CN113801228B

CN113801228B - CD 38-targeted single-chain antibody, fully human chimeric antigen receptor, preparation method and application

Info

Publication number: CN113801228B
Application number: CN202010555320.9A
Authority: CN
Inventors: 何玫瑰; 闵晨雨
Original assignee: Changzhou Velox Pharmaceutical Science & Technology Co ltd
Current assignee: Changzhou Velox Pharmaceutical Science & Technology Co ltd
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2023-01-17
Anticipated expiration: 2040-06-17
Also published as: CN113801228A

Abstract

The invention discloses a single-chain antibody targeting CD38, a fully human chimeric antigen receptor, a preparation method and application. The chimeric antigen receptor comprises: the kit comprises a promoter, a leader peptide, a single-chain antibody for specifically recognizing the CD38 antigen, a tag peptide segment, a hinge region, a transmembrane region, an intracellular signal region, a self-cutting peptide and a puromycin gene which are connected in sequence. The scFv sequence capable of recognizing the CD38 with moderate affinity is selected, can specifically and efficiently recognize target cells over-expressing CD38 antigen, does not show obvious toxic or side effect on normal cells and tissues under-expressing CD38, is high in safety, and has wide clinical application prospects.

Description

CD 38-targeted single-chain antibody, fully human chimeric antigen receptor, preparation method and application

Technical Field

The invention relates to the field of cellular immunology, in particular to a single-chain antibody of a targeting CD38, a fully human chimeric antigen receptor, a preparation method and application.

Background

Adoptive Cell Therapy (ACT) is one of the biological treatment technologies, and is a method of expanding autoimmune cells (mainly T cells) in vitro and then returning them to tumor patients for treatment, which is considered as the 4 th treatment mode following surgery, radiotherapy and chemotherapy and is widely used in clinical treatment. The main major applications of adoptive cell therapy are: the combination of lymphokine activated killer cells (LAK) and IL-2 has certain curative effect on treating late malignant tumor; the Tumor Infiltrating Lymphocytes (TIL) clinical test has better effect on treating metastatic melanoma; cytokine induced killer Cells (CIK) are currently subjected to many clinical tests in China, and have remarkable effects on tumors such as liver cancer and lung cancer. However, the three treatment methods require activation of T cells, and two activation signals are required for T cell activation, namely, a first signal for activation of TCR-CD3 and MHC-I molecules on the surface of the T cells is combined to determine the killing activity of the T cells on tumor cells; the binding of costimulatory molecules on the surface of T cells to the corresponding ligands is the second signal to activate, determining T cell proliferation. However, the tumor cells and the tumor microenvironment can down regulate MHC and ligand molecules, thereby inhibiting the killing activity of T cells on the tumor cells. Therefore, there are two main ways to perform genetic modification: gene transduction TCRs (T cell receptors) and Chimeric Antigen Receptors (CARs).

Chimeric Antigen Receptors (CAR), which are artificial receptors that mimic TCR function, consist of an antigen recognition domain, a hinge region, and a transmembrane region, connected in sequence, and an intracellular signaling domain, usually the CD3 zeta chain, or linked to one or more costimulatory molecules, such as 4-1bb, cd28. When an antigen (receptor) on the surface of a tumor cell is bound to an antibody (ligand) of a chimeric antigen receptor, a signal is transmitted into the cell through a hinge region and a transmembrane region, an intracellular signal region converts the signal into an activation signal, an effector cell is activated, and the effector cell proliferates to produce a cytokine to kill the tumor cell. Although CAR-T treatment of tumors has shown some tumor clearance, and CD19-CAR treatment in particular has made a breakthrough, CAR-T treatment still has a series of problems to be solved by further exploration: 1. lack of ideal tumor antigens as targets; 2. tumor-mediated immunosuppression; 3. serious toxic side effects of CAR-T therapy.

Multiple Myeloma (MM), a malignant tumor with abnormal proliferation of plasma cells, an incurable hematological malignancy, can affect the production of white blood cells, red blood cells and stem cells in the bone marrow. Plasma cells that have become cancerous in the bone marrow are called myeloma cells, and they proliferate in large quantities in the bone marrow, replace normal cells in the bone marrow, destroy the normal immune system, inhibit the normal hematopoietic function, and further impair the functions of bones and soft tissues (nerves, muscles, etc.). In the united states, multiple myeloma is the second largest malignancy of the blood system following non-hodgkin's lymphoma, accounting for approximately 1% of all malignancies, 13% of hematologic malignancies, and 2% of deaths due to malignancies. The mean age of onset is 62 years in men, 61 years in women, and only 2% of patients under 40 years of age.

CD38 is uniformly highly expressed on multiple myeloma cells, while it is poorly expressed on normal lymphocytes and bone marrow cells, as well as on some tissues of non-hematopoietic origin, and is a specific antigen for multiple myeloma. Because of the high expression of CD38 on MM, CD 38-targeted immunotherapy theoretically has a safer and more effective therapeutic effect, and the development of CD 38-targeted drugs such as (e.g., daratumumab, isatuximab, MOR 202) has been carried out abroad, but the efficacy is limited and safety problems still occur; therefore, there is a need to find new specific and more effective CD38 targeting therapies, and although some target CARs are being tested, there are relatively few CARs for MM therapy, further engineering tests are still needed for stability and safety of CAR performance, and how to select more stable and suitable scFv (single chain antibody) is also an unsolved key issue in CAR research, so that a safer and more effective CD38-CAR targeting development and preparation is necessary.

Disclosure of Invention

The invention aims to solve the problems of stability and safety of a targeting drug targeting CD38 in the prior art.

In order to achieve the above objects, the present invention provides a single-chain antibody that recognizes CD38 antigen moderately and specifically, the single-chain antibody comprising: a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises three complementarity determining regions: HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 comprises SEQ ID NO: 2; the amino acid sequence of the HCDR2 comprises SEQ ID NO: 3; the amino acid sequence of the HCDR3 comprises SEQ ID NO: 4; the light chain variable region comprises three complementarity determining regions: LCDR1, LCDR2, LCDR3, the amino acid sequence of LCDR1 includes SEQ ID NO: 5; the amino acid sequence of the LCDR2 comprises SEQ ID NO: 6; the amino acid sequence of the LCDR3 comprises SEQ ID NO:7, or a fragment thereof.

The invention also provides a CD 38-targeted fully human chimeric antigen receptor, which comprises a single-chain antibody specifically recognizing the CD38 antigen, wherein the amino acid sequence of the single-chain antibody comprises the amino acid sequence shown in SEQ ID NO:1, or a variant of SEQ ID NO:1 with 95-99% identity.

Preferably, the chimeric antigen receptor comprises: the promoter, the leader peptide, the single-chain antibody for specifically recognizing the CD38 antigen, the tag peptide segment, the hinge region, the transmembrane region and the intracellular signal domain are connected in sequence.

Preferably, the promoter is derived from EF1 alpha, and the nucleic acid sequence is shown as SEQ ID NO:8 is shown in the specification; the leader peptide is derived from GM-CSF, and the amino acid sequence is shown as SEQ ID NO:9 is shown in the figure; the amino acid sequence of the tag peptide segment is shown as SEQ ID NO:10 is shown in the figure; the hinge region and the transmembrane region are CD 8-derived hinge region and transmembrane region, and the amino acid sequences are respectively shown as SEQ ID NO:11 and SEQ ID NO:12 is shown in the specification; the intracellular signaling domain comprises: CD28, 4-1BB and CD3 zeta, the amino acid sequences are respectively shown in SEQ ID NO: 13. SEQ ID NO:14 and SEQ ID NO: shown at 15.

Preferably, the chimeric antigen receptor is further connected with a self-cutting peptide T2A and a puromycin gene, and the amino acid sequence of the self-cutting peptide T2A is shown as SEQ ID NO:16 is shown in the figure; the amino acid sequence of the puromycin gene is shown as SEQ ID NO: shown at 17.

The invention also provides a polynucleotide for coding the CD 38-targeted fully human chimeric antigen receptor, wherein the chimeric antigen receptor comprises a single-chain antibody specifically recognizing the CD38 antigen, and the nucleic acid sequence of the single-chain antibody comprises the nucleotide sequence shown in SEQ ID NO:18, or a variant of SEQ ID NO:18 with 95-99% identity.

The invention also provides a preparation method of the CD 38-targeted fully human chimeric antigen receptor based on a pMD19-T vector, which comprises the following steps:

(1) Synthesizing a nucleotide sequence for coding the chimeric antigen receptor to obtain an EF1 alpha-SP-CD 38-CAR-T2A-Puro gene segment; the gene fragment comprises a promoter, a leader peptide, a single-chain antibody for specifically recognizing a CD38 antigen, a tag peptide fragment, a hinge region, a transmembrane region, an intracellular signal region, T2A and a puromycin gene;

(2) Carrying out double enzyme digestion on the EF1 alpha-SP-CD 38-CAR-T2A-Puro gene fragment by using restriction enzymes BamH-I and SalI to obtain a target fragment; and (2) digesting the pMD19-T vector by using restriction enzymes BamH-I and SalI, and then connecting the digested target fragment and the pMD19-T vector by using T4 ligase to obtain a pMD19-T plasmid capable of expressing the chimeric antigen receptor.

The invention also provides a pMD19-T plasmid capable of expressing the chimeric antigen receptor, and the pMD19-T plasmid is prepared by the preparation method of the CD 38-targeted fully human chimeric antigen receptor based on the pMD19-T vector.

The invention also provides a T cell expressing the CD 38-targeted fully human chimeric antigen receptor.

The invention also provides application of the CD 38-targeted fully human chimeric antigen receptor in preparing a medicament for inhibiting or treating a malignant hematological disease or a solid tumor capable of expressing CD 38.

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

(1) The invention selects the scFv sequence which has moderate affinity and can recognize the CD38, can specifically and efficiently recognize the target cell of the high expression CD38, has no obvious toxic or side effect on the cell and the tissue of the low expression CD38, and has better immune tolerance and higher safety.

(2) In the traditional preparation process, the CAR-T cells used for treatment are often in the terminal differentiation stage, have short survival time and are difficult to exert long-acting killing function, so the treatment effect is poor. The CD28 and 41BB costimulatory signals adopted by the invention obviously inhibit the terminal differentiation of T cells and promote the generation of memory type T cells with longer survival time. The transgenic modified CAR-T cell has stronger and more durable killing capacity and is expected to improve the treatment effect of solid tumors.

(3) The invention ensures that the extracellular recognition region and the intracellular signal region are well combined by utilizing the sequences of the CD8 hinge region and the transmembrane region, and ensures the function of the CAR-T cell.

(4) The non-viral vector pMD19-T electrotransfection system is adopted, so that potential safety hazards caused by using a viral vector are avoided, the safety and the effectiveness are higher, and the method is more suitable for clinical treatment.

Drawings

FIG. 1 is a block diagram of a fully human CD38-CAR (hCD 38 CAR).

FIG. 2 is a flow chart of transfection efficiency of fully human CD38-CAR-T cells.

FIG. 3 is the results of the cytotoxicity of fully human CD38-CAR-T cells against CD 38-overexpressing RPMI-8226 tumor cells and Daudi tumor cells.

FIG. 4 is the cytotoxicity assay of fully human CD38-CAR-T cells on normal PBMC cells naturally expressing CD 38.

FIG. 5 is a graph of fully human CD38-CAR-T cells controlling mouse tumor growth.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.

The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental reagents and materials involved are conventional biochemical reagents and materials unless otherwise specified.

Definition of terms

An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds, or an antigen-binding portion thereof. Antibodies include single chain antibodies.

The "heavy chain" consists of a heavy chain variable region (VH) and a heavy chain constant region.

The "light chain" consists of a light chain variable region (VL) and a light chain constant region.

Single-chain antibody fragment (scFv): the heavy chain variable region and the light chain variable region of the antibody are connected by a short peptide (linker) with 15-20 amino acids.

The "heavy chain variable region" and "light chain variable region" can be further subdivided into hypervariable regions, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions termed "framework regions" (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRI, CDRl, FR2, CDR2, FR3, CDR3, FR4, the variable regions of the heavy and light chains contain binding domains that can interact with antigens. In the invention, CDRL, CDR2 and CDR3 of a heavy chain variable region are respectively expressed as HCDR1, HCDR2 and HCDR3; CDRL, CDR2, CDR3 of the light chain variable region are denoted as LCDR1, LCDR2 and LCDR3, respectively.

"CAR-T" refers to a chimeric antigen receptor T cell. The scFv for recognizing tumor-associated antigen and an intracellular signal domain 'immunoreceptor tyrosine activation motif' are subjected to in vitro gene recombination to generate recombinant plasmids, the recombinant plasmids are transfected into T cells of a patient in vitro by a transfection technology to enable the T cells of the patient to express a tumor antigen receptor, and the transfected T cells are purified and amplified in a large scale and are called chimeric antigen receptor T cells.

"95-99% identity" refers to homology or similarity of greater than 95% (optimally greater than 98%). It is known to those skilled in the art that mutation of one or a few amino acids or nucleotides in a sequence does not, in some cases, substantially alter the biological function of the sequence. The mutation of amino acid or nucleotide also includes the modification of amino acid or nucleotide sequence, and the biological function is the same as or equivalent to the sequence provided by the present invention, and this type of sequence is within the protection scope of the present invention.

The nucleic acid sequence for coding scFv provided by the invention is shown as SEQ ID NO:18, the amino acid sequence of the corresponding scFv is shown in SEQ ID NO:1 is shown. SEQ ID NO:2 to SEQ ID NO:7 is the sequence shown in SEQ ID NO:1.

Specific screening methods for the sequences of the present invention can be found in patent document CN201911234429.6 (title of the invention: fully human antibodies or antibody fragments against CD19, chimeric antigen receptor, and application). The following examples are for nucleic acid molecules comprising SEQ ID NO:1 (scFv) was tested for chimeric antigen receptor.

1. CAR structure

The basic structure of the CD38-CAR (human chimeric antigen receptor targeting CD 38) is composed of an EF1 alpha promoter (promoter), a leader peptide (SP), a single-chain antibody (scFv) specifically recognizing CD38 antigen, a tag peptide segment (Myc-tag), a human CD8a molecule Hinge region (CD 8 Hinge), a transmembrane region (CD 8 TM), a human CD28 molecule intracellular region (CD 28), a human 4-1BB molecule intracellular region (4-1 BB), a human CD3 zeta molecule intracellular region (CD 3 zeta), a 2A self-cleavage peptide (T2A) and a puromycin (puromycin) gene which are connected in series in sequence, and the structure formed by the series connection is shown as hCD38 CAR in figure 1. The tandem structure is denoted hereinafter by EF 1. Alpha. -SP-CD 38-CAR-T2A-Puro.

2. CAR sequence synthesis and vector construction

The CAR coding sequence is synthesized by Kinsley of Jiangsu, and the obtained EF1 alpha-SP-CD 38-CAR-T2A-Puro structural DNA sequence (shown in figure 1) is inserted into a pMD19-T plasmid through enzyme digestion connection to construct a non-viral electrotransformation expression plasmid pMD19-T-EF1 alpha-SP-CD 38-CAR-T2A-Puro, namely the pMD19-T plasmid capable of expressing a chimeric antigen receptor.

3. Electrotransformation expression plasmid extraction

pMD19-T-EF1 alpha-SP-CD 38-CAR-T2A-Puro was transferred into E.coli and amplified in E.coli in large quantities. Then, plasmid DNA of Escherichia coli was extracted using an endotoxin-free plasmid macrodrawing kit.

4. T cell transfection and purification

Separating lymphocytes from human serum, and adding T lymphocyte culture fluid [ CTS ] ^TM AIM V ^TM SFM (RUO), thermo, cargo number: a3021002]The irradiated PBMC cells, the OKT-3, the hIL-2 and the endotoxin-free plasmid DNA are subjected to expansion culture, and the plasmid pMD19-T-EF1 alpha-SP-CD 38-CAR-T2A-Puro is transfected by adopting an electrotransformation modeTransferring into T cells to obtain T lymphocytes with EF1 alpha-SP-CD 38-CAR-T2A-Puro gene, namely CAR-T cells.

5. T cell CAR expression efficiency

After 7 days of T cell transfection, puromycin was added at a certain concentration to the T lymphocyte culture broth. After 20 days of T cell transfection, T cell surface CAR expression was examined by flow cytometry. The results are shown in FIG. 2. Figure 2 shows that the CAR expression positive rate reached about 100%, demonstrating the success of CAR expression plasmid construction and electrotransformation plasmid.

6. Long-acting killing effect of CAR-T cells on tumor cells over-expressing related antigens

To determine the better sustained killing of CD38-CAR-T cells, the experiments were performed using target cells carrying the fluorescence signal of luciferase. The ratio of effective target (effector cell: target cell, E: T) 4:1 and 1:1, T cells (effector cells) were co-incubated with CD 38-positive target cells (Daudi-Fluc, RPMI-8226-Fluc) or CD 38-negative target cells (K562-Fluc), respectively, for 72 hours (as shown in fig. 3). After the completion of the co-incubation, a substrate for fluorescence signal of luciferase (Perkinelmer, cat # 122799) was added, and then the apoptosis of the target cells was analyzed by a microplate reader. The results show that the CAR-T Cells have strong killing capacity on tumor Cells (CD 38+ Cells) over-expressing CD38, and have little influence on CD38 negative Cells, which indicates that the CAR-T Cells have strong specific killing capacity.

7. Toxic side effects of CAR-T cells on normal cells that underexpress the relevant antigen

To further determine the toxic side effects of CD38-CAR-T cells (pMD-T-CD 38-scfv-003-CAR T cells in FIG. 4) on normal cells with low expression of CD38, we previously stained human Peripheral Blood Mononuclear Cells (PBMC) using the CellTrace (TM) blood Cell Proliferation Kit (Thermo, cat # C34571). The ratio of effective target to target (effector cells: target cells, E: T) 4: 1. 1: 1T cells (effector cells) were incubated with normal PBMC cells that underexpress CD38 for 72 hours (as shown in FIG. 4). After the end of the co-incubation, the cells were harvested by centrifugation and co-incubated with anti-CD 3 and anti-CD 38 antibodies, and finally PBMC cells were analyzed for CD 38-positive apoptosis by flow cytometry. The results show that CAR-T cells have very low toxic side effects on normal cells that under express CD 38.

8. Animal experiments to verify CAR-T cell function

Immunodeficient mice were inoculated via tail vein with 5X 10 ⁵ 7 days after each Daudi cell, 1X 10 cells were injected into the tail vein ⁷ Individual CAR-T cells were treated. At 0,7 days of treatment, tumor signals were observed using a small animal in vivo imaging device. As shown in figure 5, tumor signals were significantly reduced 7 days after CAR-T cell injection, suggesting that CAR-T cells have good tumor killing ability.

In conclusion, the scFv sequence or the chimeric antigen receptor provided by the invention has the capacity of recognizing CD38 with moderate affinity, can efficiently recognize target cells, and has low toxic and side effects and high safety on cells with low expression of CD 38.

While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.

Sequence listing

<110> Changzhou Fislos pharmaceutical technology Ltd

<120> CD 38-targeted single-chain antibody, fully human chimeric antigen receptor, preparation method and application

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 245

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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

1 5 10 15

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

20 25 30

Ser Ser Asn Trp Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu

35 40 45

Glu Trp Ile Gly Glu Ile Tyr His Ser Gly Ser Thr Asn Tyr Asn Pro

50 55 60

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

65 70 75 80

Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Glu Asp Tyr Tyr Tyr Tyr Met Asp Val Trp Gly Lys

100 105 110

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

115 120 125

Gly Ser Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser Val

130 135 140

Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser Ser

145 150 155 160

Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly

165 170 175

Thr Ala Pro Lys Leu Leu Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly

180 185 190

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

195 200 205

Gly Ile Thr Gly Leu Gln Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly

210 215 220

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

225 230 235 240

Leu Thr Val Leu Gly

245

<210> 2

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Gly Ser Ile Ser Ser Ser Asn Trp

1 5

<210> 3

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ile Tyr His Ser Gly Ser Thr

1 5

<210> 4

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ala Arg Glu Asp Tyr Tyr Tyr Tyr Met Asp Val

1 5 10

<210> 5

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Ser Ser Asn Ile Gly Asn Asn Tyr

1 5

<210> 6

<211> 3

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asp Asn Asn

1

<210> 7

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Thr Trp Asp Ser Ser Leu Ser Ala Gly Val

1 5 10

<210> 8

<211> 546

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60

cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180

gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240

acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300

ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360

tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420

cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480

actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540

gcctac 546

<210> 9

<211> 22

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

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

20

<210> 10

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 11

<211> 55

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro

1 5 10 15

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

20 25 30

Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg

35 40 45

Gly Leu Asp Phe Ala Cys Asp

50 55

<210> 12

<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

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

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn

20 25

<210> 13

<211> 41

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 14

<211> 47

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

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

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

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

35 40 45

<210> 15

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 16

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 17

<211> 199

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala Thr Arg Asp Asp Val

1 5 10 15

Pro Arg Ala Val Arg Thr Leu Ala Ala Ala Phe Ala Asp Tyr Pro Ala

20 25 30

Thr Arg His Thr Val Asp Pro Asp Arg His Ile Glu Arg Val Thr Glu

35 40 45

Leu Gln Glu Leu Phe Leu Thr Arg Val Gly Leu Asp Ile Gly Lys Val

50 55 60

Trp Val Ala Asp Asp Gly Ala Ala Val Ala Val Trp Thr Thr Pro Glu

65 70 75 80

Ser Val Glu Ala Gly Ala Val Phe Ala Glu Ile Gly Pro Arg Met Ala

85 90 95

Glu Leu Ser Gly Ser Arg Leu Ala Ala Gln Gln Gln Met Glu Gly Leu

100 105 110

Leu Ala Pro His Arg Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val

115 120 125

Gly Val Ser Pro Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val

130 135 140

Leu Pro Gly Val Glu Ala Ala Glu Arg Ala Gly Val Pro Ala Phe Leu

145 150 155 160

Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr Glu Arg Leu Gly Phe

165 170 175

Thr Val Thr Ala Asp Val Glu Val Pro Glu Gly Pro Arg Thr Trp Cys

180 185 190

Met Thr Arg Lys Pro Gly Ala

195

<210> 18

<211> 735

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atggcacagg tgcagctggt ggagtctggc ccaggactgg tgaagccttc ggggaccctg 60

tccctcacct gcgctgtctc tggtggctcc atcagcagta gtaactggtg gagttgggtc 120

cgccagcccc cagggaaggg gctggagtgg attggggaaa tctatcatag tgggagcacc 180

aactacaacc cgtccctcaa gagtcgagtc accatatcag tagacaagtc caagaaccag 240

ttctccctga agctgagctc tgtgaccgcc gcggacacgg ccgtgtatta ctgtgcgaga 300

gaggactact actactacat ggacgtctgg ggcaaaggga ccacggtcac cgtctcctca 360

ggcggcggcg gctctggcgg aggcggcagc ggcggcggat cccagtctgt gttgacgcag 420

ccgccctcag tgtctgcggc cccaggacag aaggtcacca tctcctgctc tggaagcagc 480

tccaacattg ggaataatta tgtatcctgg taccagcagc tcccaggaac agcccccaaa 540

ctcctcattt atgacaataa taagcgaccc tcagggattc ctgaccgatt ctctggctcc 600

aagtctggca cgtcagccac cctgggcatc accggactcc agactgggga cgaggccgat 660

tattactgcg gaacatggga tagcagcctg agtgctgggg tgttcggcgg agggaccaag 720

ctgaccgtcc taggt 735

Claims

1. A single chain antibody that recognizes CD38 antigen moderately and specifically, said single chain antibody comprising: a heavy chain variable region and a light chain variable region;

the heavy chain variable region comprises three complementarity determining regions: HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of the HCDR1 is SEQ ID NO: 2;

the amino acid sequence of the HCDR2 is SEQ ID NO: 3;

the amino acid sequence of the HCDR3 is SEQ ID NO: 4;

the light chain variable region comprises three complementarity determining regions: LCDR1, LCDR2, LCDR3, the amino acid sequence of LCDR1 is SEQ ID NO: 5;

the amino acid sequence of the LCDR2 is SEQ ID NO: 6;

the amino acid sequence of the LCDR3 is SEQ ID NO:7, or a sequence shown in the figure.

2. A fully human chimeric antigen receptor targeting CD38, said chimeric antigen receptor comprising a single chain antibody specifically recognizing CD38 antigen, said single chain antibody having the amino acid sequence of SEQ ID NO:1.

3. the CD38 targeted chimeric antigen receptor of claim 2, wherein said chimeric antigen receptor comprises: the promoter, the leader peptide, the single-chain antibody for specifically recognizing the CD38 antigen, the tag peptide segment, the hinge region, the transmembrane region and the intracellular signal domain are connected in sequence.

4. The CD 38-targeted fully human chimeric antigen receptor of claim 3, wherein: the promoter is derived from EF1 alpha, and the nucleic acid sequence is shown as SEQ ID NO:8 is shown in the specification; the leader peptide is derived from GM-CSF, and the amino acid sequence is shown in SEQ ID NO:9 is shown in the figure; the amino acid sequence of the tag peptide segment is shown as SEQ ID NO:10 is shown in the figure; the hinge region and the transmembrane region are CD 8-derived hinge region and transmembrane region, and the amino acid sequences are respectively shown as SEQ ID NO:11 and SEQ ID NO:12 is shown in the specification; the intracellular signaling domain comprises: CD28, 4-1BB and CD3 zeta, the amino acid sequences are respectively shown in SEQ ID NO: 13. SEQ ID NO:14 and SEQ ID NO: shown at 15.

5. The CD 38-targeted fully human chimeric antigen receptor of claim 3, wherein said chimeric antigen receptor is further linked with a self-cleaving peptide T2A and a puromycin gene, wherein the amino acid sequence of said self-cleaving peptide T2A is as set forth in SEQ ID NO:16 is shown in the figure; the amino acid sequence of the puromycin gene is shown as SEQ ID NO: shown at 17.

6. A polynucleotide encoding the CD 38-targeted chimeric antigen receptor of full human origin according to any one of claims 2 to 5, wherein the chimeric antigen receptor comprises a single-chain antibody specifically recognizing the CD38 antigen, and the nucleic acid sequence of the single-chain antibody is SEQ ID NO:18.

7. the method for preparing a fully human chimeric antigen receptor targeting CD38 based on a pMD19-T vector according to any one of claims 2 to 5, comprising the steps of:

(1) Synthesizing a nucleotide sequence for coding the chimeric antigen receptor to obtain an EF1 alpha-SP-CD 38-CAR-T2A-Puro gene segment; the gene segment comprises a promoter, a leader peptide, a single-chain antibody for specifically recognizing the CD38 antigen, a tag peptide segment, a hinge region, a transmembrane region, an intracellular signal region, T2A and a puromycin gene;

(2) Carrying out double enzyme digestion on the EF1 alpha-SP-CD 38-CAR-T2A-Puro gene fragment by using restriction enzymes BamH-I and SalI to obtain a target fragment; and (2) digesting the pMD19-T vector by using restriction enzymes BamH-I and SalI, and then connecting the digested target fragment with the pMD19-T vector through T4 ligase to obtain the pMD19-T plasmid capable of expressing the chimeric antigen receptor.

8. A pMD19-T plasmid capable of expressing the chimeric antigen receptor of any one of claims 2 to 5, wherein the pMD19-T plasmid is prepared by the method for preparing the CD 38-targeted fully human chimeric antigen receptor of claim 7 based on the pMD19-T vector.

9. A T cell expressing the CD 38-targeted fully human chimeric antigen receptor of any one of claims 2 to 5.

10. Use of a CD 38-targeting fully human chimeric antigen receptor according to any one of claims 2 to 5 for the preparation of a medicament for inhibiting or treating a hematological malignancy or a solid tumor capable of expressing CD 38.