CN109593137B

CN109593137B - Construction and application of novel CD20-CAR vector of anti-CD 20 antibody

Info

Publication number: CN109593137B
Application number: CN201811652891.3A
Authority: CN
Inventors: 杨林; 游凤涛
Original assignee: Persongen Biotherapeutics Suzhou Co ltd
Current assignee: Persongen Biotherapeutics Suzhou Co ltd
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2023-04-14
Anticipated expiration: 2038-12-29
Also published as: CN109593137A

Abstract

The invention provides the construction and application of a novel CD20-CAR vector based on a humanized CD20 antibody. Specifically, the present invention provides a chimeric antigen receptor whose antigen-binding domain (i.e., scFv) includes an antibody heavy chain variable region shown in SEQ ID No.1 and an antibody light chain variable region shown in SEQ ID No. 2. The CD20-CAR-T cells constructed by using the CD20-CAR vector constructed by the invention show very good killing effect in vitro.

Description

Construction and application of novel CD20-CAR vector of anti-CD 20 antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to construction and application of a novel CD20-CAR vector based on a humanized CD20 antibody.

Background

In recent years, chimeric antigen receptor T cell (CAR-T) therapy, as a powerful novel adoptive immunotherapy technology, has shown very effective therapeutic effects, and is used for treating various solid and hematological cancers, especially CD19-CAR-T therapy against B cell lymphoblastic leukemia and lymphoma, which shows very good therapeutic effects clinically, but for CD19 negative B cell tumors, CD19-CAR-T is not effective, and CD20 is also a good target of B cell tumors, so that CD20-CAR-T can be used for targeted therapy of CD20 positive B cell leukemia and lymphoma, and CD20-CAR-T cells constructed with humanized CD20 antibody sequences can reduce immunogenicity generated after entering into the body and improve therapeutic effects compared with murine CD20 sequences.

Therefore, there is an urgent need in the art to develop a novel CD20-CAR vector based on a humanized CD20 antibody.

Disclosure of Invention

The invention aims to provide construction and application of a novel CD20-CAR vector based on a humanized CD20 antibody.

In a first aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR) (sequence) whose antigen-binding domain (i.e., scFv) comprises an antibody heavy chain variable region shown in SEQ ID No.1 and an antibody light chain variable region shown in SEQ ID No. 2.

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQRLEWMGAIYPGNGDTSYNQKFKGRVTITADTSASTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS(SEQ ID No.:1)

DIQLTQSPSFLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIKRTV(SEQ ID No.:2)

In another preferred embodiment, the antigen binding domain (scFv) of the chimeric antigen receptor is represented by formula I or formula II:

V _H －V _L ,(I)；V _L －V _H ,(II)

wherein, V _H Is an antibody heavy chain variable region; v _L Is an antibody light chain variable region; "-" is a linker peptide or peptide bond.

In another preferred embodiment, the amino acid sequence of the connecting peptide is shown in SEQ ID NO. 3.

GGGGSGGGGSGGGGS(SEQ ID No.:3)

In another preferred embodiment, the chimeric antigen receptor has the structure shown in the following formula:

L－scFv－H－TM－C－CD3ζ

wherein the content of the first and second substances,

l is an optional Leader sequence (i.e. signal peptide sequence);

scFv is an antigen binding domain;

h is a hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

CD3 ζ is a cytoplasmic signaling sequence derived from CD3 ζ;

the antigen binding domains and "-" are as described above, respectively.

In another preferred embodiment, the sequence of L is shown in SEQ ID No. 4.

MLLLVTSLLLCELPHPAFLLIP(SEQ ID No.:4)

In another preferred embodiment, the sequence of H is as shown in SEQ ID No. 5.

ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID No.:5)

In another preferred embodiment, the sequence of TM comprises the transmembrane region from ICOS, preferably the sequence of TM is as shown in SEQ ID No. 6.

WLPIGCAAFVVVCILGCILICWL(SEQ ID No.:6)

In another preferred embodiment, the costimulatory signal molecule comprises an ICOS-derived costimulatory signal molecule, and/or a 4-1 BB-derived costimulatory signal molecule.

In another preferred embodiment, the amino acid sequence of the ICOS-derived costimulatory signal molecule is shown in SEQ ID No. 7.

TKKKYSSSVHDPNGEY MFMRAVNTAKKSRLTDVTL(SEQ ID No.:7)

In another preferred embodiment, the amino acid sequence of the co-stimulatory signaling molecule from 4-1BB source is shown in SEQ ID NO. 8.

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID No.:8)

In another preferred embodiment, the sequence of CD3 ζ is as shown in SEQ ID No. 9.

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID No.:9)

In another preferred embodiment, the sequence of the chimeric antigen receptor is as shown in SEQ ID No. 10.

MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQRLEWMGAIYPGNGDTSYNQKFKGRVTITADTSASTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVSGGGGSGGGGSGGGGSDIQLTQSPSFLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGTSFTLTISSLQPEDFA

TYYCQQWTSNPPTFGGGTKVEIKRTVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL

HNHYTQKSLSLSLGKWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID No.:10)

In a second aspect of the invention there is provided a nucleic acid molecule encoding a Chimeric Antigen Receptor (CAR) according to the first aspect of the invention.

In another preferred embodiment, said nucleic acid molecule comprises a nucleic acid sequence encoding said hinge region selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 5;

(b) A polynucleotide having a sequence as set forth in SEQ ID No. 11;

GAGAGCAAGTACGGACCTCCTTGTCCTCCTTGTCCAGCTCCAGAGTTTGAGGGCGGACCTAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTTCCAGAGCACCTACAGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGACATCGCAGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAACTACAAGACCACACCTCCAGTGCTGGATAGCGACGGAAGCTTCTTCCTGTACAGCCGGCTGACCGTGGATAAAAGCCGCTGGCAGGAGGGCAACGTGTTCAGTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTCGGCAAG(SEQ ID NO.:11)

(c) A polynucleotide having a nucleotide sequence having 90% or more (preferably 95% or more) homology with the sequence shown in SEQ ID No. 11 and encoding the amino acid sequence shown in SEQ ID No. 5;

(d) A polynucleotide complementary to any one of the polynucleotides of (a) - (c).

In another preferred embodiment, said nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane region of ICOS selected from the group consisting of seq id no:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 6;

(b) A polynucleotide having a sequence as set forth in SEQ ID No. 12;

TGGCTGCCTATTGGTTGCGCAGCTTTCGTCGTCGTCTGCATCCTGGGTTGCATCCTGATTTGCTGGCTG(SEQ ID NO.:12)

(c) A polynucleotide having a nucleotide sequence having 90% or more (preferably 95% or more) homology to the sequence shown in SEQ ID No. 12 and encoding the amino acid sequence shown in SEQ ID No. 6;

In another preferred embodiment, the nucleic acid molecule comprises a co-stimulatory signaling molecule coding sequence comprising an ICOS-derived co-stimulatory signaling molecule coding sequence, and/or a 4-1 BB-derived co-stimulatory signaling molecule coding sequence, wherein

The ICOS-derived costimulatory signal molecule coding sequence is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 7;

(b) A polynucleotide having a sequence as set forth in SEQ ID No. 13;

ACCAAGAAGAAGTACAGCTCTAGCGTGCACGACCCTAACGGCGAGTACATGTTCATGCGGGCCGTCAACACCGCCAAAAAGAGCCGGCTGACCGACGTGACACTG(SEQ ID NO.:13)

(c) A polynucleotide having a nucleotide sequence having 90% or more (preferably 95% or more) homology to the sequence shown in SEQ ID No. 13 and encoding the amino acid sequence shown in SEQ ID No. 7;

(d) A polynucleotide complementary to a polynucleotide of any one of (a) - (c);

the coding sequence of the 4-1BB derived costimulatory signal molecule is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 8;

(b) A polynucleotide having a sequence as shown in SEQ ID No. 14;

AAGAGGGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCAGTGCAGACAACACAGGAGGAAGACGGCTGCTCTTGCAGGTTCCCAGAGGAGGAGGAGGGCGGTTGCGAGCTG(SEQ ID NO.:14)

(c) A polynucleotide having a nucleotide sequence having 90% or more (preferably 95% or more) homology with the sequence shown in SEQ ID No. 14 and encoding the amino acid sequence shown in SEQ ID No. 8;

In another preferred embodiment, said nucleic acid molecule comprises a nucleic acid sequence encoding an intracellular signaling domain of said CD3 ζ selected from the group consisting of seq id nos:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 9;

(b) A polynucleotide having a sequence as set forth in SEQ ID No. 15;

AGAGTGAAGTTCAGCAGAAGCGCCGACGCTCCAGCTTATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGGAGAGAGGAATACGACGTGCTGGACAAGAGGAGGGGCAGAGATCCCGAGATGGGAGGCAAGCCCAGAAGAAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATTGGCATGAAGGGCGAGAGAAGGAGGGGCAAAGGCCACGACGGACTGTATCAGGGCCTGTCTACCGCCACCAAGGATACCTACGACGCTCTGCACATGCAGGCTCTGCCTCCTAGA(SEQ ID NO.:15)

(c) A polynucleotide having a nucleotide sequence having 90% or more (preferably 95% or more) homology with the sequence shown in SEQ ID No. 15 and encoding the amino acid sequence shown in SEQ ID No. 9;

In another preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 10;

(b) A polynucleotide having a sequence as shown in SEQ ID No. 16;

(c) A polynucleotide having a nucleotide sequence homology of 95% or more (preferably 98% or more) to the sequence shown in SEQ ID No. 16 and encoding an amino acid sequence shown in SEQ ID No. 10;

In another preferred embodiment, the nucleic acid molecule is isolated.

In another preferred embodiment, the sequence of the nucleic acid molecule is as shown in SEQ ID No. 16.

ATGCTGCTGCTGGTGACCTCTCTGCTGCTCTGCGAACTGCCTCACCCAGCCTTTCTGCTGATCCCTCAGGTGCAGCTGGTGCAGTCAGGAGCCGAAGTGAAGAAGCCAGGAGCCTCAGTGAAGGTGTCTTGCAAGGCCAGCGGCTACACCTTCACCAGCTACAACATGCATTGGGTCCGGCAGACCCCAGGACAGAGACTCGAGTGGATGGGAGCCATCTACCCCGGCAACGGCGATACCAGCTACAACCAGAAGTTCAAGGGCCGCGTGACCATCACAGCCGATACAAGCGCCAGCACCGCCTACATGGAGCTGTCTAGCCTGAGGAGCGAGGACACAGCCGTGTACTATTGCGCCAGGAGCACCTACTACGGCGGCGATTGGTACTTCAACGTGTGGGGCCAGGGCACAACAGTGACAGTGAGCGGAGGAGGAGGAAGCGGAGGAGGAGGATCTGGAGGAGGCGGCAGCGATATTCAGCTGACCCAGAGCCCTTCTTTTCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACTTGCAGGGCCAGCAGCAGCGTGTCCTACATCCATTGGTACCAGCAGAAGCCAGGCAAGGCTCCTAAGCTGCTGATCTACGCCACCAGCAACCTGGCCAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGAAGCGGCACAAGCTTTACCCTGACCATCAGCTCTCTGCAGCCAGAGGACTTCGCCACCTACTACTGCCAGCAGTGGACCAGCAACCCTCCTACATTTGGCGGCGGAACCAAGGTGGAGATCAAGCGGACCGTGGAGAGCAAGTACGGACCTCCTTGTCCTCCTTGTCCAGCTCCAGAGTTTGAGGGCGGACCTAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCAGAAGTGACTTGCGTGGTGGTGGACGTGTCTCAGGAGGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCTAAGACCAAGCCCAGGGAGGAGCAGTTCCAGAGCACCTACAGGGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACCCTGCCTCCTAGCCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGACATCGCAGTCGAGTGGGAAAGCAACGGCCAGCCCGAGAACAACTACAAGACCACACCTCCAGTGCTGGATAGCGACGGAAGCTTCTTCCTGTACAGCCGGCTGACCGTGGATAAAAGCCGCTGGCAGGAGGGCAACGTGTTCAGTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTCGGCAAGTGGCTGCCTATTGGTTGCGCCGCCTTTGTCGTCGTCTGTATCCTCGGCTGCATCCTGATTTGTTGGCTGACCAAGAAGAAGTACAGCAGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGCGGGCCGTCAACACCGCCAAGAAGAGCAGGCTGACCGACGTGACACTGAAGAGGGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCAGTGCAGACAACACAGGAGGAAGACGGCTGCTCTTGCAGGTTCCCAGAGGAGGAGGAGGGCGGTTGCGAGCTGAGAGTGAAGTTCAGCAGAAGCGCCGACGCTCCAGCTTATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGGAGAGAGGAATACGACGTGCTGGACAAGAGGAGGGGCAGAGATCCCGAGATGGGAGGCAAGCCCAGAAGAAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATTGGCATGAAGGGCGAGAGAAGGAGGGGCAAAGGCCACGACGGACTGTATCAGGGCCTGTCTACCGCCACCAAGGATACCTACGACGCTCTGCACATGCAGGCTCTGCCTCCTAGATGA(SEQ ID NO.:16)

In a third aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In another preferred embodiment, the vector is a lentiviral vector.

In a fourth aspect of the invention, there is provided a host cell comprising the vector or chromosome of the third aspect of the invention integrated with an exogenous nucleic acid molecule of the second aspect of the invention.

In another preferred embodiment, the cell is an isolated cell, and/or the cell is a genetically engineered cell.

In another preferred embodiment, the cell is a mammalian cell.

In another preferred embodiment, the cell is a T cell.

In a fifth aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a chimeric antigen receptor according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a cell according to the fourth aspect of the invention.

In a sixth aspect of the invention, there is provided a use of the chimeric antigen receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, or the cell of the fourth aspect of the invention, for the manufacture of a medicament or formulation for the treatment of a tumour.

In another preferred embodiment, the tumor comprises a CD20 positive tumor.

In a seventh aspect of the invention, there is provided a method of treating a disease, comprising administering to a subject in need thereof an amount of the chimeric antigen receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, or the cell of the fourth aspect of the invention, or the pharmaceutical composition of the fifth aspect of the invention.

In another preferred embodiment, the disease is a tumor.

In an eighth aspect of the invention there is provided a method of making a CAR-T cell (CAR-modified T cell) expressing a chimeric antigen receptor according to the first aspect of the invention,

the method comprises the following steps: (ii) transfecting the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention into a T cell, thereby obtaining the CAR-T cell.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the structure of the humanized CD20-CAR vector.

Figure 2 shows a flow chart of transfection efficiency of humanized CD20-CAR-T cells.

Figure 3 shows the cytotoxicity of humanized CD20-CAR-T cells against K562 cells overexpressing CD 20.

FIG. 4 shows the cytotoxicity of humanized CD20-CAR-T cells against B-lymphoid leukemia cells (Nalm-6) and lymphoma cells (Raji) naturally expressing CD 20.

FIG. 5 shows cytokine secretion following incubation of CD20-CAR-T cells with Raji and Nalm-6 cells.

Detailed Description

The inventor of the invention has extensively and deeply studied and constructed a novel CD20-CAR vector targeting CD20 malignant tumor for the first time (third generation CAR vector). The CAR-T cell constructed by the CAR vector constructed by the humanized CD20 antibody sequence can clinically target B cell malignant tumors (B cell leukemia and lymphoma) of a CD20 antigen, can avoid the immunogenicity of the CD20-CAR-T cell constructed by the murine CD20scFv in a human body, improves the clinical curative effect, and lays a foundation for better targeted treatment of the B cell derived leukemia and lymphoma. In addition, the CD20-CAR-T cells constructed by using the CD20-CAR vector constructed by the invention show very good killing effect in vitro. The present invention has been completed based on this finding.

Chimeric antigen receptors

The invention provides Chimeric Antigen Receptors (CARs) comprising an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding member (also referred to as an antigen-binding domain). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. The costimulatory signaling region refers to a portion of the intracellular domain that includes the costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.

A linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an ectodomain or a cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets CD 20. The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, to be driven to death, or to otherwise be affected, and causing the patient's tumor burden to shrink or be eliminated. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain, and the CD3 zeta signaling domain.

In one embodiment, a CD20 targeting CAR of the invention comprises the specific signaling domains of the invention (transmembrane region of ICOS, intracellular signaling domains of 4-1BB and CD3 ζ in tandem). The signaling domains of the invention significantly increase the anti-tumor activity and in vivo persistence of CAR-T cells compared to other means of CD 20-targeted CARs.

In a preferred embodiment of the invention, the amino acid sequence of the Chimeric Antigen Receptor (CAR) provided by the invention is as follows:

the coding DNA sequence is as follows:

antigen binding domains

In one embodiment, the CAR of the invention comprises a target-specific binding element referred to as an antigen-binding domain. The antigen binding domain of the CAR of the invention is a specific binding element that targets CD 20.

In a preferred embodiment of the invention, the antigen binding domain comprises the heavy chain variable region and the light chain variable region of an anti-CD 20 antibody.

In another preferred embodiment, the amino acid sequence of the antibody heavy chain variable region is as follows:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQTPGQRLEWMGAIYPGNGDTSYNQKFKGRVTITADTSASTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGQGTTVTVS(SEQ ID NO.:1)；

in another preferred embodiment, the amino acid sequence of the variable region of the antibody light chain is as follows:

DIQLTQSPSFLSASVGDRVTITCRASSSVSYIHWYQQKPGKAPKLLIYATSNLASGVPSRFSGSGSGTSFTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIKRTV(SEQ ID NO.:2)；

in a preferred embodiment of the present invention, the amino acid sequence of the linkage between the heavy chain variable region and the light chain variable region is as follows:

GGGGSGGGGSGGGGS(SEQ ID NO.:3)

hinge region and transmembrane region

For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domains may be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with other members of the receptor complex.

In a preferred embodiment of the invention, the hinge region comprises the following amino acid sequence:

the coding DNA sequence is as follows:

in a preferred embodiment of the invention, the transmembrane region of the CAR of the invention is an ICOS-derived transmembrane region.

In a preferred embodiment of the invention, the amino acid sequence of the ICOS-derived transmembrane region is as follows:

WLPIGCAAFVVVCILGCILICWL(SEQ ID NO.:6)

the coding DNA sequence is as follows:

intracellular domains

The intracellular domains in the CAR of the invention include the signaling domain of ICOS, the signaling domain of 4-1BB, and the signaling domain of CD3 zeta.

Preferably, the intracellular signaling domain of ICOS comprises the amino acid sequence:

TKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL(SEQ ID NO.:7)

the coding DNA sequence is as follows:

preferably, the intracellular signaling domain of 4-1BB comprises the amino acid sequence:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID No.:8)

the coding DNA sequence is as follows:

preferably, the intracellular signaling domain of CD3 ζ comprises the amino acid sequence:

the coding DNA sequence is as follows:

carrier

The invention also provides DNA constructs encoding the CAR sequences of the invention.

Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The present invention also provides a vector into which the DNA construct of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, since they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus, in that they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, molecular cloning. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Generally, suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584, WO01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp apart, and activity begins to decline. Depending on the promoter, it appears that the individual elements may function cooperatively or independently to initiate transcription.

An example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1 α (EF-1 α). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV 40) early promoter, the mouse mammary cancer virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr (Epstein-Barr) virus immediate early promoter, the rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch that is capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

To assess the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, the reporter gene is the following: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., ui-Tei et al, 2000febs letters 479. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Generally, the construct with the minimum of 5 flanking regions that showed the highest level of reporter gene expression was identified as the promoter. Such promoter regions can be linked to reporter genes and used to evaluate the ability of an agent to modulate promoter-driven transcription.

Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell by any method known in the art, e.g., mammalian, bacterial, yeast or insect cells. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., sambrook et al (2001, molecular cloning. A preferred method for introducing the polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. Pat. nos. 5,350,674 and 5,585,362.

Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).

In the case of non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for use to introduce nucleic acids into host cells (ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained as a suspension in the lipid, contained in or complexed with a micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in bilayer structures, either as micelles or with a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fatty droplets that occur naturally in the cytoplasm as well as such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Therapeutic applications

The invention includes cells (e.g., T cells) transduced with a Lentiviral Vector (LV) encoding a CAR of the invention. The transduced T cells can elicit a CAR-mediated T-cell response.

Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering to a mammal a T cell expressing a CAR of the invention.

In one embodiment, the invention includes a class of cell therapies in which T cells are genetically modified to express a CAR of the invention, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing tumor cells of the recipient. Unlike antibody therapy, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the CAR-modified T cell induces an immune response specific to the antigen binding domain in the CAR. For example, anti-CD 20 CAR-T cells elicit a specific immune response against CD 20-expressing cells.

Although the data disclosed herein specifically disclose lentiviral vectors comprising an anti-CD 20scFv, a human Fc hinge region, an ICOS transmembrane and intracellular region, and 4-1BB and CD3 zeta signaling domains, the invention should be construed to include any number of variations on each of the construct components.

Treatable cancers include tumors that are not vascularized or have not substantially vascularized, as well as vascularized tumors. The cancer may comprise a non-solid tumor (such as a hematological tumor, e.g., leukemia and lymphoma) or may comprise a solid tumor. The types of cancer treated with the CAR of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemias or lymphoid malignancies, benign and malignant tumors, e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematological) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythrocytic leukemias), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, hodgkin's disease, non-hodgkin's lymphoma (indolent and higher forms), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

A solid tumor is an abnormal mass of tissue that generally does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

The CAR-modified T cells of the invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) Expanding the cell, ii) introducing a nucleic acid encoding the CAR into the cell, and/or iii) cryopreserving the cell.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cell may be allogeneic, syngeneic (syngeneic) or xenogeneic with respect to the recipient.

In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods of in vivo immunization to elicit an immune response against an antigen in a patient.

Generally, cells activated and expanded as described herein are useful for the treatment and prevention of diseases arising in immunocompromised individuals. In particular, the CAR-modified T cells of the invention are used to treat CCL. In certain embodiments, the cells of the invention are used to treat a patient at risk of developing CCL. Accordingly, the invention provides a method of treating or preventing CCL, comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, a pharmaceutical composition of the invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibiting effective amount", or "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, extent of infection or metastasis and individual variability of the condition of the patient (subject). It is possible to generally point out: pharmaceutical compositions comprising T cells described herein can be in the range of 10 ⁴ To 10 ⁹ Dosage of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Doses of individual cells per kg body weight (including all integer values within those ranges) are administered. T cell compositionsMultiple administrations at these doses are also possible. Cells can be administered by using infusion techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng.J.of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject composition may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) any number of relevant treatment modalities, including but not limited to treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatment for PML patients. In further embodiments, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In a further embodiment, the cell composition of the invention is administered to the patient in conjunction with (e.g., prior to, concurrently with, or subsequent to) bone marrow transplantation with a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject receives an injection of the expanded immune cells of the invention. In an additional embodiment, the expanded cells are administered pre-or post-surgery.

The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage rates for human administration can be effected in accordance with accepted practice in the art. Typically, 1X 10 may be administered per treatment or per course of treatment ⁶ 1 to 10 ¹⁰ The modified T cells of the invention (e.g., CAR-T19 cells) are administered to a patient, for example, by intravenous infusion.

The main advantages of the invention include:

1) The novel CD20-CAR vectors of the humanized CD20 antibodies of the present invention can target B cell malignancies of the CD20 antigen;

2) The novel CD20-CAR vector of the humanized CD20 antibody can avoid the immunogenicity generated in a human body by CD20-CAR-T cells constructed by murine CD20scFv, and improve the clinical curative effect;

3) The CD20-CAR-T cells constructed by using the CD20-CAR vector constructed by the invention show very good killing effect in vitro.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1.

Humanized CD20-CAR-T transfection efficiency

The human Fc antibody of the invention detects the CAR positive rate of CD20-CAR-T cells, and the result is shown in figure 2, and the positive rate of humanized CD20-CAR-T is 28.49%.

Example 2.

Cytotoxicity of humanized CD20-CAR-T cells on CD20 overexpressing K562 cells

The inventors constructed CD 20-overexpressing K562 (K562-CD 20) cells as target cells and evaluated the killing function of humanized CD20-CAR-T cells in vitro with a potency to target ratio of 0.5, 1, 5, 1, 10. K562 cells were negative target cells and T cells were control effector cells.

The results are shown in fig. 3, compared with the control T cells, the CD20-CAR-T cells have significant specific killing effect on the K562-CD20 cells (CD 20 positive) but no specific killing effect on the K562 cells (CD 20 negative), which indicates that the constructed humanized CD20-CAR-T cells can generate specific cytotoxicity on the overexpressed CD20 positive K562-CD20 cells in vitro but have no specific cytotoxicity on the control K562 cells.

Example 3.

Cytotoxicity of humanized CD20-CAR-T cells against B-lymphoblastic leukemia cells (Nalm-6) and lymphoma cells (Raji) naturally expressing CD20

We performed in vitro killing experiments on B-lymphocytic leukemia (Nalm-6) and lymphoma (Raji) cells with the constructed humanized CD20-CAR-T cells at a potency to target ratio of 0.5, 1, 5. T cells were control effector cells.

The results are shown in fig. 4, and compared with the control T cells, the CD20-CAR-T cells have obvious specific killing effect on both Raji and Nalm-6, which indicates that the constructed humanized CD20-CAR-T cells have obvious cytotoxicity on the Raji and Nalm-6 cells which are positive for CD20 in vitro.

Example 4.

Cytokine secretion after incubation of CD20-CAR-T cells with Raji and Nalm-6 cells

The inventors further analyzed the release of cytokines after incubation of CD20-CAR-T cells with Raji and Nalm-6 cells, with the effective target ratio of CD20-CAR-T cells to target cells being 1 and 5 for the 1,t cell group as control group, and the cytokines tested were IL-2, IFN- γ, TNF- α, granzyme B.

Results as shown in fig. 5, T cells produced substantially no or very low cytokine production after co-incubation with target cells, whereas CD20-CAR-T cells produced significant cytokine release after incubation with Raji and Nalm-6 cells, demonstrating that CD20-CAR-T cells are specifically activated by CD 20-positive tumor cells.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Bosheng Ji medicine technology (Suzhou) Co., ltd

<120> construction and application of novel CD20-CAR vector of anti-CD 20 antibody

<130> P2018-2464

<160> 16

<170> PatentIn version 3.5

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

1 5 10 15

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

20 25 30

Asn Met His Trp Val Arg Gln Thr Pro Gly Gln Arg Leu Glu Trp Met

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser

115 120

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

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

20 25 30

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

35 40 45

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

50 55 60

Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105

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

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

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Ala Phe Leu Leu Ile Pro

20

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Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

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

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

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

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

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Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly

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Cys Ile Leu Ile Cys Trp Leu

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Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr

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Val Thr Leu

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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

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Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

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

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

1 5 10 15

Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Gln Ser Gly Ala Glu

20 25 30

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

35 40 45

Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val Arg Gln Thr Pro Gly

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Ala Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp

115 120 125

Tyr Phe Asn Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Gly Gly

130 135 140

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

145 150 155 160

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

165 170 175

Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile His Trp Tyr

180 185 190

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

195 200 205

Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

210 215 220

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

225 230 235 240

Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr Phe Gly Gly

245 250 255

Gly Thr Lys Val Glu Ile Lys Arg Thr Val Glu Ser Lys Tyr Gly Pro

260 265 270

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

275 280 285

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

290 295 300

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

305 310 315 320

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

325 330 335

Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser

340 345 350

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

355 360 365

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

370 375 380

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

385 390 395 400

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

405 410 415

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

420 425 430

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

435 440 445

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

450 455 460

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

465 470 475 480

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

485 490 495

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

500 505 510

Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His

515 520 525

Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys

530 535 540

Lys Ser Arg Leu Thr Asp Val Thr Leu Lys Arg Gly Arg Lys Lys Leu

545 550 555 560

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

565 570 575

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

580 585 590

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

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

690 695 700

Pro Pro Arg

705

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<211> 687

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gagagcaagt acggacctcc ttgtcctcct tgtccagctc cagagtttga gggcggacct 60

agcgtgttcc tgttccctcc caagcccaag gacaccctga tgatcagccg gaccccagaa 120

gtgacttgcg tggtggtgga cgtgtctcag gaggaccccg aggtgcagtt caattggtac 180

gtggacggcg tggaggtgca caacgctaag accaagccca gggaggagca gttccagagc 240

acctacaggg tggtgtccgt gctgacagtg ctgcaccagg attggctgaa cggcaaggag 300

tacaagtgca aggtgtccaa caagggcctg cctagcagca tcgagaagac catcagcaag 360

gccaagggcc agcctagaga gcctcaggtg tacaccctgc ctcctagcca ggaggagatg 420

accaagaacc aggtgtccct gacttgcctc gtgaagggct tctaccccag cgacatcgca 480

gtcgagtggg aaagcaacgg ccagcccgag aacaactaca agaccacacc tccagtgctg 540

gatagcgacg gaagcttctt cctgtacagc cggctgaccg tggataaaag ccgctggcag 600

gagggcaacg tgttcagttg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 660

aagagcctga gcctgagcct cggcaag 687

<210> 12

<211> 69

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tggctgccta ttggttgcgc agctttcgtc gtcgtctgca tcctgggttg catcctgatt 60

tgctggctg 69

<210> 13

<211> 105

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

accaagaaga agtacagctc tagcgtgcac gaccctaacg gcgagtacat gttcatgcgg 60

gccgtcaaca ccgccaaaaa gagccggctg accgacgtga cactg 105

<210> 14

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

aagaggggcc ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg gccagtgcag 60

acaacacagg aggaagacgg ctgctcttgc aggttcccag aggaggagga gggcggttgc 120

gagctg 126

<210> 15

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

agagtgaagt tcagcagaag cgccgacgct ccagcttatc agcagggcca gaaccagctg 60

tacaacgagc tgaacctggg caggagagag gaatacgacg tgctggacaa gaggaggggc 120

agagatcccg agatgggagg caagcccaga agaaagaacc cccaggaggg cctgtacaac 180

gagctgcaga aggacaagat ggccgaggcc tacagcgaga ttggcatgaa gggcgagaga 240

aggaggggca aaggccacga cggactgtat cagggcctgt ctaccgccac caaggatacc 300

tacgacgctc tgcacatgca ggctctgcct cctaga 336

<210> 16

<211> 2124

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atgctgctgc tggtgacctc tctgctgctc tgcgaactgc ctcacccagc ctttctgctg 60

atccctcagg tgcagctggt gcagtcagga gccgaagtga agaagccagg agcctcagtg 120

aaggtgtctt gcaaggccag cggctacacc ttcaccagct acaacatgca ttgggtccgg 180

cagaccccag gacagagact cgagtggatg ggagccatct accccggcaa cggcgatacc 240

agctacaacc agaagttcaa gggccgcgtg accatcacag ccgatacaag cgccagcacc 300

gcctacatgg agctgtctag cctgaggagc gaggacacag ccgtgtacta ttgcgccagg 360

agcacctact acggcggcga ttggtacttc aacgtgtggg gccagggcac aacagtgaca 420

gtgagcggag gaggaggaag cggaggagga ggatctggag gaggcggcag cgatattcag 480

ctgacccaga gcccttcttt tctgagcgcc agcgtgggcg acagagtgac catcacttgc 540

agggccagca gcagcgtgtc ctacatccat tggtaccagc agaagccagg caaggctcct 600

aagctgctga tctacgccac cagcaacctg gccagcggcg tgcctagcag attcagcggc 660

agcggaagcg gcacaagctt taccctgacc atcagctctc tgcagccaga ggacttcgcc 720

acctactact gccagcagtg gaccagcaac cctcctacat ttggcggcgg aaccaaggtg 780

gagatcaagc ggaccgtgga gagcaagtac ggacctcctt gtcctccttg tccagctcca 840

gagtttgagg gcggacctag cgtgttcctg ttccctccca agcccaagga caccctgatg 900

atcagccgga ccccagaagt gacttgcgtg gtggtggacg tgtctcagga ggaccccgag 960

gtgcagttca attggtacgt ggacggcgtg gaggtgcaca acgctaagac caagcccagg 1020

gaggagcagt tccagagcac ctacagggtg gtgtccgtgc tgacagtgct gcaccaggat 1080

tggctgaacg gcaaggagta caagtgcaag gtgtccaaca agggcctgcc tagcagcatc 1140

gagaagacca tcagcaaggc caagggccag cctagagagc ctcaggtgta caccctgcct 1200

cctagccagg aggagatgac caagaaccag gtgtccctga cttgcctcgt gaagggcttc 1260

taccccagcg acatcgcagt cgagtgggaa agcaacggcc agcccgagaa caactacaag 1320

accacacctc cagtgctgga tagcgacgga agcttcttcc tgtacagccg gctgaccgtg 1380

gataaaagcc gctggcagga gggcaacgtg ttcagttgca gcgtgatgca cgaggccctg 1440

cacaaccact acacccagaa gagcctgagc ctgagcctcg gcaagtggct gcctattggt 1500

tgcgccgcct ttgtcgtcgt ctgtatcctc ggctgcatcc tgatttgttg gctgaccaag 1560

aagaagtaca gcagcagcgt gcacgacccc aacggcgagt acatgttcat gcgggccgtc 1620

aacaccgcca agaagagcag gctgaccgac gtgacactga agaggggccg gaagaagctg 1680

ctgtacatct tcaagcagcc cttcatgcgg ccagtgcaga caacacagga ggaagacggc 1740

tgctcttgca ggttcccaga ggaggaggag ggcggttgcg agctgagagt gaagttcagc 1800

agaagcgccg acgctccagc ttatcagcag ggccagaacc agctgtacaa cgagctgaac 1860

ctgggcagga gagaggaata cgacgtgctg gacaagagga ggggcagaga tcccgagatg 1920

ggaggcaagc ccagaagaaa gaacccccag gagggcctgt acaacgagct gcagaaggac 1980

aagatggccg aggcctacag cgagattggc atgaagggcg agagaaggag gggcaaaggc 2040

cacgacggac tgtatcaggg cctgtctacc gccaccaagg atacctacga cgctctgcac 2100

atgcaggctc tgcctcctag atga 2124

Claims

1. An anti-CD 20 chimeric antigen receptor, wherein the antigen binding domain of the chimeric antigen receptor, i.e., the scFv comprises an antibody heavy chain variable region as set forth in SEQ ID No.1 and an antibody light chain variable region as set forth in SEQ ID No. 2;

the structure of the chimeric antigen receptor is shown as the following formula:

L－scFv－H－TM－C－CD3ζ

wherein the content of the first and second substances,

l is an optional leader sequence, i.e. a signal peptide sequence;

scFv is an antigen binding domain;

h is a hinge region;

TM is a transmembrane domain;

c is a costimulatory signal molecule;

CD3 ζ is a cytoplasmic signaling sequence derived from CD3 ζ;

"-" is a linker peptide or peptide bond.

2. The chimeric antigen receptor of claim 1, wherein the amino acid sequence of the linker peptide is set forth in SEQ ID No. 3.

3. The chimeric antigen receptor of claim 1, wherein the sequence of L is as set forth in SEQ ID No. 4.

4. The chimeric antigen receptor of claim 1, wherein the sequence of H is as set forth in SEQ ID No. 5.

5. The chimeric antigen receptor according to claim 1, wherein the sequence of the TM comprises a transmembrane region derived from ICOS.

6. The chimeric antigen receptor of claim 5, wherein the sequence of TM is as shown in SEQ ID No. 6.

7. The chimeric antigen receptor according to claim 1, wherein the costimulatory signal molecule comprises an ICOS-derived costimulatory signal molecule, and/or a 4-1 BB-derived costimulatory signal molecule.

8. The chimeric antigen receptor of claim 7, wherein the amino acid sequence of the ICOS-derived costimulatory signal molecule is as set forth in SEQ ID No. 7.

9. The chimeric antigen receptor of claim 7, wherein the amino acid sequence of the 4-1 BB-derived costimulatory signal molecule is set forth in SEQ ID No. 8.

10. The chimeric antigen receptor of claim 1, wherein the sequence of CD3 ζ is as set forth in SEQ ID No. 9.

11. The chimeric antigen receptor of claim 1, wherein the sequence of the chimeric antigen receptor is set forth in SEQ ID No. 10.

12. A nucleic acid molecule encoding the chimeric antigen receptor of claim 1.

13. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule comprises a nucleic acid sequence encoding a hinge region selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 5;

(b) A polynucleotide having a sequence as shown in SEQ ID No. 11.

14. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane domain selected from the group consisting of seq id nos:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 6;

(b) The sequence is shown in SEQ ID NO. 12.

15. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule comprises a costimulatory signal molecule coding sequence, said costimulatory signal molecule coding sequence comprising an ICOS-derived costimulatory signal molecule coding sequence, and/or a 4-1 BB-derived costimulatory signal molecule coding sequence, wherein

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 7;

(b) A polynucleotide having a sequence as shown in SEQ ID No. 13;

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 8;

(b) A polynucleotide having a sequence as shown in SEQ ID No. 14.

16. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule comprises a nucleic acid sequence encoding an intracellular signaling domain of CD3 ζ selected from the group consisting of seq id nos:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 9;

(b) A polynucleotide having a sequence as shown in SEQ ID No. 15.

17. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of seq id no:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 10;

(b) A polynucleotide having a sequence as shown in SEQ ID No. 16.

18. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule is isolated.

19. The nucleic acid molecule of claim 12, wherein the sequence of the nucleic acid molecule is set forth in SEQ ID No. 16.

20. A vector comprising the nucleic acid molecule of claim 12.

21. The vector of claim 20, wherein said vector is a lentiviral vector.

22. A host cell comprising the vector of claim 20 or having integrated into its chromosome an exogenous nucleic acid molecule of claim 12.

23. The host cell of claim 22, wherein the cell is an isolated cell, and/or the cell is a genetically engineered cell.

24. The host cell of claim 22, wherein the cell is a mammalian cell.

25. The host cell of claim 22, wherein the cell is a T cell.

26. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 12, the vector of claim 20, or the host cell of claim 22.

27. Use of the chimeric antigen receptor of claim 1, the nucleic acid molecule of claim 12, the vector of claim 20, or the host cell of claim 22 for the preparation of a medicament or formulation for the treatment of a CD 20-positive tumor;

the CD20 positive tumor is a B lymphocyte leukemia or lymphoma.

28. A method of making a CAR-T cell expressing the chimeric antigen receptor of claim 1,

the method comprises the following steps: transferring the nucleic acid molecule of claim 12 or the vector of claim 20 into a T cell, thereby obtaining the CAR-T cell.