CN111484563A

CN111484563A - anti-CD 38 chimeric antigen receptor and application thereof

Info

Publication number: CN111484563A
Application number: CN202010362796.0A
Authority: CN
Inventors: 桑威
Original assignee: Affiliated Hospital of Xuzhou Medical University
Current assignee: Affiliated Hospital of Xuzhou Medical University
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2020-08-04
Anticipated expiration: 2040-04-30
Also published as: CN111484563B

Abstract

The invention relates to an anti-CD 38 chimeric antigen receptor (CD38-CAR) and application thereof, wherein the anti-CD 38 chimeric antigen receptor comprises an antigen binding domain (scFv) and a signaling domain, wherein the antigen binding domain (CD38scFv) is an anti-human CD38 antibody, and the signaling domain of the anti-CD 38 antibody is formed by sequentially connecting a human CD8a molecular hinge region with a transmembrane region CD8TM (CD8a-CD8TM), a human CD28 molecular intracellular region CD28(CD28), a human 4-1BB molecular intracellular region (4-1BB) and a human CD3 zeta molecular intracellular region (CD3 zeta) in series. The CD38scFv comprises a heavy chain variable region and a light chain variable region of specific sequences. The chimeric antigen receptor has higher specificity and safety, and can effectively act on CD38 positive tumors.

Description

anti-CD 38 chimeric antigen receptor and application thereof

Technical Field

The invention relates to the technical field of tumor immunotherapy and provides a method and a composition for treating human tumors. The invention obtains the fusion gene of a single-chain antibody (scFv) of a coded high-affinity hCD38, a Hinge region (CD8Hinge) and a transmembrane region (CD8TM) of a human CD8a molecule, an intracellular region (CD28) of a human CD28 molecule, an intracellular region (4-1BB) of a human 4-1BB molecule and an intracellular region (CD3 zeta) of a human CD3 zeta molecule by a genetic engineering technology, inserts the fusion gene into an expression vector, and transfers the fusion gene into a human T lymphocyte to enable the T cell to express the fusion gene. The invention also relates to polypeptides expressed by the aforementioned fusion genes, vectors, and immune cells expressing said CAR on their surface for use in immunotherapy. The modified T cells involved in the invention can target and specifically kill tumor cells positive for CD 38.

Background

CD38 is a single-chain transmembrane glycoprotein of 45kDa in size, and the overall structure can be divided into an N-terminal intracellular region, a single-pass transmembrane structure and a C-terminal extracellular region. Its extracellular region can catalyze the synthesis and degradation of cyclic adenosine diphosphate ribose (cADPR). cADPR is a nucleotide metabolite that participates in calcium mobilization of intracellular calcium stores by acting on ryanodine receptors (RyRs). In adults, CD38 is expressed in most NK cells, T cells, B cells, macrophages. There is also a small amount of CD38 expression in the pancreas, brain, kidney, and muscle.

CD38 is highly expressed on the surface of various tumors, including multiple myeloma, diffuse large B-cell lymphoma (D L BC L), chronic lymphocytic leukemia (C LL), acute lymphocytic leukemia (a LL), plasma cell leukemia (PC L), acute myelogenous leukemia (AM L), follicular lymphoma (F L), mantle cell lymphoma (MC L), lung cancer, and the like.

The anti-CD 38-CAR technology is different from traditional biological drugs such as antibody drugs, ADCs and recombinant proteins, and has the characteristics of living drugs. For example, antibody drugs need to mediate immune cells to kill tumor cells, and after CAR-T cells recognize, T cells can be directly activated, thereby directly killing tumor cells. CAR-T is a living drug, can be greatly amplified in vivo in the course of treatment of patients, and has rapid onset of action, high remission rate, and longer remission time compared with conventional antibody drug therapy.

Disclosure of Invention

The invention aims to provide a chimeric antigen receptor which has better specificity, higher safety and more obvious curative effect and application thereof, aiming at the defects of the prior art.

The invention also aims to provide a nucleic acid for encoding the chimeric antigen receptor and application thereof.

The invention also aims to provide a cell containing the chimeric antigen receptor and application thereof.

The fourth object of the present invention is to provide a humanized antibody against a CD 38-positive tumor.

In order to solve the technical problems, the technical scheme adopted by the invention is detailed as follows:

a Chimeric Antigen Receptor (CAR) comprising: an antigen binding domain (CD38scFv) and a signaling domain, wherein the antigen binding domain (CD38scFv), which is an anti-human CD38 antibody, comprises a hinge region of a human CD8a molecule in tandem with a transmembrane region, CD8TM (CD8a-CD8TM), an intracellular region CD28(CD28) of a human CD28 molecule, an intracellular region (4-1BB) of a human 4-1BB molecule, and an intracellular region (CD3 zeta) of a human CD3 zeta molecule. The chimeric antigen receptor CD38-CAR of the invention has a structure of CD38scFv-CD8a-CD8TM-CD28-4-1BB-CD3 zeta.

The anti-human CD38scfv amino acid sequence in the chimeric antigen receptor is shown in SEQ ID NO.1, or the polypeptide with 85-99% of identity with the amino acid sequence shown in SEQ ID NO. 1; the nucleotide sequence of the polypeptide is shown in SEQ ID NO.2, or the nucleotide sequence which has 85 to 99 percent of identity with the nucleotide sequence shown in SEQ ID NO. 2.

An anti-human CD38 antibody, wherein the heavy chain sequence of the antibody is shown as SEQ ID NO.17, or a polypeptide with 85-99% identity with the amino acid sequence shown as SEQ ID NO. 17; the coding sequence is shown in SEQ ID NO.18, or a nucleotide sequence with 85% -99% identity with SEQ ID NO. 18. The light chain of the antibody is shown as SEQ ID NO.19 or polypeptide with 85-99% of identity with the amino acid sequence shown as SEQ ID NO. 19; the coding sequence is shown in SEQ ID NO.20, or a nucleotide sequence with 85-99% identity with SEQ ID NO. 20.

An anti-human CD38 antibody, wherein the sequence of a heavy chain variable region HCDR1 of the antibody is shown as SEQ ID NO.11, the sequence of HCDR2 is shown as SEQ ID NO.12, the sequence of HCDR3 is shown as SEQ ID NO.13, the sequence of a light chain variable region L CDR1 of the antibody is shown as SEQ ID NO.14, the sequence of a L CDR2 is shown as SEQ ID NO.15, and the sequence of a L CDR3 is shown as SEQ ID NO. 16.

The amino acid sequence of the hinge region and the transmembrane region CD8TM of the human CD8a molecule in the chimeric antigen receptor is shown as SEQ ID NO.3, or the polypeptide which has 85 to 99 percent of identity with the amino acid sequence shown as SEQ ID NO. 3; the coding sequence is SEQ ID NO.4, or a sequence which has 85 to 99 percent of identity with the nucleotide sequence shown in SEQ ID NO. 4.

The amino acid sequence of the intracellular region CD28 of the human CD28 molecule is shown as SEQ ID NO.5, or the polypeptide with 85-99% of identity with the amino acid sequence shown as SEQ ID NO. 5; the coding sequence is SEQ ID NO.6, or a sequence which has 85 to 99 percent of identity with the nucleotide sequence shown in SEQ ID NO. 6.

The amino acid sequence of the intracellular region of the human 4-1BB molecule is shown as SEQ ID NO.7, or the polypeptide with 85-99% identity to the amino acid sequence shown as SEQ ID NO. 7; the coding sequence is SEQ ID NO.8, or a sequence which has 85 to 99 percent of identity with the nucleotide sequence shown in SEQ ID NO. 8.

The amino acid sequence of the intracellular region of the human CD3 zeta molecule is shown in SEQ ID NO.9, or the polypeptide with 85-99% identity to the amino acid sequence shown in SEQ ID NO. 9; the coding sequence is SEQ ID NO.10, or a sequence which has 85 to 99 percent of identity with the nucleotide sequence shown in SEQ ID NO. 10.

A recombinant expression vector is characterized in that the recombinant expression vector comprises coding genes of anti-human CD38 antibodies HCDR1, HCDR2 and HCDR3 and coding genes of anti-human CD38 antibodies L CDR1, L CDR2 and L CDR 3.

A recombinant expression vector comprising genes encoding the heavy and light chains of an anti-human CD38 antibody.

A recombinant expression vector, characterized in that said recombinant expression vector comprises an antigen binding domain (CD38 scFv).

A recombinant expression vector, characterized in that said recombinant expression vector comprises the antigen receptor CD38-CAR of the present invention.

A cell expressing anti-human CD38 antibodies HCDR1, HCDR2 and HCDR3 and anti-human CD38 antibodies L CDR1, L CDR2 and L CDR3 is preferably an immune cell, and more preferably an immune cell differentiated by culturing T lymphocyte, NK cell, NKT cell, macrophage, mesenchymal stem cell, hematopoietic stem cell, pluripotent stem cell or embryonic stem cell.

A cell expressing the heavy and light chains of an anti-human CD38 antibody, preferably an immune cell; further preferred are immune cells differentiated by culture of T lymphocytes, NK cells, NKT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells or embryonic stem cells.

A cell expressing a CD38scFv, preferably an immune cell; further preferred are immune cells differentiated by culture of T lymphocytes, NK cells, NKT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells or embryonic stem cells.

A cell expressing the chimeric antigen receptor CD38-CAR, preferably an immune cell; further preferred are immune cells differentiated by culture of T lymphocytes, NK cells, NKT cells, macrophages, mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells or embryonic stem cells.

A method for preparing a novel chimeric antigen receptor CD38-CAR modified T cell, the method comprising isolating and activating a T cell to be modified, and then transducing the T cell with the aforementioned expression vector.

Contains the novel chimeric antigen receptor, an expression vector and the application of the cell in preparing a medicament for treating tumors. The tumor is positive CD38 tumor, preferably myeloma and lymphoma.

Drawings

FIG. 1 shows the positive rate of T lymphocytes expressing the CD38-CAR gene obtained by transferring the plasmid pMD19-T-CD38-CAR into T cells by electrotransfection.

Fig. 2 shows the killing effect exhibited after 24 and 48 hours of co-incubation of CD38-CAR gene expressing T lymphocytes with RPMI8226 cells, where T-156 is an abbreviation for CD38-CAR gene expressing T lymphocytes of the present invention, effector cells: the target cells (E: T) are 4: 1 or 1: 1.

FIG. 3 shows the secretion of IFN- γ in the supernatant of T lymphocytes expressing the CD38-CAR gene, wherein 156-CAR T is an abbreviation for T lymphocytes expressing the CD38-CAR gene in the present invention, incubated with human multiple myeloma cells, leukemia cells, for 72 hours.

FIG. 4 shows GM CSF secreted in the supernatant of T lymphocytes expressing the CD38-CAR gene incubated with human multiple myeloma and leukemia cells for 72 hours. Wherein 156-CAR T is an abbreviation for T lymphocytes expressing the CD38-CAR gene of the present invention.

FIG. 5 shows the secretion of I L-2 in the supernatant of T lymphocytes expressing the CD38-CAR gene when incubated with human multiple myeloma and leukemia cells for 72 hours, wherein 156-CAR T is an abbreviation for T lymphocytes expressing the CD38-CAR gene in the present invention.

Fig. 6 shows the killing effect exhibited by T lymphocytes expressing the CD38-CAR gene after 24 hours and 48 hours of co-incubation with Raji cells, respectively, where T-156 is an abbreviation for T lymphocytes expressing the CD38-CAR gene of the present invention, effector cells: the target cells (E: T) are 4: 1 or 1: 1.

FIG. 7 shows the killing effect of T lymphocytes expressing the CD38-CAR gene after 24 hours and 48 hours of co-incubation with Daudi cells, respectively, wherein T-156 is an abbreviation for T lymphocytes expressing the CD38-CAR gene of the present invention, effector cells/target cells (E: T) at 4: 1 or 1: 1.

FIG. 8 shows the killing effect of T lymphocytes expressing the CD38-CAR gene after incubation with THP-1 cells for 24 hours and 48 hours, respectively, wherein T-156 is abbreviated as T lymphocytes of the CD38-CAR gene of the present invention, effector cells/target cells (E: T) were 4: 1 or 1: 1.

Detailed Description

The invention provides a chimeric antigen receptor targeting CD38, an immune effector cell and application thereof in clinical treatment of tumors, and the invention is further illustrated by combining specific examples below.

As used herein, the term "identity" of an amino acid sequence is used interchangeably with "similarity" and refers to the degree of similarity between amino acid sequences as determined by sequence alignment software such as B L AST methods and software for amino acid sequence alignment are well known to those skilled in the art.an engineered amino acid sequence can be obtained by substitution, deletion and/or addition of one or several (e.g., 1-15, e.g., 2, 3, 5, 8, 10 or 12) amino acid residues to a known amino acid sequence.for example, by engineering the CD38 antigen-binding domain of the invention as represented by SEQ ID NO.1 by conventional protein engineering means (e.g., amino acid conservative substitutions, etc.), variant sequences can be obtained that have at least 85% (e.g., 85% -99% or 90% -99% or 95% -99%) sequence identity to SEQ ID NO.1 and have substantially the same antigen-binding domain.

The term "antigen binding domain" as used herein is intended to include the variable region of a functional antibody portion, preferably an antigen binding and/or intact antibody. Antibody fragments include Fab, Fab ', F (ab') 2, Fv fragments, single chain antibody scFv single domain antibody VHH and multispecific antibodies.

T-156/156-CAR T in the figures of the present invention is an abbreviation for T lymphocytes expressing the CD38-CAR gene of the present invention, both referring to the same T cell.

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental procedures, for which specific conditions are not indicated in the examples, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, J. SammBruke et al) or according to the conditions recommended by the manufacturers. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1 CAR sequence Synthesis and vector construction

The chimeric antigen receptor is synthesized by Jiangsu Kingsrei, and the obtained DNA sequence of the anti CD38-CAR structure is inserted into a pMD19-T plasmid through enzyme digestion connection to construct a non-viral electrotransformation expression plasmid pMD19-T-CD 38-CAR.

EXAMPLE 2 electrotransformation of expression plasmid extraction

Electrotransfer plasmids carrying the pMD19-T-CD38-CAR gene were transferred into E.coli and the pMD19-T-CD38-CAR carrying the pMD19-T-CD38-CAR gene was amplified in E.coli to a large extent. Then, plasmid DNA of Escherichia coli was extracted using an endotoxin-free plasmid macrodrawing kit.

The constructed Plasmid with the pMD19-T-CD38-CAR gene is hot-shocked in a water bath at 42 ℃ for 90 seconds, transformed into escherichia coli Top10 competence, cultured overnight at 37 ℃ and 200rpm/min for 14-16 hours by using L B culture medium, and the Plasmid with the pMD19-T-CD38-CAR gene is greatly amplified in escherichia coli, the overnight-cultured bacterium liquid is centrifuged at 4000rpm for 15 minutes the next day, the supernatant is removed, and then an endotoxin-Free Plasmid macro-extraction Kit (Endo-Free Plasmid Maxi Kit, Omega, cat number: D6926) is used for extracting the pMD19-T-CD38-CAR Plasmid in escherichia coli.

Example 3T cell transfection and purification

Lymphocytes were isolated from human serum and expanded by adding T-lymphocyte culture medium (CTSTM AIM VTM SFM (RUO), Thermo, cat # A3021002), OKT-3, hI L-2 and endotoxin-free plasmid DNA, and plasmid pMD19-T-CD38-CAR was transferred to T cells by electrotransfection to obtain T lymphocytes carrying the CD38-CAR gene (CD 38-CAR-T).

Treating peripheral blood with anticoagulant, standing at room temperature for 15min, centrifuging at room temperature of 350 × g for 5min, collecting plasma, and diluting with equal volume of 1 × PBS. An equal volume of Ficoll solution was added to the upper layer slowly. Centrifuging at room temperature for 25min, and sequentially separating into platelet layer, leukocyte layer, Ficoll layer and erythrocyte layer from top to bottom. The buffy coat was carefully aspirated, transferred to a new centrifuge tube, washed 2 times with PBS, the pellet cells were counted and resuspended to a density of 2x 107/ml, and the cells were cryopreserved using 10% DMSO serum. The isolated PBMC were used directly for culture, and cultured overnight at 37 ℃ with 5% humidity and 5% carbon dioxide in a T lymphocyte culture medium (CTSTM AIM VTM SFM (RUO), Thermo, cat # A3021002) containing 10% FBS.

Nuclear transfection was performed using the plasmids extracted in the second step to generate anti-CD 38T cells (CD38-CAR-T) encoding a vector comprising CD38scFv, CD28 transmembrane region, human 4-1BB, human CD3 zeta, puromycin, and then 0.5-2. mu.g/ml puromycin for one nuclear transfection, 2X107 PBMC cells were resuspended in 100. mu.l buffer of human T cell Nuclear ofector kit (L onza, # VPA-1002) with 10. mu.g anti-CD 38-CARPiggyBac transposon vector and 5. mu.g SuperpiggyBac transposase plasmid^TMII/2b device (L onza) T cellsOne day after nuclear transfection, 50ng/ml anti-CD 3 antibody (Miltenyi, Cat. # 130-.

Example 4T cell CAR expression efficiency

After 20 days of T cell transfection, T cell surface CAR expression was examined by flow cytometry. The results are shown in FIG. 1. Figure 1 shows that CAR expression positive rate reached 100%, demonstrating the success of CAR expression plasmid construction and electroporation.

After 20 days of T cell transfection, T cell surface CAR expression was examined by flow cytometry. 3X 105T cells were removed, centrifuged at 300g for 5min at room temperature, incubated for 1h at room temperature using anti-myc-PE fluorescent antibody (Muc-Tag (9B11) Mouse mAb PEconugate, cell signaling, cat # 3739S) and then subjected to flow detection. The results are shown in FIG. 1. FIG. 1 shows that the myc-PE positive rate reached 100%, demonstrating the success of CAR expression plasmid construction and plasmid electrotransformation.

Example 5 Long-term killing of CD 38-positive RPMI-8226 tumor cells by CAR-T cells

In order to determine that CD38-CAR-T cells have better sustained killing capacity, a target cell carrying L uciference fluorescence signals is used for a killing test, T cells (effector cells) are incubated with CD38 positive target cells (RPMI-8226-Fluc) or CD38 negative target cells (K562-Fluc) respectively for 24 hours and 48 hours (as shown in FIG. 2) according to the effective target ratio (effector cells: target cells, E: T) of 4: 1 and 1: 1, after the incubation is finished, a substrate (Perkinelmer, product number: 122799) of L uciference fluorescence signals is added, and then apoptosis of the target cells is analyzed by a microplate reader.

The above-constructed T cells transduced with no plasmid, and CD38-CAR-T cells were transfected with luciferase reporter gene, respectively, at a ratio of effective target (effector cells: target cells, E: T) 4: 1 and 1: 1, co-incubation of T cells with CD38 positive target cells (RPMI-8226-Fluc) or CD38 negative target cells (K562-Fluc), respectively. Co-incubation cultures were performed for 24 hours, 48 hours using 96-well white microplates and using complete RPMI 1640 medium with 10% FBS in incubators with 5% carbon dioxide at 37 ℃, humidity, and 3 replicates per sample, respectively. Meanwhile, 1 × 104CD38 target cells were cultured in complete RPMI 1640 medium in 96-well white microtitre plates for 24 hours and 48 hours, respectively, as controls. After the experimental time had elapsed, 0.75mg/ml D-fluorescein K + salt (Perkinelmer, cat # 122799) was added and the target apoptosis was analyzed using an EnSpire Multi-mode plate reader (Perkinelmer) to read the signal immediately. The result shows (figure 2), the CD38-CAR-T cell constructed by the method has strong killing capacity on tumor cells over-expressing CD38, and has little influence on CD38 negative cells, which indicates that the CD38-CAR-T cell has strong specific killing capacity. The influence on CD38 negative cells is small, and the CAR-T cells have strong specific killing capacity.

Example 7 CART cells cause secretion of RPMI-8226 tumor cell-associated cytokines

CD38-CAR-T cells (effector cells) were co-incubated with CD38 positive target cells (RPMI-8226-Fluc) or CD38 negative target cells (K562-Fluc), respectively, for 72 hours (as shown in fig. 3). after the co-incubation was completed, GMCSF, IFN-r and I L-2 secreted in the supernatant were detected.4 x105 plasmid-untransfected T cells or CD38-CAR-T cells were co-cultured with 1x105K562(CD38 negative target cells), RPMI8226(CD38 positive cells), respectively, in a 96-well plate in 200 μ l of fresh 1640 medium containing 10% FBS, and after 72 hours, the supernatant was collected and the amounts of cytokine expression including interferon (IFN- γ), tumor necrosis factor (TNF- α), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-2 (I L-2) were detected using the Alpha L kit from PerkinElmer (as shown in fig. 5).

Example 8 Long-acting killing of CD38-CAR-T cells against CD 38-positive THP-1 tumor cells

The above-constructed T cells transduced with no plasmid, and CD38-CAR-T cells were transfected with luciferase reporter gene, respectively, at a ratio of effective target (effector cells: target cells, E: T) 4: 1 and 1: 1, co-incubation of T cells with CD38 positive target cells (THP-1-Fluc) or CD38 negative target cells (K562-Fluc), respectively. Co-incubation cultures were performed for 24 hours, 48 hours using 96-well white microplates and using complete RPMI 1640 medium with 10% FBS in incubators with 5% carbon dioxide at 37 ℃, humidity, and 3 replicates per sample, respectively. Meanwhile, 1 × 104CD38 target cells were cultured in complete RPMI 1640 medium in 96-well white microtitre plates for 24 hours and 48 hours, respectively, as controls. After the experimental time had elapsed, 0.75mg/ml D-fluorescein K + salt (Perkinelmer, cat # 122799) was added and the target apoptosis was analyzed using an EnSpire Multi-mode plate reader (Perkinelmer) to read the signal immediately. The result shows (figure 8), the CD38-CAR-T cell constructed by the method has strong killing capacity on tumor cells over-expressing CD38, and has little influence on CD38 negative cells, which indicates that the CAR-T cell has strong specific killing capacity.

Example 9 Long-term killing of CD38-CAR-T cells against CD 38-positive Daudi tumor cells

The above-constructed T cells transduced with no plasmid, and CD38-CAR-T cells were transfected with luciferase reporter gene, respectively, at a ratio of effective target (effector cells: target cells, E: T) 4: 1 and 1: 1, co-incubation of T cells with CD38 positive target cells (Daudi-Fluc) or CD38 negative target cells (K562-Fluc), respectively. Co-incubation cultures were performed for 24 hours, 48 hours using 96-well white microplates and using complete RPMI 1640 medium with 10% FBS in incubators with 5% carbon dioxide at 37 ℃, humidity, and 3 replicates per sample, respectively. Meanwhile, 1 × 104CD38 target cells were cultured in complete RPMI 1640 medium in 96-well white microtitre plates for 24 hours and 48 hours, respectively, as controls. After the experimental time had elapsed, 0.75mg/ml D-fluorescein K + salt (Perkinelmer, cat # 122799) was added and the target apoptosis was analyzed using an EnSpire Multi-mode plate reader (Perkinelmer) to read the signal immediately. The result shows (figure 7), the CD38-CAR-T cell constructed by the method has strong killing capacity on tumor cells over-expressing CD38, and has little influence on CD38 negative cells, which indicates that the CAR-T cell has strong specific killing capacity.

Example 10 Long-acting killing of CD38-CAR-T cells against CD 38-positive Raji tumor cells

The above-constructed T cells transduced with no plasmid, and CD38-CAR-T cells were transfected with luciferase reporter gene, respectively, at a ratio of effective target (effector cells: target cells, E: T) 4: 1 and 1: 1, co-incubation of T cells with CD38 positive target cells (Raji-Fluc) or CD38 negative target cells (K562-Fluc), respectively. Co-incubation cultures were performed for 24 hours, 48 hours using 96-well white microplates and using complete RPMI 1640 medium with 10% FBS in incubators with 5% carbon dioxide at 37 ℃, humidity, and 3 replicates per sample, respectively. Meanwhile, 1 × 104CD38 target cells were cultured in complete RPMI 1640 medium in 96-well white microtitre plates for 24 hours and 48 hours, respectively, as controls. After the experimental time had elapsed, 0.75mg/ml D-fluorescein K + salt (Perkinelmer, cat # 122799) was added and the target apoptosis was analyzed using an EnSpire Multi-mode plate reader (Perkinelmer) to read the signal immediately. The result shows (figure 6), the CD38-CAR-T cell constructed by the method has strong killing capacity on tumor cells over-expressing CD38, and has little influence on CD38 negative cells, which indicates that the CAR-T cell has strong specific killing capacity.

SEQUENCE LISTING

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

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

1 5 10 15

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

20 25 30

Asp Val Leu Asp Lys Arg Lys 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

65 70 75

<130>10

<170>PatentIn version 3.3

<211>237

<212>DNA

<213> Artificial Synthesis

<400>10

agggtgaagt tcagcaggag cgccgacgcc cccgcctacc agcagggcca gaaccagctg 60

tacaacgagc tgaacctggg caggagggag gagtacgacg tgctggacaa gaggaagggc 120

agggaccccg agatgggcgg caagcccagg aggaagaacc cccaggaggg cctgtacaac 180

gagctgcaga aggacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgag 237

<130>11

<170>PatentIn version 3.3

<211>5

<212>PRT

<213> Artificial Synthesis

<400>11

Ser His Gly Met His

1 5

<130>12

<170>PatentIn version 3.3

<211>17

<212>PRT

<213> Artificial Synthesis

<400>12

Leu Ile Trp Phe Asp Gly Ser Gln Lys Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<130>13

<170>PatentIn version 3.3

<211>16

<212>PRT

<213> Artificial Synthesis

<400>13

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

1 5 10 15

<130>14

<170>PatentIn version 3.3

<211>12

<212>PRT

<213> Artificial Synthesis

<400>14

Arg Ala Ser Gln Thr Val Thr Asn Ser Arg Leu Ala

1 5 10

<130>15

<170>PatentIn version 3.3

<211>7

<212>PRT

<213> Artificial Synthesis

<400>15

Asp Ala Ser Tyr Arg Ala Thr

1 5

<130>16

<170>PatentIn version 3.3

<211>9

<212>PRT

<213> Artificial Synthesis

<400>16

Gln Gln Tyr Gly Asn Ser Pro Arg Thr

1 5

<130>17

<170>PatentIn version 3.3

<211>125

<212>PRT

<213> Artificial Synthesis

<400>17

Met Ala Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro

1 5 10 15

Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Ala Leu Ser Phe Arg

20 25 30

Ser His Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ala Leu Ile Trp Phe Asp Gly Ser Gln Lys Tyr Tyr Ala Asp

50 55 60

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

65 70 75 80

Leu Phe Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Gly Cys Ile Thr Met Val Arg Gly Val His His Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val

115 120 125

<130>18

<170>PatentIn version 3.3

<211>375

<212>DNA

<213> Artificial Synthesis

<400>18

atggcacagg tacagctggt acagtctggg ggaggcgtgg tccagcctgg gaggtccctg 60

agactctcct gtgcagcgtc tgcattgagt ttcaggagtc atggcatgca ctgggtccgc 120

caggctccag gcaagggact ggagtgggtg gctcttattt ggtttgatgg aagtcagaaa 180

tattatgcag attccgtgaa gggccgattc gccatctcca gagacaattc caagaacacc 240

ttgtttctgc aaatgaacag cctgagagtc gaggacacgg ctgtgtatta ctgtgcgaga 300

ggttgtatta ctatggttcg gggagttcat cactggtact tcgatctctg gggccgtgga 360

accctggtca ccgtc 375

<130>19

<170>PatentIn version 3.3

<211>109

<212>PRT

<213> Artificial Synthesis

<400>19

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

1 5 10 15

Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Thr Asn Ser

20 25 30

Arg Leu Ala Trp Tyr Gln Glu Lys Pro Gly Leu Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Asp Ala Ser Tyr Arg Ala Thr Gly Ile Pro Asp Arg Phe Thr

50 55 60

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

65 70 75 80

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

85 90 95

Arg Thr Phe Gly Arg Gly Thr Lys Val Asp Ile Lys Arg

100 105

<130>20

<170>PatentIn version 3.3

<211>327

<212>DNA

<213> Artificial Synthesis

<400>20

gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccaggtga cagagccacc 60

ctctcctgca gggccagtca gactgttacc aacagccgct tagcctggta ccaggagaaa 120

cctggcctgg ctccgaggct cctcatctat gatgcatcct acagggccac cggcatccca 180

gacaggttca ctggcagtgg gtctgggaca gacttcactc tcattatcag cagactggag 240

cctgaagatt ttgcagtgta ttggtgtcag cagtacggta attcacctcg gacgttcggc 300

cgagggacca aagtggatat caaacgt 327

Claims

1. An anti-CD 38 chimeric antigen receptor comprising an extracellular domain capable of binding antigen, a transmembrane domain and at least one intracellular domain, characterized in that the extracellular domain is an anti-CD 38 antibody, and the sequence of the anti-CD 38 antibody is shown in SEQ ID No.1 or a sequence having 85% to 99% identity to SEQ ID No. 1.

2. An anti-CD 38 antibody, characterized in that the antibody sequence is shown in SEQ ID NO.1 or a sequence with 85% -99% identity with SEQ ID NO. 1.

3. The anti-CD 38 antibody of claim 2, wherein the coding gene is shown in SEQ ID NO.2, or a sequence with 85% -99% identity with SEQ ID NO. 2.

4. An anti-CD 38 antibody, characterized in that three CDR sequences of the heavy chain of the antibody are respectively shown as SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13, and three CDR sequences of the light chain are respectively shown as SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16.

5. An anti-CD 38 antibody, wherein the antibody heavy chain sequence is as shown in SEQ ID No.17, or a sequence having 85% to 99% identity to SEQ ID No. 17; the light chain sequence of the antibody is shown in SEQ ID NO.19, or a sequence with 85% -99% of identity with SEQ ID NO. 19.

6. The coding sequence of the heavy chain of the antibody of claim 5 is as shown in SEQ ID No.18, or a sequence with 85% -99% identity to SEQ ID No. 18; the coding sequence of the light chain is shown as SEQ ID NO.20, or a sequence which has 85-99% of identity with SEQ ID NO. 20.

7. The anti-CD 38 chimeric antigen receptor of claim 1, wherein said chimeric antigen receptor is composed of an anti-human CD38 single-chain antibody scFv, a human CD8a molecule hinge region, a transmembrane region CD8TM, a human CD28 molecule intracellular region CD28, a human 4-1BB molecule intracellular region, and a human CD3 ζ molecule intracellular region connected in series in that order.

8. The chimeric antigen receptor according to claim 7, wherein the amino acid sequence of the anti-human CD38 single-chain antibody scFv is shown as SEQ ID No.1, the amino acid sequences of the hinge region and transmembrane region CD8TM of human CD8a molecule are shown as SEQ ID No.3, the amino acid sequence of the intracellular region CD28 of human CD28 molecule is shown as SEQ ID No.5, the amino acid sequence of the intracellular region of human 4-1BB molecule is shown as SEQ ID No.7, and the amino acid sequence of the intracellular region of human CD3 zeta molecule is shown as SEQ ID No. 9.

9. A recombinant expression vector comprising a gene encoding the chimeric antigen receptor of claim 1, 6, 7 or 8; or the recombinant expression vector comprises a gene encoding the anti-CD 38 antibody of claim 2, 4 or 5.

10. An immune cell comprising the chimeric antigen receptor of claim 1, 6, 7 or 8, or comprising the anti-CD 38 antibody of claim 2, 4 or 5, further preferably an immune cell differentiated in T-lymphocyte, NK cell, NKT cell, macrophage, mesenchymal stem cell, hematopoietic stem cell, pluripotent stem cell or embryonic stem cell culture.

11. Use of an immune cell according to claim 10 for the preparation of a medicament for the treatment of a tumour, wherein said tumour is a CD38 positive tumour, preferably a myeloma, lymphoma or leukemia.