CN108409840B

CN108409840B - anti-CD 123 single-chain antibody, chimeric antigen receptor combined with same and application

Info

Publication number: CN108409840B
Application number: CN201810207761.2A
Authority: CN
Inventors: 张巍; 单娟娟; 赵文旭; 陈军; 黄霞; 赵永春; 徐艳敏; 张茜真
Original assignee: Chongqing Precision Biotech Co ltd
Current assignee: Chongqing Precision Biotech Co ltd
Priority date: 2017-05-18
Filing date: 2018-03-14
Publication date: 2020-06-23
Anticipated expiration: 2038-03-14
Also published as: CN108409840A

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to an anti-CD 123 single-chain antibody, a combined chimeric antigen receptor thereof and application thereof. The amino acid sequence of the polypeptide for identifying the CD123 antigen is shown as SEQ ID NO. 1. A chimeric antigen receptor against CD123 antigen, consisting of a polypeptide (ScFv) recognizing the CD123 antigen, a hinge region, a transmembrane region and an intracellular signal region connected in this order. After the chimeric antigen receptor of the anti-CD 123 antigen is expressed in immune cells, not only can the tumor target cells expressing the CD123 antigen be effectively eliminated, but also the tumor target cells of negative antigen (not expressing CD123) have no toxic effect; and can maintain the positive rate of a Chimeric Antigen Receptor (CAR) targeting CD123 in the cell culture process of a patient, can proliferate for a long time after the stimulation of a target antigen, and can be used for the targeted therapy of tumors.

Description

anti-CD 123 single-chain antibody, chimeric antigen receptor combined with same and application

Technical Field

The invention belongs to the field of genetic engineering, and relates to an anti-CD 123 single-chain antibody, a combined chimeric antigen receptor and application thereof, and a lentiviral vector of the chimeric antigen receptor of an anti-CD 123 antigen and application thereof.

Background

Adoptive Cell Therapy (ACT) is one of the biological treatment technologies, and a method of ex vivo expansion of autoimmune cells (mainly T cells) and then reinfusing them back to tumor patients for the purpose of treatment 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) has better effect on treating the metastatic melanoma in clinical tests; 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 need to activate T cells, and the T cell activation needs two activation signals, namely a first signal for the T cell surface TCR-CD3 to be combined with MHC-I molecules to be activated so as 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 for activation, 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. There is therefore a need for genetic engineering, mainly in two ways: gene transduction TCRs (T cell receptors) and Chimeric Antigen Receptors (CARs).

Chimeric Antigen Receptors (CARs), abbreviated as CARs, are artificial receptors that mimic TCR function and consist of an antigen recognition domain, a hinge region, and a transmembrane region, in turn linked to an intracellular signaling domain, usually the CD3 zeta chain or FcR gamma, or linked to one or more costimulatory molecules, such as 4-1BB (CD137), CD28, ICOS (CD 278). 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, thereby killing the tumor cell. Although CAR-T treatment of tumors has shown some tumor clearance, and CD19-CAR treatment in particular has made a breakthrough in the treatment of hematological tumors, CAR-T treatment still has a series of problems that need to be further explored: 1, CAR-T cells have poor ability to proliferate in vivo; 2, the artificial receptor still has certain potential safety hazard.

Acute Myelogenous Leukemia (AML) is a malignant disease of hematopoietic stem/progenitor cells of a myeloid line, is characterized by abnormal hyperplasia of primary and juvenile myelogenous cells in bone marrow and peripheral blood as a main characteristic, and is urgent in most cases, dangerous in the future, and life can be threatened if treatment is not performed in time; conventional induction chemotherapy, while capable of relieving AML, ultimately does not prevent relapse of AML and the high mortality of relapsed patients. However, conventional treatment of AML has not changed for 50 years and new changes are urgently sought.

The CD123 is also called interleukin 3 receptor α chain (IL-3R α) is highly expressed in leukemia stem cells or leukemia naive cells, is not expressed or is low expressed in normal hematopoietic stem cells, and is a leukemia-related antigen which is also a specific antigen of acute myeloid leukemia, the emergence of CD123 target is a breakthrough in AML treatment, because CD123 is highly expressed in AML, theoretically, CD123 targeted immunotherapy has safer and more effective treatment effect, foreign countries also have already carried out clinical tests of CD123 targeted drugs, but the efficacy is very limited and safety problems still occur, therefore, a new therapy which is better and more effective targeted to CD123 needs to be found, although tests of some targets are in progress at present, the number of CAR for treating AML is relatively small, further transformation tests are still needed in the aspects of stability and safety of CAR performance, and how to select more stable and suitable scFV (single-chain antibody) is also an unresolved problem in research, so that the CD123 is a more effective target for preparing the CAR is a very necessary key point in the research and development of CD 123-CAR.

At present, no good treatment scheme exists for the myeloid tumor expressing CD123, no good solution is provided for the transfection stability of the antigen chimeric receptor on T cells, and no report is provided for the influence of CD123-scFV combined with the antigen chimeric receptor on the stability of the chimeric antigen receptor cells and the curative effect on the CD123 myeloid tumor.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a chimeric antigen receptor comprising an anti-CD 123 single-chain antibody and a combination thereof and an application thereof, wherein the chimeric antigen receptor comprising a CD123-scFV of the present invention can be more stably expressed in T lymphocytes, can better eliminate tumor target cells expressing CD123 antigen, and has no toxic effect on tumor cells negative for antigen (not expressing CD 123).

In order to achieve the purpose, the technical scheme of the invention is as follows:

a polypeptide for recognizing CD123 antigen, wherein the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1.

The second objective of the invention is to provide an application of the polypeptide in preparing a single-chain antibody against CD123 antigen.

The invention also aims to provide a chimeric antigen receptor for resisting the CD123 antigen, which is formed by sequentially connecting a polypeptide (ScFv) for recognizing the CD123 antigen, a hinge region, a transmembrane region and an intracellular signal domain; the amino acid sequence of the polypeptide for recognizing the CD123 antigen contains the polypeptide shown in SEQ ID NO. 1.

The Single-chain antibody (scFv) is formed by connecting variable regions (VH and VL regions) of an antibody through a short peptide (Linker) of 15-20 amino acids, can better retain the affinity activity of the scFv to an antigen, and has the characteristics of small molecular weight, strong penetrating power, weak antigenicity and the like.

The mode of linking the heavy chain and the light chain of the single-chain antibody by the Linker is two modes of VH-Linker-VL or VL-Linker-VH. The choice of Linker may affect the stability and aggregation of single chain antibodies. The most commonly used Linker is GGGGS formed by connecting amino acid (Gly) and serine (Ser) as a unit, and one or more units are connected, so that the inventor discovers that the single-chain antibody (ScFv) for resisting the CD123 antigen disclosed by the patent can better maintain the stability of the single-chain antibody by connecting the single-chain antibody in a VL-Linker-VH connecting mode through previous exploration.

The chimeric antigen receptor needs to overcome two technical barriers, namely, search for a more stable and effective scFV for recognizing the CD123 antigen.

It is a challenge to engineer antibodies that recognize carcinoembryonic antigens so that they retain their affinity for the antigen and combine to optimally recognize the CD123 chimeric antigen receptor. The inventor selects two single-chain antibodies of CD123-scFV-1 and CD123-scFV-2, the amino acid sequence is shown as SEQ ID NO:1 or SEQ ID NO:2, and the nucleotide sequence is shown as SEQ ID NO:12 or SEQ ID NO:13 to carry out the research of CAR combination.

In the protection of the chimeric antigen receptor, three groups of chimeric antigen receptors under other combination ideas are eliminated, and only the groups protected by the invention have unexpected technical effects.

The optimal chimeric antigen receptor combination is screened by keeping the antigen recognition sequence unchanged and randomly combining a hinge region, a transmembrane region and an inclusion signal region; alternatively, the hinge, transmembrane and internal envelope signal regions are retained and different antigen recognition sequences (scFV) are screened. The results of the method are random, and only when properly configured, the affinity is likely to be comparable to that of the original mouse antibody.

The combined mode of the partial protective CAR can play a role in stably expressing T lymphocytes from patients after being tested, has better capacity of eliminating tumor cells, and is used for adoptive cell therapy aiming at malignant hematological diseases.

Further, the amino acid sequence of the hinge region is shown as SEQ ID NO. 3 or SEQ ID NO. 4.

Furthermore, the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 5 or SEQ ID NO. 6.

Further, the intracellular signal domain is a CD28 and/or CD137 and/or CD3 zeta chain; the amino acid sequence of the CD28 is shown as SEQ ID NO. 7, the amino acid sequence of the CD137 is shown as SEQ ID NO. 8, and the amino acid sequence of the CD3 zeta chain is shown as SEQ ID NO. 9.

When the antigen (receptor) on the surface of the tumor cell is combined with the antibody (ligand) of the chimeric antigen receptor, the signal is transmitted to the inside of the cell through a hinge region and a transmembrane region, an intracellular signal domain converts the signal into an activation signal, an effector cell is activated, and the effector cell proliferates and produces cytokines so as to kill the tumor cell. Chimeric antigen receptors have advantages over TCR engineering: (1) specificity: antibodies (ligands) specifically recognize antigens (receptors); (2) the efficiency is high: the mismatch between the transgenic TCR and the endogenous TCR of the patient can not occur; (3) non-MHC-I restriction: the immune escape caused by the MHC-I molecules under the down-regulation of tumor cells and tumor microenvironment can be overcome without the combination with the MHC-I molecules; (4) the antigen selection range is wide: the antigen can be carbohydrate, lipid, and protein.

Furthermore, the amino acid sequence of the chimeric antigen receptor is formed by sequentially connecting SEQ ID NO.1, SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 in series.

Furthermore, the nucleotide sequence of the chimeric antigen receptor is formed by sequentially connecting SEQ ID NO.12, SEQ ID NO. 14, SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20 in series.

The nucleic acid sequence of the hinge region is shown as SEQ ID NO. 14 or SEQ ID NO. 15; the nucleic acid sequence of the transmembrane region is shown as SEQ ID NO. 16 or SEQ ID NO. 17; the intracellular signal domain is a CD28 and/or CD137 and/or CD3 zeta chain; the nucleic acid sequence of the CD28 is shown as SEQ ID NO. 18, the nucleic acid sequence of the CD137 is shown as SEQ ID NO. 19, and the nucleic acid sequence of the CD3 zeta chain is shown as SEQ ID NO. 20.

The chimeric antigen receptor nucleic acid sequence is formed by sequentially and serially combining the single-chain antibody, the hinge region, the transmembrane region and the nucleic acid sequence of the envelope signal region.

The invention also aims to provide a preparation method of the lentiviral vector of the chimeric antigen receptor, which specifically comprises the following steps:

1) gene sequence of chimeric antigen receptor for the synthesis of anti-CD 123 antigen: synthesizing different single-chain antibodies (ScFv), a hinge region, a transmembrane region and an intracellular signal domain which sequentially contain a leader peptide and an anti-human anti-CD 123 antigen; the hinge region nucleic acid sequence is shown as SEQ ID NO. 14 or SEQ ID NO. 15, the transmembrane region nucleic acid sequence is shown as SEQ ID NO. 16 or SEQ ID NO. 17, and the intracellular signal domain nucleic acid sequence is shown as SEQ ID NO. 18 andor SEQ ID NO. 19 andor SEQ ID NO. 20;

2) construction of a lentiviral vector expressing a chimeric antigen receptor: designing a primer, wherein the nucleotide sequence of a forward primer is shown as SEQ ID NO.21, the nucleotide sequence of a reverse primer is shown as SEQ ID NO.22, and performing PCR amplification by taking the sequence of the chimeric antigen receptor as a template to obtain a DNA fragment;

and carrying out double digestion on the gene sequence of the DNA fragment by using restriction endonucleases NheI and SalI, simultaneously carrying out enzyme digestion on a lentiviral expression vector pCDH-EF1 by using the restriction endonucleases NheI and SalI, and then connecting the plum-cut target fragment and the lentiviral expression vector fragment by using T4 ligase to obtain the lentiviral vector for expressing the chimeric antigen receptor.

Further, after step 2), the lentiviral vector is packaged and purified.

The invention also aims to provide a lentivirus vector obtained by the preparation method.

The lentivirus vector obtained by the method has high positive expression rate, is stable in the cell culture process of patients and does not cause the reduction of the positive rate with the passage of time. Thus, the T cell infected with the lentiviral vector has a function of killing the target cell.

The invention also aims to provide a T cell infected by the lentiviral vector.

The invention also aims to provide application of the T cell in preparing a medicament for treating malignant hematological diseases. The chimeric antigen receptor of the invention can be used for adoptive cell therapy for myeloid tumors. After the chimeric antigen receptor for resisting the CD123 antigen is expressed in immune cells, the chimeric antigen receptor can effectively eliminate tumor target cells expressing the CD123 antigen, and has no toxic effect on tumor cells of negative antigens (not expressing the CD 123); and can maintain the positive rate of a Chimeric Antigen Receptor (CAR) targeting CD123 in the cell culture process of a patient, can proliferate for a long time after the stimulation of a target antigen, and can be used for the targeted therapy of tumors.

Furthermore, the cells of the hematological malignancy can express CD123. CD123, also called interleukin 3 receptor α chain (IL-3R α), is highly expressed in leukemia stem cells or leukemia naive cells, is not expressed or is low expressed in normal hematopoietic stem cells, is a leukemia-associated antigen and is a specific antigen of acute myeloid leukemia.

Further, the hematologic malignancies are acute myeloid leukemia, acute lymphocytic leukemia and B-cell chronic lymphocytic proliferative disease expressing CD 123.

The invention also aims to provide application of the polypeptide of SEQ ID NO.1 in preparing a medicament for treating malignant hematological diseases.

Further, the cells of hematological malignancies can express CD 123.

In addition, returning to the origin end of the invention, the invention also aims to provide the application of the polypeptide of SEQ ID NO.1 in preparing a vector for accurately capturing cells capable of expressing CD123 hematologic malignancies.

In addition to cell therapy, radiotherapy may also be used. The amino acid sequence of the single-chain antibody (ScFv) for resisting the CD123 antigen is shown as SEQ ID NO. 1.

The invention also aims to provide application of the amino acid sequence shown as SEQ ID NO.1 in preparing an antigen recognition domain in the CAR-T framework.

In general, after the scFv-CAR chimeric antigen receptor disclosed by the invention is expressed in immune cells, the positive rate of a CD 123-targeting Chimeric Antigen Receptor (CAR) in a patient cell culture process can be maintained, the proliferation and tumor killing capacity of CAR-T can be enhanced, and the scFv-CAR chimeric antigen receptor has no toxic or side effect on antigen-negative cells and can be used for targeted therapy of tumors.

The invention has the beneficial effects that:

1) after the chimeric antigen receptor for resisting the CD123 antigen is expressed in immune cells, the chimeric antigen receptor can effectively eliminate tumor target cells expressing the CD123 antigen, and has no toxic effect on tumor cells of negative antigens (not expressing the CD 123); and can maintain the positive rate of a Chimeric Antigen Receptor (CAR) targeting CD123 in the cell culture process of a patient, can proliferate for a long time after the stimulation of a target antigen, and can be used for the targeted therapy of tumors.

2) The chimeric antigen receptor provided by the invention is high in humanization degree through modification, and can effectively reduce the immunogenicity of the CAR and enhance the persistence and safety of the CAR-T in vivo.

3) The chimeric antigen receptor provided by the invention can be stably expressed in T lymphocytes, particularly patient-derived T lymphocytes, and can be used for preparing a medicament for treating the hematological malignancy and adoptive cell therapy for the hematological malignancy.

Drawings

FIG. 1 is a diagram of the structure of a different CD 123-CAR.

FIG. 2 is the transfection efficiency (CAR-positive rate) of the different CD123-CAR in FIG. 1 for transfection of normal donor T lymphocytes; the results show that CD123-CAR-3 transfects T cells with the worst efficiency.

FIG. 3 shows the flow test results and statistics of different CD123-CAR-T cells in FIG. 2, CD4/CD 8.

FIG. 4 shows FCM detecting the expression level of CD123 in tumor cells; wherein the THP-1-Luc highly expresses CD123, and the U937-Luc moderately expresses CD123 as positive cells; Raji-Luc low expressing CD123 as negative cells. (all cells stably transformed luciferase to allow for the detection of CAR-T killing ability).

FIG. 5 is an in vitro killing activity assay of each CD123-CAR-T cell of FIG. 3; panel A shows the killing ability of CD123-CAR-T to the target cells of FIG. 4 at 6 small aged target ratios of 5:1,1:1, respectively; panel B shows the killing ability of CD123-CAR-T on the target cells of FIG. 4 at 24 hours 5:1,1: 1; the CAR-T cell CD123-CAR-1 and CD123-CAR-2 constructed by the CD123-scFV-1 have relatively good capacity of eliminating tumor cells.

FIG. 6 is a graph of cytokine release detection after killing of tumor cells by different CAR-T cells; collecting supernatant of each group of killed CD123-CAR-T in the experiment of FIG. 5 and killing for 24 hours, and detecting secretion of IFN gamma and IL-2 by ELISA, wherein the A is secretion detection of IFN gamma, and the B is secretion of IL-2; the results show that consistent with the results of in vitro killing, the CD123-CAR-1 and CD123-CAR-2 have higher factor secretion, while the CD123-CAR-3 and CD123-CAR-4 have higher factor release for killing negative cells, and the high non-specific killing has potential risk when being used for in vivo experiments.

FIG. 7 is the proliferative capacity of CD123-CAR-T cells under target cell stimulation without the addition of exogenous cytokines; the CAR-T cell constructed by using the CD123-scFV-1 has the strongest proliferation capacity.

FIG. 8 is a CD123-CAR-T cell killing activity assay in vitro.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental procedures, in the preferred embodiments, which do not specify specific conditions, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers. The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1 construction of chimeric antigen receptor viruses

To screen for better CAR combinations requires the construction of the following 6 chimeric antigen receptors, respectively:

CD123-CAR-1，CD123-CAR-2，CD123-CAR-3，CD123-CAR-4，CD123-CAR-5，CD123-CAR-6。

1. the gene sequence of the chimeric antigen receptor for targeting CD123 containing different scFVs is synthesized to sequentially contain a leader peptide (also called signal peptide) (abbreviated as LP), different single-chain antibodies ScFv against human CD123 antigen, an IgG4 hinge region (abbreviated as IgG4), a CD28 transmembrane region or a CD8 transmembrane region (abbreviated as TM), and the structure of the chimeric antigen receptor is shown in FIG. 1.

2. Construction of Lentiviral vectors expressing chimeric antigen receptors

1) The following primers were designed and synthesized by the biosciences of tsingry, tokyo, the specific primers were as follows:

primer 1: 5' -aggctagcatgggatggagctgtatcat-3' (SEQ ID NO.21), the NheI restriction endonuclease site is underlined;

primer 2: 5' -gattgtcgacttagcgagggggcagggcctgcatgtga-3' (SEQ ID NO.22), the SalI restriction enzyme site is underlined.

Then taking the sequences shown in SEQ ID NO.21 and SEQ ID NO.22 as primers, taking each synthesized chimeric antigen receptor sequence as a template to carry out PCR amplification, adding the sample according to the instruction of KOD FXNEO DNA polymerase (purchased from TOYOBO company), and carrying out pre-denaturation at 95 ℃ for 5min under the PCR reaction condition; denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 68 ℃ for 30s, for 30 cycles. The amplification products were identified on a 1% agarose gel. The results showed that about 2000bp DNA fragment was obtained by amplification, and then the DNA fragment was recovered by using a recovery kit (Promega corporation) according to the protocol to obtain chimeric antigen receptor, and the recovered DNA fragment was sequenced by Nanjing Kingsry Biotech.

The cloned gene sequence encoding the chimeric antigen receptor was digested simultaneously with restriction enzymes NheI and SalI (from Thermo) and the lentiviral expression vector pCDH-EF1 (from addendum Plasmid #72266) as described in the specification. The enzyme digestion product is separated by agarose gel electrophoresis, DNA fragment is recovered by an agarose gel DNA fragment recovery kit, and then the target fragment and the vector fragment are connected by T4 ligase (purchased from Promega company) to obtain the lentiviral vector expressing the chimeric antigen receptor, which is named as Lv-scFv (CEA). Mu.l of the lentiviral vector was transformed into Escherichia coli TOP10, cultured at 30 ℃ for 16h, and then the monoclonal was picked up, cultured at 30 ℃ for 12h, and then the plasmid was extracted using a plasmid extraction kit (Invitrogen), the specific method being described in the specification.

The CD123-CAR-1, CD123-CAR-2, CD123-CAR-3, CD123-CAR-4, CD123-CAR-5, CD123-CAR-6 lentiviral vectors were constructed as above, respectively;

3. packaging of lentiviruses

The method for packaging lentivirus of this example employs calcium phosphate method, which comprises culturing 293T cells in DMEM medium containing 10% (wt) FBS to a preferred state, and then culturing 293T cells at 1 × 10%⁵/cm²The cell confluency is transferred to 1 culture bottle with 75cm2 to be cultured for 22h, and the cell confluency is ensured to be 70-80% during transfection; then, replacing the liquid with a preheated DMEM medium containing 2% (wt) FBS, and culturing for 2h for later use; 680 μ l ddH₂Adding 20 mu g of lentiviral vector, 20 mu g of pMDLg/pRRE plasmid, 20 mu g of pRSV-Rev, 10 mu g of pMD2.G plasmid and 100 mu l of 2.5mM CaCl2 into a 15ml centrifuge tube, uniformly mixing, dropwise adding 2 × HBS into the mixed solution by using a pipette after uniformly mixing, simultaneously blowing and uniformly mixing by using a 10ml pipette, standing at room temperature for 15min after uniformly mixing, dropwise adding the mixed solution into the prepared cells, continuously culturing for 12-16h, replacing the culture solution by a DMEM culture medium containing 10% FBS (wt), continuously culturing for 48h and 72h, collecting cell supernatants and purifying the viruses.

4. Lentivirus purification

Collecting the virus supernatant in a 50ml centrifuge tube, and centrifuging for 10min at 3000 r/min; then filtering with a filter membrane, and centrifuging the filtrate to a new 50ml centrifuge tube; then adding 50% by mass of PEG6000 and 4M NaCl respectively according to the amount of virus supernatant, then fixing the volume with medical saline until the final concentration of PEG6000 is 8.5% and the final concentration of NaCl is 0.3M, standing in a refrigerator at 4 ℃ for 90min after fixing and dissolving, and reversing and mixing evenly 30 min/time; then centrifuging at 4 deg.C and 5000r/min for 30min, discarding supernatant, resuspending virus with DMEM medium, subpackaging with 1.5ml EP tube, 40 μ l per tube, and storing at-80 deg.C for use. The prepared viruses were named CD123-CAR-1, CD123-CAR-2, CD123-CAR-3, CD123-CAR-4, CD123-CAR-5, CD123-CAR-6, respectively.

5. Lentivirus titer determination

1) Viral infection of 293T cells

Before infection, 293T cells are paved in a 24-well plate, 1 mu l of purified virus is taken, diluted 10 times by medical normal saline, 1 mu l of Polybrene (Polybrene) solution is added into each well of the cells, 1 mu l and 9 mu l of diluted virus solution are respectively added into the 293T cells, the solution is replaced by DMEM medium containing 10% FBS (wt) after 24h, and the cells are collected and extracted by centrifugation for 5min at 1000r/min after 72h of infection.

2) Extraction of genome

The genome extraction Kit was QIAamp DNA Blood Mini Kit purchased from Qiagen (cat # 511004), and was used according to the Kit instructions.

3) qRT-PCR determination of viral titre

The reaction system is as follows, Premix Ex TaqTM II (2 ×) 10. mu.l, upstream primer (GAGup) 1. mu.l, downstream primer (GAGdn) 1. mu.l, extracted genome 1. mu.l, RNase-Free dH₂O7 μ l, at least 3 replicate wells per sample, standard. Amplification was then performed as follows: pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, after the reaction is finished, analyzing data by using analysis software, and calculating the virus titer according to a standard curve.

Example 2 detection of transfection efficiency of chimeric antigen receptor in T cells

1) Isolation of human peripheral blood mononuclear cells

Collecting about 60ml of peripheral blood by using a blood collection tube added with anticoagulant, subpackaging the collected peripheral blood into 30ml of 50ml centrifuge tubes respectively, and adding 7.5ml of hydroxyethyl starch for dilution; naturally settling for about 30min at room temperature (18-25 ℃), collecting upper layer plasma, and centrifuging the collected upper layer plasma for 15min at 1400 rpm/min; then, resuspending the precipitate with physiological saline, adding the precipitate to lymphocyte separation liquid according to the volume ratio of 1:1, performing gradient centrifugation at 400g/min, setting the centrifuge deceleration as 1, and centrifuging for 20 min; after centrifugation, the centrifuging tube divide into from top to bottom: a first layer: a plasma layer; a second layer: a layer of annular opalescent lymphocytes; and a third layer: a transparent separation liquid layer; a fourth layer: a layer of red blood cells; and (3) taking the second white lymphocyte layer, washing the second white lymphocyte layer for 2 times by using normal saline, centrifuging the second white lymphocyte layer for 10min at 400g/min for the first time and 1100rpm/min for the second time for 5min, re-suspending the cells by using the normal saline, and adding an RPMI1640 complete culture medium containing 10% FBS for culture to obtain the human peripheral blood mononuclear cells.

2) Lentiviral vector infection of T lymphocytes

Culturing newly prepared mononuclear cell PBMC with RPMI1640 complete culture medium containing 10% fetal calf serum, and adding anti-CD 3 monoclonal antibody for activation on day 1; the first 3 days of lentivirus infection; adding a lentiviral vector corresponding to 5MOI, and taking uninfected T lymphocytes as a blank control; after 24h, the medium was replaced with RPMI1640 complete medium containing 500IU/ml recombinant human IL-2, and the culture was continued for 10-20 days. The results are shown in FIG. 2, and several CAR structures can well transfect T lymphocytes.

Example 3 detection of the Positive Rate of CAR molecules and the ratio of CD4 to CD8

CD123-CAR-T cells cultured for 11 days at 300g/min were centrifuged for 5min, the supernatant was discarded and resuspended in 1ml of PBS, the procedure was repeated twice, 100ul of PBS cells were added with flow detection antibodies labeled CD3, CD4, CD8(BD Co.) and Protein-L (Beijing Yiqianshengzhou Co.) and incubated for 30 minutes at four degrees, PBS was washed 3 times, mouse anti-Protein-L antibody labeled with AF647 (Nanjing Kingjinsry Co.) was added and washed twice at four degrees for 30 minutes with PBS, and 200ul of PBS-labeled cells were added after the supernatant was discarded and then the flow detection results were obtained as shown in Table 1 and FIG. 3.

TABLE 1 flow assay results and statistics for different CD123-CAR-T cells CD4/CD8

CAR name	CD4(％)	CD8(％)	PL(％)(10d)	Cell viability (%)
					CD123-CAR-1	7.7	82.9	76.89	94
CD123-CAR-2	4.0	80.8	65.09	91
					CD123-CAR-3	10.3	80.1	43.68	90
CD123-CAR-4	4.2	85.5	53.68	90

Example 4FCM detection of expression levels of CD123 in tumor cells

1) Target cell Luc construction

The method comprises the following steps of transfecting a luciferase-expressing lentiviral vector into a tumor target cell to be tested by using an in vitro transfection mode.

2) Construction of exogenous high-expression CD123 tumor cell strain

In order to better verify the killing capacity of CD123-CAR-T cells with different combinations, tumor cells which do not express CD123 are used for constructing cells with high expression of CD123 through an exogenous transfection mode, and as a result, as shown in FIG. 4, two strains of cells with high expression of CD123 both meet the high expression requirement, and cell experiments can be carried out.

3) Detection of CD123 positive rate of different tumor target cells

Tumor cells were centrifuged at 300g/min for 5min, the supernatant was discarded and resuspended in 1ml PBS, the procedure was repeated twice, 100ul PBS cells were incubated for 30min at four degrees with CD123 labeled flow detection antibody (purchased from BD), and PBS washed twice and resuspended in 200ul PBS for detection on the upper machine. As a result, the expression of CD123 on the cell surfaces of Raji-Luc, U937-Luc and THP-1-Luc was detected respectively as shown in FIG. 4, and the results showed that Raji-Luc does not express CD123, U937-Luc moderately expresses CD123 (56.73% expression), and THP-1-Luc highly expresses CD123 (99.85% expression).

Example 5 detection of respective CD123-CAR-T cell killing Activity in vitro

The killing ability of chimeric antigen receptors of different scFVs to target cells is achieved by an ACEA xCELLigence RTCAM instrument and a luciferase analysis method respectively.

1) Luciferase method: and (2) laying a 96-well plate, wherein the tumor cells (THP-1, U937)1 x 10^4 expressing the CD123 marked with the firefly luciferase are laid per well, target cells (tumor cells) are laid according to different effect-target ratios (1:2, 1:1, 5:1 and 10:1), the target cells are placed in a cell culture box for 37 degrees and are respectively cultured for 6 hours, and the killing capacity of the CD123-CAR on the target cells is indirectly detected by detecting the quantity of the residual target cells through detecting the fluorescence degree of the luciferase after 24 hours. The results are shown in FIG. 5: the CD123-CAR-1 and CD123-CAR-2 have high killing activity and high specificity on tumor cells, and only kill tumor cells expressing CD123, while few tumor cells with negative CD123 are killed. Table 2 is the statistics of the killing results of fig. 5.

Table 2: percent killing of CD123-CAR-T cells on CD 123-positive tumor cells at different time periods and different effective target ratios

2) ACEA xCELLigence RTCA MP Instrument: the experimental steps are carried out according to the instruction of the instrument; target cells (constructed CD123 expressing tumor cells and negative control cells) were plated at 2-5 x 10 a 4 per well in a 96-well plate equipped with the instrument the first day, the tumor cells attached to the bottoms of the wells are recorded every 15 minutes by taking the electrical resistance index as data, corresponding CAR-T cells are paved into each well according to a pre-designed effective target ratio after 24 hours, the electrical resistance index of the CAR-T cells paved into each well after 24 hours is recorded, supernatants of each group are collected after 24 hours to be subjected to enzyme-linked immunosorbent assay (ELISA) detection, and the electrical resistance index analysis result of each group is analyzed by an instrument, wherein the CAR-T cell killing rate is equal to baseline electrical resistance index-real-time electrical resistance index, the specific result analysis is that 1 represents an initial cell index, the cell index is higher than 1, such as 2 represents 2 times of tumor cell proliferation, and the cell index is lower than 1, such as 0.3, such as 70% of tumor cell killing; if the cell indexes are both higher than 1 but lower than the T-con and medium group indexes, the CAR has an inhibitory effect on the proliferation of tumor cells but cannot well eliminate the tumor cells. The result is shown in figure 8, the chimeric antigen receptor constructed by CD123-scFV1 as the antigen recognition area has better specificity and high activity of killing CD123 positive tumor cells, and the combination of the preferred CD123-CAR-5, namely CD123-scFV-1-CD8 hinge-CD28TM-CD28-CD137-CD3 chimeric antigen and the receptor has higher effectiveness and safety. The results are shown in Table 3 below:

table 3: killing capacity statistics of CAR-transfected T cells of different scFV and hinge (hinge) structure combinations

Example 6: cytokine detection

1) Cytokine detection was performed by the method of Elisa using a kit of the company BD.

2) And (3) diluting the standard: the sample dilution method is described in the product specification.

3) And standard product holes are formed in the enzyme-labeled coating plate, 50 mu l of standard products with different concentrations are sequentially added, and each concentration is provided with 2 parallel holes.

4) Sample adding: respectively arranging blank holes (the blank reference hole is not added with a sample, an enzyme labeling reagent and a biotin labeled antibody, and the rest steps are operated in the same way) and sample holes to be detected; 40 mul of sample is firstly added into the sample hole to be detected on the enzyme-labeled coated plate, and then 10 mul of biotin-labeled antibody is added. Adding a sample to the bottom of the hole of the enzyme label plate, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;

5) and (3) incubation: sealing the plate with a sealing plate film, and then incubating for 30 minutes at 37 ℃;

6) preparing liquid: diluting 30 times of the concentrated washing liquid with 30 times of distilled water for later use;

7) washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 30 seconds, then discarding, repeating the steps for 5 times, and patting dry;

8) adding an enzyme: adding 50 mul of enzyme-labeled reagent into each hole except for blank holes;

9) and (3) incubation: the operation is the same as 3;

10) washing: the operation is the same as 5;

11) and (4) terminating: adding 50 mul of stop solution into each hole to stop the reaction;

12) and (3) determination: the absorbance (OD value) of each well was measured sequentially at a wavelength of 450nm with blank air conditioning of zero.

The results are shown in FIG. 6, wherein CD123-CAR-1 and CD123-CAR-2 have high secretion of IFN-gamma and IL-2 for killing tumor cells expressing CD123, and have good specificity for negative cells. The results are shown in Table 4:

table 4: cytokine secretion following killing of tumor cells by different combinations of CAR-transfected T cells

	CD123-CAR-1	CD123-CAR-2	CD123-CAR-3	CD123-CAR-4	con
						IFN-γ	5550.827	3772.863	1759.522	1497.438	289.7076
IL-2	1025.895	1706.253	1284.273	366.5646	484.0675

Example 7 proliferative Capacity of CD123-CAR-T cells under target cell stimulation without addition of exogenous cytokines

To mimic the in vivo environment, increasing fold of CD123-CAR-T cells were observed by stimulating CD123-CAR-T cells by target cells without the addition of exogenous cytokines. Normally cultured CD123-CAR-T cells are 300g/min, centrifuged for 5min, the supernatant is discarded and resuspended in 2ml PBS, the centrifugation step is repeated for 2 times, the cells are diluted to 1 x 10^6/ml by fresh culture medium without exogenous cytokines, 24-well plate target cells are plated at 2 x 10^5/500ul per well, the effective target ratio is 1:1, stimulation is performed once every 7 days, and cell counting is performed 3-4 days after stimulation, and the total number of cells is counted to draw a proliferation curve on 7 days. As shown in FIG. 7, CD123-CAR-1 has the strongest proliferation ability after activation by CD 123-positive tumor cells in the absence of exogenous cytokine stimulation. Table 5 statistics of the proliferation fold of CAR-T cells at 14 days of stimulation

Table 5: CAR-T cell proliferation fold after stimulation of CAR-T cells by CD123 positive tumor cells

	CD123-CAR-1	CD123-CAR-2	CD123-CAR-3	CD123-CAR-4	con
						Fold of proliferation	5.72	2.83	1.52	1.56	1.56

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

<110> Chongqing precision Biotechnology Co., Ltd

<120> anti-CD 123 single-chain antibody, chimeric antigen receptor combined with same and application

<160>22

<170>PatentIn version 3.3

<210>1

<211>240

<212>PRT

<213>Artificial

<220>

<223>CD123-scFv-1

<400>1

Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr LeuAla Ala Ser Pro

5 10 15

Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser

20 25 30

Lys Asp Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys

35 40 45

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

50 55 60

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

65 70 75

Ser Ser Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln

80 85 90

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

95 100 105

Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu

110 115 120

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

125 130 135

Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser

140 145 150

Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln Arg

155 160 165

Pro Asp Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp

170 175 180

Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu

185 190 195

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

200 205 210

Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Asn

215 220 225

Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

230 235 240

<210>2

<211>247

<212>PRT

<213>Artificial

<220>

<223>CD123-scFV-2

<400>2

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

5 10 15

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

20 25 30

Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Ser Phe

35 40 45

Lys Trp Met Gly Trp Ile Asn Thr Tyr Thr Gly Glu Ser Thr Tyr

50 55 60

Ser Ala Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser

65 70 75

Ala Ser Thr Ala Tyr Leu His Ile Asn Asp Leu Lys Asn Glu Asp

80 85 90

Thr Ala Thr Tyr Phe Cys Ala Arg Ser Gly Gly Tyr Asp Pro Met

95 100 105

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Ser

110 115 120

Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

125 130 135

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

140 145 150

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

155 160 165

Asp Asn Tyr Gly Asn Thr Phe Met His Trp Tyr Gln Gln Lys Pro

170 175 180

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu

185 190 195

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

200 205 210

Phe Thr Leu Thr Ile Asn Pro Val Glu Ala Asp Asp Val Ala Thr

215 220 225

Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Pro Thr Phe Gly Ala

230 235 240

Gly Thr Lys Leu Glu Leu Lys

245

<210>3

<211>229

<212>PRT

<213>

<220>

<223> IgG4 hinge

<400>3

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

5 10 15

Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

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

35 40 45

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

50 55 60

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

65 70 75

Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

80 85 90

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

95 100 105

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

110 115 120

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

125 130 135

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

140 145 150

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

155 160 165

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

170 175 180

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

185 190 195

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met

200 205 210

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

215 220 225

Ser Leu Gly Lys

<210>4

<211>47

<212>PRT

<213>

<220>

<223> CD8 hinge

<400>4

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

5 10 15

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

20 25 30

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

35 40 45

Cys Asp

<210>5

<211>24

<212>PRT

<213>

<220>

<223> CD8 transmembrane region

<400>5

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

5 10 15

Leu Ser Leu Val Ile Thr Leu Tyr Cys

20

<210>6

<211>27

<212>PRT

<213>

<220>

<223> CD28 transmembrane region

<400>6

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

5 10 15

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210>7

<211>68

<212>PRT

<213>

<220>

<223> CD28 costimulatory signals

<400>7

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser

5 10 15

Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys

20 25 30

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

35 40 45

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

50 55 60

Arg Asp Phe Ala Ala Tyr Arg Ser

65

<210>8

<211>44

<212>PRT

<213>

<220>

<223> CD137 co-stimulation signals

<400>8

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

5 10 15

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

20 25 30

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

35 40

<210>9

<211>111

<212>PRT

<213>

<220>

<223> CD3 costimulatory signals

<400>9

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

5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75

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

80 85 90

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

95 100 105

Gln Ala Leu Pro Pro Arg

110

<210>10

<211>18

<212>PRT

<213>Artificial

<220>

<223>Linker

<400>10

Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

5 10 15

Thr Lys Gly

<210>11

<211>54

<212>DNA

<213>Artificial

<220>

<223>Linker

<400>11

ggcagcacca gcggcagcgg caaaccgggc agcggcgaag gcagcaccaa aggc 54

<210>12

<211>720

<212>DNA

<213>Artificial

<220>

<223>CD123-scFv-1

<400>12

gatgtgcaga ttacccagag cccgagctat ctggcggcga gcccgggcga aaccattacc 60

attaactgcc gcgcgagcaa aagcattagc aaagatctgg cgtggtatca ggaaaaaccg 120

ggcaaaacca acaaactgct gatttatagc ggcagcaccc tgcagagcgg cattccgagc 180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctggaaccg 240

gaagattttg cgatgtatta ttgccagcag cataacaaat atccgtatac ctttggcggc 300

ggcaccaaac tggaaattaa ggcagcacca gcggcagcgg caaaccgggc agcggcgaag 360

gcagcaccaa aggcacaggt gcagctgcag cagccgggcg cggaactggt gcgcccgggc 420

gcgagcgtga aactgagctg caaagcgagc ggctatacct ttaccagcta ttggatgaac 480

tgggtgaaac agcgcccgga tcagggcctg gaatggattg gccgcattga tccgtatgat 540

agcgaaaccc attataacca gaaatttaaa gataaagcga ttctgaccgt ggataaaagc 600

agcagcaccg cgtatatgca gctgagcagc ctgaccagcg aagatagcgc ggtgtattat 660

tgcgcgcgcg gcaactggga tgattattgg ggccagggca ccaccctgac cgtgagcagc 720

<210>13

<211>741

<212>DNA

<213>Artificial

<220>

<223>CD123-scFV-2

<400>13

gatattgtgc tgacccagag cccggcgagc ctggcggtga gcctgggcca gcgcgcgacc 60

attagctgcc gcgcgagcga aagcgtggat aactatggca acacctttat gcattggtat 120

cagcagaaac cgggccagcc gccgaaactg ctgatttatc gcgcgagcaa cctggaaagc 180

ggcattccgg cgcgctttag cggcagcggc agccgcaccg attttaccct gaccattaac 240

ccggtggaag cggatgatgt ggcgacctat tattgccagc agagcaacga agatccgccg 300

acctttggcg cgggcaccaa actggaactg aaaggcagca ccagcggcag cggcaaaccg 360

ggcagcggcg aaggcagcac caaaggccag attcagctgg tgcagagcgg cccggaactg 420

aaaaaaccgg gcgaaaccgt gaaaattagc tgcaaagcga gcggctatat ttttaccaac 480

tatggcatga actgggtgaa acaggcgccg ggcaaaagct ttaaatggat gggctggatt 540

aacacctata ccggcgaaag cacctatagc gcggatttta aaggccgctt tgcgtttagc 600

ctggaaacca gcgcgagcac cgcgtatctg catattaacg atctgaaaaa cgaagatacc 660

gcgacctatt tttgcgcgcg cagcggcggc tatgatccga tggattattg gggccagggc 720

accagcgtga ccgtgagcag c 741

<210>14

<211>687

<212>DNA

<213>

<220>

<223> IgG4 hinge

<400>14

gaaagcaaat atggcccgcc gtgcccgccg tgcccggcgc cggaatttct gggcggcccg 60

agcgtgtttc tgtttccgcc gaaaccgaaa gataccctga tgattagccg caccccggaa 120

gtgacctgcg tggtggtgga tgtgagccag gaagatccgg aagtgcagtt taactggtat 180

gtggatggcg tggaagtgca taacgcgaaa accaaaccgc gcgaagaaca gtttaacagc 240

acctatcgcg tggtgagcgt gctgaccgtg ctgcatcagg attggctgaa cggcaaagaa 300

tataaatgca aagtgagcaa caaaggcctg ccgagcagca ttgaaaaaac cattagcaaa 360

gcgaaaggcc agccgcgcga accgcaggtg tataccctgc cgccgagcca ggaagaaatg 420

accaaaaacc aggtgagcct gacctgcctg gtgaaaggct tttatccgag cgatattgcg 480

gtggaatggg aaagcaacgg ccagccggaa aacaactata aaaccacccc gccggtgctg 540

gatagcgatg gcagcttttt tctgtatagc cgcctgaccg tggataaaag ccgctggcag 600

gaaggcaacg tgtttagctg cagcgtgatg catgaagcgc tgcataacca ttatacccag 660

aaaagcctga gcctgagcct gggcaaa 687

<210>15

<211>141

<212>DNA

<213>

<220>

<223> CD8 hinge

<400>15

aagcccacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 60

cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 120

gggctggact tcgcctgcga c 141

<210>16

<211>72

<212>DNA

<213>

<220>

<223> CD8 transmembrane region

<400>16

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210>17

<211>87

<212>DNA

<213>

<220>

<223> CD28 transmembrane region

<400>17

gaattcttct gggtgctggt cgtggtgggt ggcgtgctgg cctgctacag cctgctggtg 60

acagtggcct tcatcatctt ttgggtg 87

<210>18

<211>204

<212>DNA

<213>

<220>

<223> CD28 costimulatory signals

<400>18

ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60

gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120

aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180

cgcgacttcg cagcctatcg ctcc 204

<210>19

<211>132

<212>DNA

<213>

<220>

<223> CD137 co-stimulation signals

<400>19

gttaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 60

caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 120

tgtgaactga ga 132

<210>20

<211>333

<212>DNA

<213>

<220>

<223> CD3 costimulatory signals

<400>20

gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 60

aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 120

gaccctgaga tggggggaaa gccgagaagg aagaaccctc aggaaggcct gtacaatgaa 180

ctgcagaaag ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg 240

aggggcaagg ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac 300

gacgcccttc acatgcaggc cctgccccct cgc 333

<210>21

<211>28

<212>DNA

<213>Artificial

<220>

<223> Forward primer

<400>21

aggctagcat gggatggagc tgtatcat 28

<210>22

<211>38

<212>DNA

<213>Artificial

<220>

<223> reverse primer

<400>22

gattgtcgac ttagcgaggg ggcagggcct gcatgtga 38

Claims

1. A chimeric antigen receptor for CD123 antigen, which is characterized in that the chimeric antigen receptor is formed by sequentially connecting a polypeptide for recognizing CD123 antigen, a hinge region, a transmembrane region and an intracellular signal region; the amino acid sequence of the chimeric antigen receptor for resisting the CD123 antigen is formed by sequentially connecting SEQ ID NO.1, SEQ ID NO. 3, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 in series, or formed by sequentially connecting SEQ ID NO.1, SEQ ID NO. 4, SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9 in series.

2. A chimeric antigen receptor lentiviral vector comprising the anti-CD 123 antigen of claim 1.

3. A T cell infected with the lentiviral vector of claim 2.