CN109651511A

CN109651511A - A kind of Chimeric antigen receptor and its application targeting BCMA

Info

Publication number: CN109651511A
Application number: CN201811598951.8A
Authority: CN
Inventors: 李光超; 罗敏; 曾剑华; 郭锦涛; 莫文俊
Original assignee: Guangzhou Bio Gene Technology Co Ltd
Current assignee: Guangzhou Bio Gene Technology Co Ltd
Priority date: 2018-12-26
Filing date: 2018-12-26
Publication date: 2019-04-19
Anticipated expiration: 2038-12-26
Also published as: CN109651511B

Abstract

The present invention relates to a kind of targeting BCMA Chimeric antigen receptor, the Chimeric antigen receptor includes can be in conjunction with the extracellular domain, transmembrane domain and at least one intracellular domain of antigen, wherein the extracellular domain is anti-BCMA single domain antibody；Wherein, the amino acid sequence of the anti-BCMA single domain antibody is selected from: (a) amino acid sequence as shown in SEQ ID NO.1；Or, (b) amino acid sequence as shown in SEQ ID NO.1 is formed by one or more replacing, adding or deleting for amino acid, can specific bond in Chimeric antigen receptor, have in conjunction with BCMA and inducing T cell signal transduction function variant.Chimeric antigen receptor of the invention has smaller clinical side effects and higher safety, can effectively reduce solid tumor stove, effectively improve the therapeutic effect of tumour.

Description

BCMA (brain cell activating antigen) targeted chimeric antigen receptor and application thereof

Technical Field

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

Background

With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T-cell immunotherapy (CAR-T) is one of the most promising tumor immunotherapy. Generally, a chimeric antigen receptor CAR consists of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. Typically, the CAR comprises a Single chain fragment variable (scFv) of an antibody or a binding domain specific for a Tumor Associated Antigen (TAA), which is coupled to the cytoplasmic domain of a T cell signaling molecule by a hinge and a transmembrane region. The most common lymphocyte activation moiety comprises a T cell costimulatory domain in tandem with a T cell effector function triggering (e.g., CD3 ζ) moiety. CAR-mediated adoptive immunotherapy allows CAR-transplanted T cells to directly recognize TAAs on target tumor cells in a non-HLA-restricted manner.

The vast majority of patients with B-cell malignancies and multiple myeloma are significant contributors to cancer death. The response of B cell malignancies to various forms of treatment is mixed. The traditional methods for treating B cell malignancies have toxic and side effects, but the immunotherapy using antibodies against CD19, CD20, CD22, CD23, CD52, etc. has general effects. As technology developed, treatment began using cells modified to express Chimeric Antigen Receptors (CARs), but the therapeutic effect of the established antigen binding domains used in CARs was unpredictable, rendering the therapeutic effect unstable.

The use of BCMA antigens as the extracellular domain for the treatment of B-cell related diseases is disclosed, but the antigen binding domain binds too strongly, and CAR T cells induce large-scale cytokine release, resulting in a potentially lethal immune response that is considered a "cytokine storm"; however, if the antigen binding domain binds too weakly, CAR T cells fail to exhibit sufficient therapeutic efficacy in clearing cancer cells. In the prior art, CN 106687483 a discloses humanized anti-BCMA chimeric antigen receptor therapy for cancer by administering a method of expressing CAR genetically modified T cells to achieve treatment of diseases associated with expression of B cell maturation antigen protein (BCMA). CN 107207598A discloses BCMA chimeric antigen receptors for use in adoptive T cell therapy of B cell related conditions.

Thus, in addition to the advantages of antibody therapy in creating a chimeric antigen receptor targeting BCMA, the selection of an antibody with moderate binding to the antigen binding domain must provide a more effective therapeutic option for B cell-related diseases.

Disclosure of Invention

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

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

in one aspect, the invention provides a chimeric antigen receptor targeting BCMA comprising an extracellular domain capable of binding an antigen, a transmembrane domain, and at least one intracellular domain, wherein the extracellular domain is an anti-BCMA single domain antibody;

wherein the amino acid sequence of the anti-BCMA single domain antibody is selected from the group consisting of:

(a) an amino acid sequence shown as SEQ ID NO. 1; or,

(b) the amino acid sequence shown as SEQ ID NO.1 is formed by substitution, addition or deletion of one or more amino acids, can be specifically combined on a chimeric antigen receptor, and has variants with the functions of combining BCMA and inducing T cell signal transduction.

In the invention, the inventor finds an anti-BCMA single-domain antibody by screening a large amount of BCMA antibodies and analyzing whether the antibodies can be combined with human Fc segments or not through affinity, and can remarkably improve the treatment effect of the chimeric antigen receptor by combining the anti-BCMA single-domain antibody with an extracellular domain of the chimeric antigen receptor.

In a specific embodiment, the amino acid sequence shown in SEQ ID NO.1 is as follows: qvqvlsglgqaggslrlsscalgrttfsdhtlgwfrqapgkerefvgaisggstyyadsgrftisrdkaknylqmnslkpeddvaycaaaddrydrywgqgtqvtvss.

According to the invention, the amino acid sequence formed by substituting, adding or deleting one or more amino acids of the amino acid sequence shown in SEQ ID NO.1 has at least 90% of identity, preferably 95%, and more preferably 98% of identity with the amino acid sequence shown in SEQ ID NO. 1.

In a particular embodiment, the variant has 92% identity to SEQ ID No.1, still has the ability to specifically bind to a chimeric antigen receptor, has the ability to bind BCMA and induce T cell signaling.

In a particular embodiment, the variant has 94% identity to SEQ ID No.1, still has the ability to specifically bind to a chimeric antigen receptor, has the ability to bind BCMA and induce T cell signaling.

In a particular embodiment, the variant has 95% identity to SEQ ID No.1, still has the ability to specifically bind to a chimeric antigen receptor, has the ability to bind BCMA and induce T cell signaling.

In a particular embodiment, the variant has 96% identity to SEQ ID No.1, still has the ability to specifically bind to a chimeric antigen receptor, has the ability to bind BCMA and induce T cell signaling.

In a particular embodiment, the variant has 98% identity to SEQ ID No.1, still has the ability to specifically bind to a chimeric antigen receptor, has the ability to bind BCMA and induce T cell signaling.

In a particular embodiment, the variant has 99% identity to SEQ ID No.1, still has the ability to specifically bind to a chimeric antigen receptor, has the ability to bind BCMA and induce T cell signaling.

According to the invention, the nucleotide sequence of the anti-BCMA single domain antibody comprises a sequence shown as SEQ ID NO.2, and the nucleotide sequence shown as SEQ ID NO.2 is as follows: caggtgcagctggtagagtctgggggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgtaccttcagtgaccataccctgggctggttccgccaggctcccgggaaggagcgtgagtttgtaggagctatttcctggagtggtggtagcacatactatgcagactccgtgagcggccgattcaccatctctcgagacaaggccaagaacacgggctatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcagcagccgacgatcgctatagtgactatcgctactggggccaggggacccaggtcaccgtctcctca are provided.

According to the invention, the nucleotide sequence of said anti-BCMA single domain antibody comprises a variant having at least 85%, for example 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, preferably 90%, more preferably 95% identity to the sequence indicated in SEQ ID No.2, which expresses a protein having the ability to specifically bind to a chimeric antigen receptor, having the function of binding to BCMA and inducing T-cell signaling.

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 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 chimeric antigen receptor of the present invention further comprises a promoter, wherein the promoter 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 chimeric antigen receptor of the present invention.

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

According to the invention, the chimeric antigen receptor is a CD8 α signal peptide, which binds to the extracellular domain of BCMA antigen, the CD8 α hinge region, the CD8 α transmembrane domain, the 4-1BB signaling domain and the CD3 zeta signaling domain in tandem.

According to the invention, the amino acid sequence of the chimeric antigen receptor comprises a sequence shown as SEQ ID NO.3, and the amino acid sequence shown as SEQ ID NO.3 is specifically as follows: MALPVTALLLPLALLLHAARPQVQLVESGGGLVQAGGSLRLSCAASGRTFSDHTLGWFRQAPGKEREFVGAISWSGGSTYYADSVSGRFTISRDKAKNTGYLQMNSLKPEDTAVYYCAAADDRYSDYRYWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR are provided.

According to the invention, the amino acid sequence of the chimeric antigen receptor has at least 90% identity, for example, can be a variant with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, preferably 95%, more preferably 98% identity, to the sequence indicated in SEQ ID No. 3.

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

The nucleic acid is specifically shown as SEQ ID NO.4 and specifically as follows: atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgccctcaggtgcagctggtagagtctgggggaggattggtgcaggctgggggctctctgagactctcctgtgcagcctctggacgtaccttcagtgaccataccctgggctggttccgccaggctcccgggaaggagcgtgagtttgtaggagctatttcctggagtggtggtagcacatactatgcagactccgtgagcggccgattcaccatctctcgagacaaggccaagaacacgggctatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcagcagccgacgatcgctatagtgactatcgctactggggccaggggacccaggtcaccgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcagggtgaagttttctcggagcgccgatgcaccagcatatcagcagggacagaatcagctgtacaacgagctgaatctgggcaggcgcgaggagtacgacgtgctggataagcggagaggcagagatcccgagatgggaggcaagccaaggaggaagaaccctcaggagggcctgtataatgagctgcagaaggacaagatggccgaggcctactctgagatcggcatgaagggagagcggagaaggggcaagggacacgatggcctgtatcagggcctgagcacagccaccaaggacacctacgatgcactgcacatgcaggccctgccacctagg are provided.

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 disease associated with expression of B cell maturation antigen protein, such as multiple myeloma, hodgkin's lymphoma, leukemia or glioblastoma.

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

(1) according to the invention, a large amount of BCMA antibodies are screened, and a new anti-BCMA antibody which can be properly combined with BCMA antigen is found, so that a better treatment effect is achieved, and a more effective treatment selection is provided for B cell related diseases;

(2) the special structure of the alpaca single domain antibody (VHH) in the chimeric antigen receptor is the currently known minimum unit (only 15KDa) capable of combining with a target antigen, and can recognize some conventional epitopes, because the molecular weight is small, the CDR3 region is longer, the loop is extended outwards, the conventional antibody can recognize no recognized epitope, and in addition, the special structure has the advantages of good stability, weak immunogenicity to human and the like;

(3) the chimeric antigen receptor has the expression positive rate of more than 32 percent, has specific killing effect on BCMA positive cells, almost does not kill BCMA negative cells, 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 is a schematic diagram of the lentiviral expression vector pLVX-EF1 α -G8 CAR-Km elements;

FIG. 3 is a plasmid map of lentiviral expression vector pLVX-EF1 α -G8 CAR-Km;

FIG. 4 is a graph of the results of flow assay of CAR-T expression, wherein FIG. 4(a) is the G8 CAR expression ratio of untransfected T cells, FIG. 4(b) is the G8 CAR expression ratio of chimeric antigen receptors of the invention, FIG. 4(c) is the FMC63CAR expression ratio of untransfected T cells, and FIG. 4(d) is the FMC63CAR expression ratio of FMC63 CAR-T;

FIG. 5 is a CART killing target cell profile;

FIG. 6 is a graph showing the result 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, a chimeric antigen receptor against BCMA (G8 CAR) was constructed, as shown in the sequence diagram of fig. 1, comprising a signal peptide sequence (Leader) of CD8 α, a single domain antibody sequence (Anti-BCMA VHH) that specifically binds to BCMA antigen, a Hinge region (Hinge) and Transmembrane region sequence (Transmembrane) of CD8 α, a 4-1BB co-stimulatory domain sequence and a CD3 zeta signaling domain sequence, as follows:

the amino acid sequence (SEQ ID NO.5) of the CD8 α signal peptide (leader) is MALPVTALLLPLALLLHAARP;

the nucleotide sequence (SEQ ID NO.6) of the CD8 α signal peptide (leader) is ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCA CGCAGCACGCCCT;

the amino acid sequence (SEQ ID NO.7) of the hinge region (hinge) of CD8 α is TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD;

the nucleotide sequence (SEQ ID NO.8) of the CD8 α hinge region (hinge) is ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT;

the amino acid sequence (SEQ ID NO.9) of the CD8 α transmembrane region (TM) is IYIWAPLAGTCGVLLLSLVITLYC;

the nucleotide sequence (SEQ ID NO.10) of the CD8 α transmembrane region (TM) is ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC;

amino acid sequence of 4-1BB intracellular co-stimulatory domain (ICD) (SEQ ID NO. 11): KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL, respectively;

4-1BB Intracellular Costimulatory Domain (ICD) (SEQ ID NO. 12): AAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTG, respectively;

amino acid sequence of the zeta-signaling domain of CD3 (SEQ ID NO. 13): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR, respectively;

nucleotide sequence of the CD3 zeta signaling domain (SEQ ID NO. 14): AGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG are provided.

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

(1) Synthesizing a G8 CAR sequence by using a whole gene, carrying out double enzyme digestion on the G8 CAR synthesized by using EcoRI and BamHI and an empty vector, carrying out enzyme digestion for 30min in water bath at 37 ℃, carrying out DNA electrophoresis by using 1.5% agarose gel, and then purifying and recovering by using an agarose gel kit of Tiangen;

(2) ligation of pCDH-EF1-MCS vector to G8 CAR gene fragment:

the linking system is as follows:

assembly	Addition amount (μ l)
		PCDH-EF1-MCS vector	2(50ng)
G8 CAR gene	10(150ng)
		T4 DNA ligation buffer	2
T4 DNA ligase (NEB)	1
		dd H₂O	5
In all	20

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 anti-BCMA chimeric antigen receptor lentiviral expression vector pLVX-EF1 α -G8 CAR-Km are shown in FIG. 2, and the vector map is shown in FIG. 3.

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, mixing uniformly, standing for 15 minutes, adding the mixed solution into a T75 culture bottle paved with 293T cells, mixing lightly, and culturing at 37 ℃ with 5% CO₂Culturing 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.

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; absorbing 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 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 G8 CAR was measured 5 days after infection and G8 CAR expression was measured by flow cytometry using FITC-laboratory Human BCMA (cat No. BCA-HF2H1, Acrobiosystems) for binding to G8 VHH, the results are shown in fig. 4(a) -fig. 4 (b); from the results of fig. 4(a) -4 (b), it was shown that the positive expression rate of G8 CAR was 32.95%.

The expression of FMC63CAR was also detected by flow cytometry using FITC-Labeled Human CD19(20-291) Protein, Fc Tag (cat # CD9-HF251, Acrobiosystems) for binding to FMC63scFv, the results are shown in FIGS. 4(c) -4 (d); from the results of fig. 4(c) -4 (d), it was shown that FMC63CAR expression positive rate was 55.55%.

Example 6: cytotoxicity assays

Cytotoxicity assay experiments were performed with G8 CAR and CAR constructed with anti-CD 19 scFv (from FMC63 clone) and blank control NC (untransfected T cells) as controls, as follows:

detecting toxicity of CAR-T cells on K562, K562-CD19 (K562 cells stably expressing CD19), K562-BCMA (K562 cells stably expressing BCMA), and RPMI 8226 cells using LDH;

centrifuging various cells, washing with serum-free phenol red-free RPMI1640 medium for several times, counting, and collecting 1 × 10⁶50. mu.L of each of K562, K562-CD19, K562-BCMA and RPMI 8226 cells was plated in a 96-well plate as target cells, and the ratio of the target cells: effector cells 1:1 untransfected T cells and CAR-T cells were added separately at 37 ℃ with 5% CO₂Culturing in a cell culture box with certain humidity for 12 h; adding lysis solution as positive control, centrifuging for 5min at 250g, collecting 100 μ L culture supernatant per well, adding into new 96-well plate, adding 20 μ L reaction solution, and placing in dark room for reaction for 20-30 min.

And (3) measuring by a microplate reader at 590nm, and calculating the dissolution percentage according to the 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 application number 201510362935.5, CD33 specific chimeric antigen receptor and application thereof.

The results are shown in fig. 5, that G8 CAR-T cells were able to specifically kill BCMA positive cells (K562-BCMA and RPMI 8226), while there was little killing ability on BCMA negative cells (K562 and K562-CD 19); in addition, FMC63CAR-T cells were able to specifically kill CD19 positive K562-CD19 cells, while having little killing ability on CD19 negative cells (K562 and RPMI 8226).

Example 7: 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 mixed at 5X 10⁵Cells/well were seeded in 24-well plates. At a rate of 5X 10 per hole⁵Adding CAR-T and untransfected T cell (T mock) into the cells respectively, supplementing a 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. 6.

As can be seen from FIG. 6, the IFN- γ cytokine levels in the co-culture supernatants of G8 CAR-T cells and BCMA positive cells (K562-BCMA and RPMI 8226) were significantly higher than in the group of BCMA negative cells (K562 and K562-CD 19).

In conclusion, compared with other chimeric antigen receptors and other tumor antigens, the CART has better effect, and the invention finds a novel anti-BCMA antibody which can be properly combined with the BCMA antigen to achieve better treatment effect, thereby providing a more effective treatment option for B cell related diseases.

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> BCMA-targeted chimeric antigen receptor and application thereof

<130>2018

<160>14

<170>PatentIn version 3.3

<210>1

<211>119

<212>PRT

<213> artificially synthesized sequence

<400>1

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

1 5 10 15

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

20 25 30

Thr Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 4045

Gly Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Thr Gln Val Thr Val Ser Ser

115

<210>2

<211>357

<212>DNA

<213> artificially synthesized sequence

<400>2

caggtgcagc tggtagagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggacg taccttcagt gaccataccc tgggctggtt ccgccaggct 120

cccgggaagg agcgtgagtt tgtaggagct atttcctgga gtggtggtag cacatactat 180

gcagactccg tgagcggccg attcaccatc tctcgagaca aggccaagaa cacgggctat 240

ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagccgac 300

gatcgctata gtgactatcg ctactggggc caggggaccc aggtcaccgt ctcctca 357

<210>3

<211>363

<212>PRT

<213> artificially synthesized sequence

<400>3

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 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu

20 25 30

Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg

35 40 45

Thr Phe Ser Asp His Thr Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys

50 55 60

Glu Arg Glu Phe Val Gly Ala Ile Ser Trp Ser Gly Gly Ser Thr Tyr

65 70 75 80

Tyr Ala Asp Ser Val Ser Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala

85 90 95

Lys Asn Thr Gly Tyr LeuGln Met Asn Ser Leu Lys Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Ala Ala Ala Asp Asp Arg Tyr Ser Asp Tyr Arg

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

355 360

<210>4

<211>963

<212>DNA

<213> artificially synthesized sequence

<400>4

atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60

cctcaggtgc agctggtaga gtctggggga ggattggtgc aggctggggg ctctctgaga 120

ctctcctgtg cagcctctgg acgtaccttc agtgaccata ccctgggctg gttccgccag 180

gctcccggga aggagcgtga gtttgtagga gctatttcct ggagtggtgg tagcacatac 240

tatgcagact ccgtgagcgg ccgattcacc atctctcgag acaaggccaa gaacacgggc 300

tatctgcaaa tgaacagcct gaaacctgag gacacggccg tttattactg tgcagcagcc 360

gacgatcgct atagtgacta tcgctactgg ggccagggga cccaggtcac cgtctcctca 420

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 480

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 540

gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 600

ctgtcactgg ttatcaccct ttactgcagg gtgaagtttt ctcggagcgc cgatgcacca 660

gcatatcagc agggacagaa tcagctgtac aacgagctga atctgggcag gcgcgaggag 720

tacgacgtgc tggataagcg gagaggcaga gatcccgaga tgggaggcaa gccaaggagg 780

aagaaccctc aggagggcct gtataatgag ctgcagaagg acaagatggc cgaggcctac 840

tctgagatcg gcatgaaggg agagcggaga aggggcaagg gacacgatgg cctgtatcag 900

ggcctgagca cagccaccaa ggacacctac gatgcactgc acatgcaggc cctgccacct 960

agg 963

<210>5

<211>21

<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

20

<210>6

<211>63

<212>DNA

<213> artificially synthesized sequence

<400>6

atggcactgc cagtgacagc cctgctgctg ccactggccc tgctgctgca cgcagcacgc 60

cct 63

<210>7

<211>45

<212>PRT

<213> artificially synthesized sequence

<400>7

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>8

<211>135

<212>DNA

<213> artificially synthesized sequence

<400>8

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210>9

<211>23

<212>PRT

<213> artificially synthesized sequence

<400>9

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

1 5 10 15

Leu Val Ile Thr Leu Tyr Cys

20

<210>10

<211>72

<212>DNA

<213> artificially synthesized sequence

<400>10

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210>11

<211>42

<212>PRT

<213> artificially synthesized sequence

<400>11

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

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210>12

<211>126

<212>DNA

<213> artificially synthesized sequence

<400>12

aagagaggca ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg ccccgtgcag 60

acaacccagg aggaggacgg ctgcagctgt cggttcccag aggaggagga gggaggatgt 120

gagctg 126

<210>13

<211>112

<212>PRT

<213> artificially synthesized sequence

<400>13

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

<210>14

<211>336

<212>DNA

<213> artificially synthesized sequence

<400>14

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 chimeric antigen receptor targeting BCMA, comprising an extracellular domain capable of binding an antigen, a transmembrane domain, and at least one intracellular domain, wherein said extracellular domain is an anti-BCMA single domain antibody;

(a) an amino acid sequence shown as SEQ ID NO. 1; or,

2. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the amino acid sequence shown in SEQ ID No.1 is a variant having at least 90%, preferably 95%, more preferably 98% identity with the amino acid sequence shown in SEQ ID No.1 by substitution, addition or deletion of one or more amino acids;

preferably, the nucleotide sequence of said anti-BCMA single domain antibody comprises a variant having the sequence as shown in SEQ ID No.2, or having at least 85%, preferably 90%, further preferably 95% identity thereto.

3. The chimeric antigen receptor according to claim 1 or 2, wherein the transmembrane domain is a CD28 transmembrane domain and/or a CD8 α transmembrane domain;

preferably, the intracellular domain further comprises an intracellular costimulatory signaling domain and/or a CD3 zeta signaling domain;

preferably, the co-stimulatory signaling domain is any one of or a combination of at least two of the human 4-1BB intracellular region, the human CD28 intracellular region, the human CD27 intracellular region, the human OX40 intracellular region, the human CD30 intracellular region, the human CD40 intracellular region or the human OX40 intracellular region, preferably the human 4-1BB intracellular region;

preferably, the extracellular domain and transmembrane domain are connected by a hinge region;

preferably, the hinge region comprises an IgG1 hinge region and/or a CD8 α hinge region;

preferably, the chimeric antigen receptor further comprises a signal peptide, preferably a CD8 α signal peptide or a secretor signal peptide.

4. The chimeric antigen receptor according to any one of claims 1-3, wherein the chimeric antigen receptor comprises a signal peptide, an extracellular domain that binds BCMA antigen, a hinge region, a transmembrane domain, a costimulatory signaling domain, and a CD3 zeta signaling domain in tandem;

preferably, the chimeric antigen receptor is a CD8 α signal peptide, binding the extracellular domain of BCMA antigen, a CD8 α hinge region, a CD8 α transmembrane domain, a 4-1BB signaling domain, and a CD3 zeta signaling domain in tandem;

preferably, the amino acid sequence of the chimeric antigen receptor comprises the sequence shown as SEQ ID No.3 or a variant thereof having at least 90% identity, preferably 95%, more preferably 98% identity.

5. A nucleic acid encoding the chimeric antigen receptor of any one of claims 1-4.

6. A viral vector comprising the nucleic acid of claim 5;

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

7. A T cell expressed by transfection of a nucleic acid sequence encoded by the chimeric antigen receptor of any one of claims 1-4 into the T cell;

preferably, the transfection is by transfection into T cells by viral vectors and/or eukaryotic expression plasmids, preferably by viral vectors.

8. A recombinant lentivirus comprising the recombinant lentivirus of claim 6, which is obtained by co-transfecting a mammalian cell with the viral vector and a packaging helper plasmid;

preferably, the mammalian cell is any one of 293 cell, 293T cell or 293F cell or a combination of at least two thereof.

9. A composition comprising the chimeric antigen receptor of any one of claims 1-4 and/or the recombinant lentivirus of claim 8.

10. Use of the chimeric antigen receptor of any one of claims 1 to 4, the recombinant lentivirus of claim 8 or the composition of claim 9 for the preparation of chimeric antigen receptor T cells, immune cells or in a medicament for the treatment of tumors;

preferably, the tumor is a B cell malignancy.