji-CD19-KO

In order to explore the potential of monomer BAFF-CAR-T and trimer TriBAFF-CAR-T in treating CD19 negative recurrent tumors, the CRISPR-cas9 technology is adopted to knock out the expression of CD19 in Raji cells, and the sgRNA (SEQ ID NO:11) targeting CD19 is constructed into a lentivirus LentiCRISPRv2 vector by referring to the instruction of Zhang LentiCRISPRV2 vector, wherein the LentiCRISPRV2 plasmid is a large fragment recovered after being cut by BsmBI endonuclease, and a connecting system is shown in Table 4;

SEQ ID NO:11：5’-CAGTCCTATGAGGATATGAG-3’；

TABLE 4 connection System

Composition (I)	Dosage of
		Linearized LentiCRISPRV2 vector	1μL(50ng)
Double-stranded DNA corresponding to sgRNA	1μL
		T4 DNA ligase (NEB)	1μL
2 Xquick ligation buffer (NEB)	5μL
		ddH₂O	3μL

LentiCRISPRRv 2-CD19-sgRNA packaging lentivirus of lentivirus vector is used for infecting Raji cells, and puromycin (5 mu g/mL) is used for screening knockout stable transfectants.

FIG. 9 shows that CD19 was knocked out in Raji cells and Raji cells Raji-CD19-KO, which knock out CD19, were successfully constructed.

Example 10 potential of TriBAFF-CAR-T for treatment of CD 19-negative relapsed tumors

To verify the killing effect of monomeric BAFF-CAR-T and trimeric TriBAFF-CAR-T on CD 19-depleted tumor target cells, this example performed cytotoxicity assay experiments with Raji-CD19-KO cells as tumor target cells, CD19-CAR-T, BAFF-CAR-T and TriBAFF-CAR-T, respectively, and untransfected T cells (Mock T) as blank control.

The results are shown in FIG. 10, that CD19-CAR-T cells lost killing ability to the target cells due to deletion of CD19 antigen from Raji-CD19-KO cells; both BAFF-CAR-T and TriBAFF-CAR-T can effectively kill Raji-CD19-KO cells, wherein TriBAFF-CAR-T cells show better killing capability. These results indicate that TriBAFF-CAR-T cells can be used to treat patients who relapse negative to CD19-CAR-T cell therapy.

In conclusion, the treatment effect of CD19 CAR-T therapy is greatly weakened by tumor recurrence caused by CD19 antigen deletion, CD19 negative recurrence is an important problem, soluble BAFF is modified, the binding capacity of BAFF and BAFF-R is retained, the proliferation capacity of B tumor cells caused by BAFF activation is deleted, the obtained modified BAFF is used as an antigen binding domain to prepare CAR molecules, and the constructed CAR-T cells have a remarkable targeted killing effect on BAFF-R positive tumor cells, especially CD19 antigen deleted tumor cells, so that an effective means is provided for treating B cell malignant tumor recurrence caused by CD19 antigen deletion.

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> chimeric antigen receptor and uses thereof

<130> 20200527

<160> 11

<170> PatentIn version 3.3

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

1 5 10 15

Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe

20 25 30

Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys

35 40 45

Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly

50 55 60

Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln

65 70 75 80

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

85 90 95

Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala

100 105 110

Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn

115 120 125

Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys

130 135 140

Leu Leu

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

1 5 10 15

Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe

20 25 30

Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys

35 40 45

Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly

50 55 60

Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln

65 70 75 80

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

85 90 95

Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala

100 105 110

Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn

115 120 125

Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys

130 135 140

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

145 150 155 160

Thr Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile

165 170 175

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

180 185 190

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

195 200 205

Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr

210 215 220

Ala Met Gly His Leu Ile Gln Arg Lys Lys Gly Gly Ser Leu Val Thr

225 230 235 240

Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser

245 250 255

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

260 265 270

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

275 280 285

Thr Phe Phe Gly Ala Leu Lys Leu Leu Gly Gly Gly Gly Ser Ala Val

290 295 300

Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile Ala

305 310 315 320

Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro

325 330 335

Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn

340 345 350

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

355 360 365

Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys

370 375 380

Lys Gly Gly Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro

385 390 395 400

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

405 410 415

Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln

420 425 430

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

435 440 445

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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

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

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

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

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Ser Leu Val Ile Thr Leu Tyr Cys

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

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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

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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

100 105 110

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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 Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln

20 25 30

Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys

35 40 45

Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser

50 55 60

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

65 70 75 80

Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met

85 90 95

Gly His Leu Ile Gln Arg Lys Lys Gly Gly Ser Leu Val Thr Leu Phe

100 105 110

Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr

115 120 125

Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395

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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 Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln

20 25 30

Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys

35 40 45

Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser

50 55 60

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

65 70 75 80

Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met

85 90 95

Gly His Leu Ile Gln Arg Lys Lys Gly Gly Ser Leu Val Thr Leu Phe

100 105 110

Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr

115 120 125

Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala

130 135 140

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

145 150 155 160

Phe Gly Ala Leu Lys Leu Leu Gly Gly Gly Gly Ser Ala Val Gln Gly

165 170 175

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

180 185 190

Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu

195 200 205

Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile

210 215 220

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

225 230 235 240

Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Gly

245 250 255

Gly Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr

260 265 270

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

275 280 285

Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser

290 295 300

Leu Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu Leu Gly Gly

305 310 315 320

Gly Gly Ser Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys

325 330 335

Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser

340 345 350

Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu

355 360 365

Glu Glu Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe

370 375 380

Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His

385 390 395 400

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

405 410 415

Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala

420 425 430

Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro

435 440 445

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

450 455 460

Ala Leu Lys Leu Leu Ser Gly Gly Gly Ser Asp Pro Thr Thr Thr Pro

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

690 695

<210> 10

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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 Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Thr Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

450 455 460

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

465 470 475 480

Gln Ala Leu Pro Pro Arg

485

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<212> DNA

<213> Artificial sequence

<400> 11

cagtcctatg aggatatgag 20

Claims

1. A chimeric antigen receptor characterized by,

the chimeric antigen receptor is composed of a CD8 alpha signal peptide, an engineered BAFF monomer, a CD8 alpha hinge region, a CD8 alpha transmembrane domain, 4-1BB and a CD3 zeta in tandem; the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 8; and/or

The chimeric antigen receptor consists of a CD8 alpha signal peptide, an engineered BAFF trimer, a CD8 alpha hinge region, a CD8 alpha transmembrane domain, 4-1BB and a CD3 zeta in tandem; the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID NO. 9.

2. A coding gene encoding the chimeric antigen receptor of claim 1.

3. A lentiviral vector comprising the coding gene of claim 2.

4. A recombinant lentivirus, wherein the recombinant lentivirus is a mammalian cell transfected with the lentiviral vector of claim 3 and a helper plasmid.

5. The recombinant lentivirus of claim 4, wherein the mammalian cells comprise any one of 293 cells, 293T cells or 293F cells or a combination of at least two thereof.

6. The recombinant lentivirus of claim 5, wherein the mammalian cell is a 293T cell.

7. A CAR-T cell expressing the chimeric antigen receptor of claim 1.

8. The CAR-T cell of claim 7, wherein the coding gene of claim 2 is integrated into the genome of the CAR-T cell.

9. The CAR-T cell of claim 8, wherein the CAR-T cell comprises the lentiviral vector of claim 3 and/or the recombinant lentivirus of any one of claims 4-6.

10. A pharmaceutical composition comprising any one of the chimeric antigen receptor of claim 1, the coding gene of claim 2, the lentiviral vector of claim 3, the recombinant lentivirus of any one of claims 4-6, or the CAR-T cell of any one of claims 7-9, or a combination of at least two thereof.

11. The pharmaceutical composition of claim 10, further comprising any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient, or diluent.

12. Use of the chimeric antigen receptor of claim 1, the coding gene of claim 2, the lentiviral vector of claim 3, the recombinant lentivirus of any one of claims 4-6, the CAR-T cell of any one of claims 7-9, or the pharmaceutical composition of claim 10 or 11 for the manufacture of a medicament for the treatment of a disease;

the disease is a B cell malignancy.

13. The use of claim 12, wherein the disease comprises recurrence of a B cell malignancy resulting from loss of CD19 antigen.