CN108409840A

CN108409840A - The Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof and application

Info

Publication number: CN108409840A
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 Biological Technology Co Ltd
Current assignee: Chongqing Precision Biological Technology Co Ltd
Priority date: 2017-05-18
Filing date: 2018-03-14
Publication date: 2018-08-17
Anticipated expiration: 2038-03-14
Also published as: CN108409840B

Abstract

The invention belongs to genetic engineering fields, and in particular to a kind of Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof and application.The amino acid sequence such as SEQ ID NO of the polypeptide of the identification CD123 antigens of the present invention:Shown in 1.The Chimeric antigen receptor of anti-CD123 antigens, by identifying the polypeptide (ScFv) of CD123 antigens, hinge area, transmembrane region and intracellular signal domain are sequentially connected composition.After the Chimeric antigen receptor of anti-CD123 antigens is expressed in immunocyte, the tumour target cell of expression CD123 antigens not only can be effectively removed, and does not have toxic effect to the tumour cell of Negative antigens (not expressing CD123)；And it is able to maintain that Chimeric antigen receptor (the chimeric antigen receptor of targeting CD123, CAR) the positive rate in patient's cell cultivation process, it can be proliferated for a long time after target antigen stimulation, can be used in the targeted therapy of tumour.

Description

The Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof and application

Technical field

The invention belongs to genetic engineering fields, are related to the Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof and answer With further relating to slow virus carrier and the application of the Chimeric antigen receptor of anti-CD123 antigens.

Background technology

Adoptive cellular treatment (adoptive cell therapy, ACT) is one kind of biological therapy technology, to exempting from self Epidemic disease cell (mainly T cell) carries out amplification in vitro, is then fed back to tumor patient to reach the method for therapeutic purposes, quilt The 4th kind of therapeutic modality being considered after operation, Radiotherapy chemotherapy, by extensive use in clinical treatment.Adoptive cellular treatment is wide General application mainly：Lymphatic circulation (lymphokine activated killer cell, LAK) with IL-2 combination therapy late malignant tumours achieve certain curative effect；Tumor infiltrating lymphocyte (tumor infiltrating Lymphocytes, TIL) treatment metastasis melanin tumor achieves preferable effect in clinical test；Cytokine induction kills Hinder cell (cytokine induced killer cell, CIK), it is domestic at present to carry out more clinical test, to liver cancer, lung The tumours such as cancer achieve significant effect.But above-mentioned three kinds of therapies are both needed to activating T cell at present, T cell activation needs I molecule of TCR-CD3 and MHC- of two kinds of activation signals, i.e. T cell surface is combined into the first signal of activation, determines T cell pair The killing activity of tumour cell；The costimulatory molecules on T cell surface are combined into the second signal of activation with respective ligand, determine T Cell Proliferation.But tumour cell, tumor microenvironment can lower MHC, ligand molecular, to inhibit T cell to kill tumour cell Wound activity.Therefore it needs that genetic modification can be carried out, there are mainly two types of modes：Gene transfer TCR (T cell receptor, TCR) With Chimeric antigen receptor (chimeric antigen receptor, CAR).

Chimeric antigen receptor (chimeric antigen receptor) abbreviation CAR, be simulate TCR functions it is artificial by Body is sequentially connected and is formed by antigen recognition domain, hinge area and transmembrane region and intracellular signal domain, and intracellular signal domain is usually CD3 ζ chains Or FcR γ, or be connected with one or more costimulatory molecules, such as 4-1BB (CD137), CD28, ICOS (CD278).Tumour cell When the antigen (receptor) on surface is combined with the antibody (ligand) of Chimeric antigen receptor, signal is transmitted by hinge area and transmembrane region To intracellular, intracellular signal domain converts the signal to activation signals, activation effect cell, and effector cell's proliferation generates cell factor To killing tumor cell.Although CAR-T treatments tumour has shown certain tumor clearance ability, especially CD19- Treatments of the CAR in terms of hematological system tumor made breakthrough progress, but CAR-T treatments are still asked there is also a series of Topic is still needed to further explore and be solved：Proliferative capacity is poor in vivo for 1, CAR-T cell；2, due to being that artificial receptors still have centainly Security risk.

Acute myeloid leukaemia (Acute myeloid leukemia, AML) is the pernicious disease of medullary system hematopoietic stem/progenitor Disease, using suddenly heavy, the prognosis as the main feature majority of cases state of an illness of original and inmature marrow cell paraplasm in marrow and peripheral blood Dangerous, being such as not treated in time often can threat to life；Although conventional induction chemother can alleviate AML but can not finally prevent The recurrence of AML and the high death rate of patients with recurrent.However the conventional therapy of AML does not have change for 50 years, is badly in need of finding new Change.

CD123 is also known as interleukin-13 receptor alpha chain (IL-3R α) height and is expressed in leukemic stem cells or leukaemia juvenile cell, Do not expressed or low expression in normal haematopoetic, be leukemia-associated antigen be also acute myeloid leukaemia specificity it is anti- It is former.The appearance of CD123 targets is the new breakthrough of AML treatments；Since CD123 is expressed in AML high, theoretically using CD123 as target Immunization therapy there is safer and more effective therapeutic effect, it is external also to have carried out using CD123 as the targeted drug of target Clinical test, but effect is very limited and still has the generation of safety issue；Therefore searching specificity is needed more preferably more to have The new treatment of the targeting CD123 of effect, is carrying out although having the CAR of some target spots testing at present, for treating AML CAR it is relatively fewer, further reforming test is still needed in the stability of CAR performances and safety, how to be selected more stable Suitable scFV (single-chain antibody) is also unsolved critical issue in CAR researchs, thus it is a kind of it is safer and more effective with The research and development preparation that CD123 is the CD123-CAR of target spot is extremely necessary.

At present for the medullary system tumour of expression CD123 there are no therapeutic scheme well, antigen Chimerical receptor is in T cell The stability of transfection also shows CD123-scFV combination antigen Chimerical receptors to embedding also without good solution without report Close the stability of antigen receptor cell and the influence to CD123 medullary system tumor efficiencies.

Invention content

In view of this, one of the objects of the present invention is to provide a kind of inosculating antibodies of anti-CD123 single-chain antibodies and combinations thereof Original receptor and application, what the Chimeric antigen receptor comprising CD123-scFV of the invention can be more stable is expressed in T lymphocytes, The tumour target cell that expression CD123 antigens can preferably be removed, to the no poison of the tumour cell of Negative antigens (not expressing CD123) Property effect.

To achieve the above object, the technical scheme is that：

Identify the polypeptide of CD123 antigens, the amino acid sequence such as SEQ ID NO of the polypeptide:Shown in 1.

The second object of the present invention is to provide a kind of above-mentioned polypeptide and is preparing answering for the single-chain antibody of anti-CD123 antigens With.

The present invention also aims to provide a kind of Chimeric antigen receptor of anti-CD123 antigens, by identifying CD123 antigens Polypeptide (ScFv), hinge area, transmembrane region and intracellular signal domain are sequentially connected composition；The ammonia of the polypeptide of the identification CD123 antigens Base acid sequence contains above-mentioned SEQ ID NO:Polypeptide described in 1.

Single-chain antibody (Single-chain variable fragment (scFv)) is variable region (VH and the VL by antibody Area) it is formed by connecting by the small peptide (Linker) of 15-20 amino acid, scFv can preferably retain its affine work to antigen Property, and have the characteristics that molecular weight is small, penetration power is strong and antigenicity is weak.

The mode of Linker connection single-chain antibody heavy chains and light chain is VH-Linker-VL or VL-Linker-VH two Kind mode.The selection of Linker may influence the stability and concentration class of single-chain antibody.Preceding most common Linker be with The GGGGS that propylhomoserin (Gly) and serine (Ser) connection are formed is unit, and one or more units connect to be formed, and inventor passes through The single-chain antibody (ScFv) of anti-CD123 antigens described in early period exploration discovery this patent connects energy with VL-Linker-VH connection types The enough stability for preferably keeping single-chain antibody.The Linker amino acid sequences such as SEQ ID NO.31 or nucleic acid sequence are such as SEQ ID NO.32。

The Chimeric antigen receptor needs to go beyond two technology barriers, first, finding more stable effective identification CD123 The scFV of antigen.

The antibody of identification carcinomebryonic antigen how is transformed, enables the affine activity for preferably retaining it to antigen, and Best identification CD123 Chimeric antigen receptors are combined as, are a problems.Inventor team is not having more effective technique prompt In the case of build CAR (Chimeric antigen receptor) using different combination by a series of reforming tests and screened, Inventor has selected two single-chain antibodies of CD123-scFV-1 and CD123-scFV-2, amino acid sequence such as SEQ ID NO:1 or SEQ ID NO:Shown in 2, nucleotide sequence such as SEQ ID NO:12 or SEQ ID NO:The research of CAR combinations is carried out shown in 13.

In the protection Chimeric antigen receptor, we also eliminated the chimeric antigen under three groups of other combination thinkings by Body, and this several groups only protected by the present invention, have unexpected technique effect.

The method for screening best Chimeric antigen receptor combination, it is constant to be to maintain antigen recognition sequence, to hinge area, cross-film Area and packet intracellular signaling domain carry out random combine；Or it is to maintain hinge area, transmembrane region and packet intracellular signaling domain are constant, and screening is not Same antigen recognition sequence (scFV).There are randomnesss for the result of method, and only in the case where being arranged to work as, affinity is It is possible that suitable with the affinity of original mouse antibodies.

The combination of the CAR of this part protection can play the T for stablizing and being expressed in patient source after test Lymphocyte, and with the ability for preferably removing tumour cell, treated for the adoptive cellular for malignant hematologic disease.

Further, the amino acid sequence of hinge area such as SEQ ID NO:3 or SEQ ID NO:Shown in 4.

Further, the amino acid sequence of the transmembrane region such as SEQ ID NO:5 or SEQ ID NO:Shown in 6.

Further, the intracellular signal domain is CD28 and/or CD137 and/or CD3 ζ chains；The amino acid sequence of the CD28 Such as SEQ ID NO:The amino acid sequence of 7, the CD137 such as SEQ ID NO:8, the amino acid sequence such as SEQ of the CD3 ζ chains ID NO:9。

When the antigen (receptor) of tumor cell surface is combined with the antibody (ligand) of the Chimeric antigen receptor, pass through hinge Sequence and transmembrane region transmit signals to intracellular, and intracellular signal domain converts the signal to activation signals, activation effect cell, effect Cell Proliferation generates cell factor to killing tumor cell.Chimeric antigen receptor has more advantage compared with TCR transformations：(1) special Property：Antibody (ligand) specific recognition antigen (receptor)；(2) efficient：Be not in transgenosis TCR and patient's endogenous TCR Mispairing occurs；(3) non-MHC- I is restricted：It need not be combined with I molecules of MHC-, tumour cell, tumor microenvironment can be overcome to lower Immunologic escape caused by I molecules of MHC-；(4) antigen range of choice is wide：Antigen can be carbohydrate, lipid, albumen.

Further, the amino acid sequence of the Chimeric antigen receptor is by SEQ ID NO.1, SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8 and SEQ ID NO:9 sequences are sequentially connected in series.

Further, the nucleotide sequence of the Chimeric antigen receptor is by SEQ ID NO.12, SEQ ID NO:14、SEQ ID NO:18、SEQ ID NO:19 and SEQ ID NO:20 sequences are sequentially connected in series.

The nucleic acid sequence of the hinge area such as SEQ ID NO:14 or SEQ ID NO:Shown in 15；The nucleic acid of the transmembrane region Sequence such as SEQ ID NO:16 or SEQ ID NO:Shown in 17；The intracellular signal domain is CD28 and/or CD137 and/or CD3 ζ Chain；The nucleic acid sequence of the CD28 such as SEQ ID NO:The nucleic acid sequence of 18, the CD137 such as SEQ ID NO:19, the CD3 The nucleic acid sequence of ζ chains such as SEQ ID NO:20.

The Chimeric antigen receptor nucleic acid sequence is by the single-chain antibody, the hinge area, the transmembrane region, institute The nucleic acid sequence for the packet intracellular signaling domain stated, which is sequentially connected in series, to be composed.

The present invention also aims to provide a kind of preparation method of the slow virus carrier of the Chimeric antigen receptor, tool Body includes the following steps：

1) gene order of the Chimeric antigen receptor of the Chimeric antigen receptor of anti-CD123 antigens is synthesized：Before synthesis contains successively Lead different single-chain antibody (ScFv), hinge area, transmembrane region and the intracellular signal domains of peptide, anti-human anti-CD123 antigens；The hinge Area's nucleic acid sequence such as SEQ ID NO:14 or SEQ ID NO:15, transmembrane region nucleic acid sequence such as SEQ ID NO:16 or SEQ ID NO:17, intracellular signal domain nucleic acid sequence such as SEQ ID NO:18 and/or SEQ ID NO:19 and/or SEQ ID NO:20；

2) slow virus carrier of structure expression Chimeric antigen receptor：Design primer, the nucleotide sequence such as SEQ of forward primer ID NO:Shown in 21, the nucleotide sequence such as SEQ ID NO of reverse primer:Shown in 22, with the sequence of the Chimeric antigen receptor PCR amplification is carried out for template, obtains DNA fragmentation；

By the gene order of DNA fragmentation restriction enzyme NheI and SalI double digestion, while in restricted Enzyme cutting NheI and SalI digestion Lentiviral pCDH-EF1, target fragment and slow virus expression after then cutting plum carry Body segment is attached by T4 ligases, obtains the slow virus carrier of expression Chimeric antigen receptor.

Further, it after step 2), packs and purifies the slow virus carrier.

The present invention also aims to provide the slow virus carrier obtained by a kind of preparation method.

The slow virus carrier is obtained under the method, the positive expression rate of such slow virus carrier is high, It is very stable in patient's cell cultivation process, and positive rate can will not be caused to decline over time.Then, with described slow The T cell of viral vector infection, such T cell have the function of killing target cell.

The present invention also aims to provide a kind of T cell of the slow virus carrier infection.

The present invention also aims to provide a kind of T cell in the drug for being used to prepare treatment malignant hematologic disease Application.The Chimeric antigen receptor of the present invention can be used for treating for the adoptive cellular of medullary system tumour.It is of the present invention After the Chimeric antigen receptor of anti-CD123 antigens is expressed in immunocyte, expression CD123 antigens not only can be effectively removed Tumour target cell, and there is no toxic effect to the tumour cell of Negative antigens (not expressing CD123)；And it is able to maintain that targeting The positive of the Chimeric antigen receptor (chimeric antigen receptor, CAR) of CD123 in patient's cell cultivation process Rate can be proliferated after target antigen stimulation, can be used in the targeted therapy of tumour for a long time.

Further, the cell of the malignant hematologic disease can express CD123.CD123 is also known as interleukin-13 receptor alpha chain (IL- 3R α) height is expressed in leukemic stem cells or leukaemia juvenile cell, and it is not expressed or low expression in normal haematopoetic, is Leukemia-associated antigen is also the specific antigen of acute myeloid leukaemia.The appearance of CD123 targets is the new prominent of AML treatments It is broken；Since CD123 is expressed in AML high, theoretically by the immunization therapy of target of CD123 there is safer and more effective treatment to imitate Fruit.

Further, the malignant hematologic disease is the acute myeloblastic leukemia for expressing CD123, acute lymphoblastic leukemia And B cell chronic lymphocytic proliferative diseases.

The present invention also aims to provide a kind of SEQ ID NO:Polypeptide described in 1 is being used to prepare treatment hematologic Application in the drug of disease.

Further, the cell of the malignant hematologic disease can express CD123.

In addition, returning to the source of invention, the present invention also aims to provide a kind of SEQ ID NO:Polypeptide described in 1 It is being used to prepare the application precisely captured in capable of expressing the carrier of CD123 Hematologic Malignancy Cells.

In addition to cell therapy, radiation treatment can be used for.The ammonia of the single-chain antibody (ScFv) of the anti-CD123 antigens Base acid sequence is as shown in SEQ ID NO.1.

It is a kind of such as SEQ ID NO the present invention also aims to provide:Amino acid sequence shown in 1 is preparing CAR-T bones The application of antigen recognition domain in frame.

It generally speaking, not only can be with after scFV-CAR Chimeric antigen receptors of the present invention are expressed in immunocyte Maintain the Chimeric antigen receptor (chimeric antigen receptor, CAR) of targeting CD123 in patient's cell cultivation process In positive rate and the proliferation of CAR-T can be reinforced and kill the ability of tumour, and to the cytotoxic pair of antigen negative Effect, can be used in the targeted therapy of tumour.

The beneficial effects of the present invention are：

1) after the Chimeric antigen receptor of anti-CD123 antigens provided by the invention is expressed in immunocyte, can not only have The tumour target cell of the removing expression CD123 antigens of effect, and there is no toxicity to the tumour cell of Negative antigens (not expressing CD123) Effect；And it is able to maintain that the Chimeric antigen receptor (chimeric antigen receptor, CAR) of targeting CD123 in patient Positive rate in cell cultivation process can be proliferated after target antigen stimulation, can be used in the targeting of tumour for a long time Treatment.

2) provided by the invention high by the Chimeric antigen receptor humanization degree of transformation, it can be effectively reduced CAR's Immunogenicity, the lasting and safeties of enhancing CAR-T in vivo.

3) Chimeric antigen receptor provided by the invention can stablize the T leaching for being expressed in T lymphocytes especially patient source Bar cell can be used for preparing the adoptive cellular in the drug for the treatment of malignant hematologic disease for malignant hematologic disease and treat.

Description of the drawings

Fig. 1 is different CD123-CAR structure charts.

Fig. 2 is the transfection efficiency (CAR positive rates) that difference CD123-CAR transfects normal donors' T lymphocytes in Fig. 1； As a result show that the efficiency of CD123-CAR-3 transfecting T cells is worst.

Fig. 3 is difference CD123-CAR-T cells CD4/CD8 flow cytometer detections result and statistical result in Fig. 2.

Fig. 4 is expressions of the FCM detection CD123 in tumour cell；Wherein THP-1-Luc high expresses CD123, U937- Luc moderates express CD123, as positive cell；CD123 is as negative cells for Raji-Luc low expressions.(all cells surely turn Luciferase so as to carry out CAR-T kill ability detection)

Fig. 5 is each CD123-CAR-T cells in vitro killing activity detection in Fig. 3；Scheme A and indicates 6 small timeliness target scores of killing It Wei 5:1,1:Killing abilities of the CD123-CAR-T to the target cell of Fig. 4 when 1；Scheme B to indicate 24 hours 5:1,1:CD123- when 1 Killing abilities of the CAR-T to the target cell of Fig. 4；CAR-T the cells CD123-CAR-1, CD123- built by CD123-scFV-1 CAR-2 has the relatively good ability for removing tumour cell.

Fig. 6 is the detection of cytokine release after different CAR-T cell killings tumour cells；Every group is collected in Fig. 5 experiments The CD123-CAR-T of killing kills 24 hours supernatants, and the secretion of IFN γ and IL-2 is detected using ELISA, and figure A is IFN γ Secretion detection, the secretion that figure B is IL-2；As a result, CD123-CAR-1 and CD123-CAR-2 consistent with Cytotoxicity in vitro result are shown There is higher cytokine secretion, and CD123-CAR-3 and CD123-CAR-4 has killing to have the higher factor to discharge negative cells, it is non- Specific killing height has potential risk for experiment in vivo.

Fig. 7 is the proliferative capacity for being not added with exogenous cytokines CD123-CAR-T cells in the case where target cell stimulates；It utilizes The CAR-T ability of cell proliferation of CD123-scFV-1 structures is most strong.

Fig. 8 detects for CD123-CAR-T cells in vitro killing activities.

Specific implementation mode

Hereinafter reference will be made to the drawings, and the preferred embodiment of the present invention is described in detail.Tool is not specified in preferred embodiment The experimental method of concrete conditions in the establishment of a specific crime, usually according to normal condition, such as Molecular Cloning:A Laboratory guide (third edition, J. Pehanorm Brookers etc. Write) described in condition, or according to the normal condition proposed by manufacturer.Illustrated embodiment is in order to preferably in the present invention Appearance illustrates, but is not that present disclosure is only limitted to illustrated embodiment.So those skilled in the art according to Foregoing invention content carries out nonessential modifications and adaptations to embodiment, still falls within protection scope of the present invention.

The structure of embodiment 1 Chimeric antigen receptor virus

It is respectively necessary for building following 6 Chimeric antigen receptor to screen better CAR combinations：

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

1. the gene order synthesis of the Chimeric antigen receptor of targeting CD123 of the synthesis containing different scFV contains leader peptide successively Different single-chain antibody ScFv, the IgG4 hinge areas (abbreviation IgG4) of (also known as signal peptide) (abbreviation LP), anti-human CD123 antigens, CD28 transmembrane regions or CD8 transmembrane regions (referred to as TM), structure is as shown in Figure 1.

2. the slow virus carrier of structure expression Chimeric antigen receptor

1) following primer is designed, and it is synthesized by Nanjing Jin Sirui biotechnologies company, specific primer is as follows：

Primer 1：5’-aggctagcaTgggatggagctgtatcat-3 ' (SEQ ID NO.21), underscore limit for NheI Property restriction enzyme site processed；

Primer 2：5’-gattgtcgacTtagcgagggggcagggcctgcatgtga-3 ' (SEQ ID NO.22), lower stroke Line is SalI restriction endonuclease sites.

Then using sequence shown in SEQ ID NO.21 and SEQ ID NO.22 as primer, each chimeric antigen of above-mentioned synthesis by Body sequence is that template carries out PCR amplification, and reaction system is by KOD FX NEO archaeal dna polymerases (being purchased from TOYOBO companies) specification Sample-adding, PCR reaction conditions are 95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 10s, 55 DEG C of annealing 15s, 68 DEG C of extension 30s, 30 are followed Ring.Amplified production is identified with 1% Ago-Gel.The results show that amplification obtains the DNA fragmentation of about 2000bp, then Carry out DNA fragmentation recycling with QIAquick Gel Extraction Kit (Promega companies), specific method is shown in specification, recycling obtain chimeric antigen by DNA recycling segment is sent Nanjing Jin Sirui biotechnologies company to be sequenced by body.

Gene order the restriction enzyme NheI and SalI of the encoding chimeric antigen receptor that clone obtains (are purchased from Thermo companies) double digestion, while (purchase is extremely with restriction enzyme NheI and SalI digestion Lentiviral pCDH-EF1 Addgene Plasmid#72266), endonuclease reaction by specification carries out.Digestion products are used after agarose gel electrophoresis detaches Agarose gel DNA fragment QIAquick Gel Extraction Kit carry out DNA fragmentation recycling, then by target fragment and carrier segments connected by T4 It connects enzyme (being purchased from Promega companies) to be attached, obtains the slow virus carrier of expression Chimeric antigen receptor, be named as Lv-scFv (CEA).Slow virus carrier is taken into 5 μ l conversion Escherichia coli TOP10,30 DEG C are cultivated picking monoclonal after 16h, the monoclonal of picking Plasmid extraction kit (Invitrogen companies) is used to extract plasmid after cultivating 12h under the conditions of 30 DEG C, specific method is shown in explanation Book.

CD123-CAR-1, CD123-CAR-2, CD123-CAR-3, CD123-CAR-4 are built respectively according to method as above, CD123-CAR-5, CD123-CAR-6 slow virus carrier；

3. the packaging of slow virus

The present embodiment packs slow virus and uses calcium phosphate method, is as follows：With the DMEM cultures containing 10% (wt) FBS Base culture 293T cells, until preferable states；Then by 293T cells with 1 × 10⁵/cm²Density reach the culture bottle of 1 75cm2 Middle culture 22h ensures that cell confluency degree is 70-80% when transfection；It is changed again with the DMEM culture mediums containing 2% (wt) FBS of preheating Liquid cultivates 2h, spare；By 680 μ l ddH₂O, 20 μ g slow virus carriers, 20 μ g pMDLg/pRRE plasmids, 20 μ g pRSV-Rev Be added in 15ml centrifuge tubes, be uniformly mixed with 10 μ g pMD2.G plasmids, 100 μ l 2.5mM CaCl2, after mixing with pipettor to 2 × HBS is added dropwise in mixed liquor, while mixing is blown and beaten with 10ml pipettors, is stored at room temperature 15min after mixing, then will mix It closes liquid to be added dropwise in the cell of above-mentioned preparation, continues after cultivating 12-16h, replaced with the DMEM culture mediums containing 10%FBS (wt) Liquid is trained, continue to collect cell conditioned medium respectively after cultivating 48h, 72h and is used for viral purification.

4. the purifying of slow virus

Viral supernatants are collected in 50ml centrifuge tubes, 3000r/min centrifuges 10min；Then membrane filtration, filtrate from The heart is to new 50ml centrifuge tubes；Then according to viral supernatants amount, be separately added into PEG6000 that mass fraction is 50% and 4MNaCl, then the PEG6000 final concentration of 0.3M of final concentration of 8.5%, NaCl are settled to medical brine, it is fixed it is molten after in 4 DEG C of ice Case stands 90min, 30min/ reverse mixing；Then 30min is centrifuged under the conditions of 4 DEG C, 5000r/min, abandons most supernatant, used Virus is resuspended in DMEM culture mediums, and 1.5mlEP pipes dispense, often 40 μ l of pipe, and -80 DEG C save backup.Obtained virus is respectively designated as CD123-CAR-1, CD123-CAR-2, CD123-CAR-3, CD123-CAR-4, CD123-CAR-5, CD123-CAR-6.

5. the titer determination of slow virus

1) virus infection 293T cells

293T cells are taken into purified viral 1 μ l to spread in 24 orifice plates before infection, dilute 10 with medical saline Times, then 1 μ l polybrenes (Polybrene) solution is added into every hole cell, then by the virus liquid point after 1 μ l and 9 μ l dilutions It is not added in 293T cells, changes liquid with the DMEM culture mediums containing 10%FBS (wt) afterwards for 24 hours, infect after 72h in 1000r/min items 5min is centrifuged under part to collect cell, extracts genome.

2) genome is extracted

Genome extraction agent box is that QIAamp DNA Blood Mini Kit are purchased from Qiagen companies (article No. 511004) it, is operated by kit specification.

3) qRT-PCR measures virus titer

Reaction system is as follows：Premix Ex TaqTM II (2 ×) 10 μ l, 1 μ l of sense primer (GAG up), downstream primer (GAGdn) 1 μ l, 1 μ l, the RNase-Free dH of genome of extracting₂7 μ l of O, each sample, standard items at least three repeating hole. Then it is expanded by following procedure：95 DEG C of pre-degeneration 30s, 95 DEG C of denaturation 5s, 60 DEG C of annealing 30s, 72 DEG C extend 30s, reaction After, with analysis software data, virus titer is calculated according to standard curve.

The detection of 2 Chimeric antigen receptor of embodiment transfection efficiency in T cell

1) separation of human peripheral blood mononuclear cell

Peripheral blood about 60ml is acquired with the heparin tube added with anti-coagulants, each 30ml of 50ml centrifuge tubes is sub-packed in, 7.5ml is added Hydroxyethyl starch dilutes；Room temperature (18~25 DEG C) natural subsidence about 30min, collect upper plasma, by the upper plasma of collection in 15min is centrifuged under the conditions of 1400rpm/min；Then it is resuspended and is precipitated with physiological saline, be by volume 1:1 is added to lymphocyte point In chaotropic, gradient centrifugation, 400g/min, centrifuge reduction of speed is set as 1, centrifuges 20min；After centrifugation, centrifuge tube from top to bottom divides For：First layer：Plasma layer；The second layer：Cyclic annular milky buffy coat；Third layer：Transparent separation liquid layer；4th layer：It is red thin Born of the same parents' layer；Second layer white buffy coat is taken, is used in combination brine 2 times, first time 400g/min, centrifuges 10min, second Secondary 1100rpm/min centrifuges 5min, and cell is resuspended in physiological saline, and 1640 complete mediums of RPMI containing 10%FBS are added Culture, obtains human peripheral blood mononuclear cell.

2) slow virus carrier infects T lymphocytes

With 1640 complete medium cultures of the RPMI freshly prepd mononuclearcell PBMC containing 10% fetal calf serum, the 1st day CD 3-resisting monoclonal antibody activation is added；Carry out slow-virus infection within first 3 days；The corresponding slow virus carriers of 5MOI are added, are uninfected by T lymphocytes are as blank control；It is complete that culture medium is changed to the RPMI1640 containing 500IU/ml recombinant human il-2s afterwards for 24 hours Culture medium continues culture 10-20 days.Testing result is as shown in Fig. 2, several CAR structures can preferably transfect T lymphocytes.

The detection of 3 CAR molecules positive rate of embodiment and CD4, CD8 ratio

The CD123-CAR-T cell 300g/min of culture 11 days centrifuge 5min, are resuspended with 1ml PBS after abandoning most supernatant, weight Duplicate step uses 100ul PBS cells that label CD3, CD4, CD8 (BD companies) and Protein-L (Beijing justice is added afterwards twice Stick up Divine Land company) four degree of flow cytometer detection antibody be incubated 30 minutes, PBS is washed 3 times, and the mouse for adding AF647 labels is anti- Protein-L antibody (Nanjing Jin Sirui companies), four degree are incubated 30 minutes PBS and wash twice, and are added after abandoning most supernatant 200ulPBS carries out flow cytometer detection result as shown in table 1 and Fig. 3 after the cell marked is resuspended.

1 difference CD123-CAR-T cells CD4/CD8 flow cytometer detections result of table and statistical result

CAR titles	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

Expressions of 4 FCM of the embodiment detection CD123 in tumour cell

1) target cell Luc is built

The slow virus carrier of transfection expression luciferase is to the tumour target cell tested in the way of in-vitro transfection It is interior.

2) external source height expression CD123 tumor cell lines structure

In order to preferably verify various combination CD123-CAR-T cells killing ability, using not expressing the swollen of CD123 Oncocyte constructs the cell of high expression CD123 by way of external source transfection, and as a result as Fig. 4 shows, two Expression of Plant Height CD123 are thin Born of the same parents reach high expression and require, and can carry out cell experiment.

3) different tumour target cell CD123 positive rates detections

Tumour cell 300g/min centrifuges 5min, is resuspended with 1ml PBS after abandoning most supernatant, repeats this step and use afterwards twice PBS washes twice weight after four degree of flow cytometer detection antibody (the being purchased from BD) incubation 30 minutes of label CD123 is added in 100ulPBS cells Upper machine testing is suspended from 200ul PBS.The results are shown in Figure 4, and to have detected Raji-Luc, U937-Luc and THP-1-Luc respectively thin The CD123 of cellular surface is expressed, and as a result shows that Raji-Luc does not express CD123, U937-Luc moderates express CD123 (56.73% tables Up to), THP-1-Luc high expresses CD123 (99.85% expression).

5 each CD123-CAR-T cells in vitro killing activity of embodiment detects

The Chimeric antigen receptor of different scFV passes through ACEA xCELLigence RTCA respectively to the killing ability of target cell The method of MP instruments and luciferase assay is completed.

1) fluorescein enzyme process：96 orifice plate bed boards are marked with the tumour cell of the expression CD123 of firefly luciferase (THP-1, U937) 1*10^4 is per hole bed board, according to different effect target ratios (1:2,1:1,5:1,10:1) bed board target cell (tumour Cell), it is positioned over 37 degree of cell incubator and co-cultures respectively 6 hours, 24 hours fluorescences by detecting luciferase detect Killing abilities of the amount indirect detection CD123-CAR of remaining target cell to target cell.The results are shown in Figure 5：CD123-CAR-1 and CD123-CAR-2 to tumour cell have high killing activity and high specificity, just for expression CD123 tumour cell into Row killing, and the nearly no killing of tumour cell of CD123 feminine genders.Table 2 is the statistical data of Fig. 5 Mortaility results.

Table 2：Different time sections difference imitates killing percentage of the target ratio CD123-CAR-T cells to CD123 positive tumor cells Than

2) ACEA xCELLigence RTCA MP instruments：Experimental procedure is carried out according to instrument specification；First day by target Cell (tumour cell and negative control cell of the expression CD123 of structure) is plated on apparatus preparation with 2-5*10^4 per hole In 96 orifice plates, the tumour cell for being attached to bottom hole is primary as every 15 minutes records of data using index of resistance, according to pre- after 24 hours The effect target first designed is spread than every hole into corresponding CAR-T cells, and record CAR-T cells spread the index of resistance into 24 hours, and 24 is small When after collect each group supernatant and carry out enzyme linked immunological (ELISA) and detect and by each group index of resistance analysis result of Instrumental Analysis, CAR-T cell killings rate=baseline electrical resistance index-real time resistance index, concrete outcome analysis, 1 indicates initiator cell index, carefully Born of the same parents' index indicates 2 times of tumor cell proliferations such as cell index higher than 1 for 2, and cell index is less than 1 if index is 0.3 expression tumour Cell is by killing 70%；If cell index is above 1 but is less than T-con and medium class indexes, indicate that CAR is thin to tumour The proliferation of born of the same parents has inhibiting effect but can not remove tumour cell well.The results are shown in Figure 8, and CD123-scFV1 is as anti- The Chimeric antigen receptor of former cog region structure has the work of the tumour cell of preferable specificity and the high killing CD123 positives Property, the inosculating antibody of preferred CD123-CAR-5, that is, CD123-scFV-1-CD8hinge-CD28TM-CD28-CD137-CD3 with by The combination of body has higher validity and safety.As a result statistics such as the following table 3：

Table 3：The killing ability statistics of the T cell of the CAR transfections of different scFV and the combination of hinge (hinge) structure

Embodiment 6：Cytokines measurement

1) method that cytokines measurement uses Elisa is carried out using BD companies kit.

2) dilution of standard items：Sample dilution mode refers to product description.

3) it is marked with quasi- sample wells on enzyme mark coating plate, sequentially adds the 50 μ l of standard items of various concentration, each concentration does 2 Parallel hole.

4) it is loaded：Set respectively blank well (blank control wells are not added with the antibody of sample, enzyme marking reagent and biotin labeling, Remaining each step operation is identical), sample to be tested hole；40 μ l of product are first loaded in sample to be tested hole on enzyme mark coating plate, then add life again The 10 μ l of antibody of object element label.Sample is added on ELISA Plate hole bottom by sample-adding, is not touched hole wall as possible, is gently shaken mixing；

5) it incubates：It is incubated 30 minutes for 37 DEG C with sealing plate film sealing plate postposition；

6) match liquid：It will be spare after 30 times of concentrated cleaning solutions, 30 times of dilutions of distilled water；

7) it washs：It carefully takes sealing plate film off, discards liquid, dry, cleaning solution is filled it up with per hole, discarded after standing 30 seconds, such as This is repeated 5 times, and pats dry；

8) enzyme：50 μ l of enzyme marking reagent are added per hole, except blank well；

9) it incubates：Operation is the same as 3；

10) it washs：Operation is the same as 5；

11) it terminates：Add 50 μ l of terminate liquid per hole, terminates reaction；

12) it measures：With blank air-conditioning zero, 450nm wavelength sequentially measures the absorbance (OD values) in each hole.

The results are shown in Figure 6 CD123-CAR-1 and CD123-CAR-2 has the tumor cytotoxicity for expressing CD123 high IFN-γ and IL-2 secretions, preferably to negative cells specificity.As a result statistics is as shown in table 4：

Table 4：Cytokine secretion after the T cell killing tumor cell of the CAR transfections of various combination

	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

Embodiment 7 is not added with the proliferative capacity of exogenous cytokines CD123-CAR-T cells in the case where target cell stimulates

In order to imitate vivo environment, CD123-CAR-T is stimulated by target cell in the case where being not added with exogenous cytokines The incremental multiple of cell observation CD123-CAR-T cells.The CD123-CAR-T cell 300g/min normally cultivated centrifuge 5min After abandon repeated centrifugation step 2 time after most supernatant 2ml PBS are resuspended, with it is fresh not plus the culture medium of exogenous cytokines dilutes Cell is to 1*10^6/ml, and 24 orifice plate target cells are per hole 2*10^5/500ul bed boards, and effect target is than 1:1, stimulation in every 7 days is primary, stimulation 3-4 afterwards carries out cell count statistics total number of cells on the 7th day and draws growth curve.CD123-CAR-1 as shown in Figure 7 is not having Under exogenous cytokines stimulation, proliferative capacity is most strong after being activated by CD123 positive tumor cells.Table 5 has counted stimulation 14 days When CAR-T cells proliferation times

Table 5：CD123 positive tumor cells stimulate CAR-T cell Proliferation multiples after CAR-T cells

	CD123-CAR-1	CD123-CAR-2	CD123-CAR-3	CD123-CAR-4	con
						Proliferation times	5.72	2.83	1.52	1.56	1.56

Finally illustrate, the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to compared with Good embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this In the right of invention.

<110>The accurate Bioisystech Co., Ltd in Chongqing

<120>The Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof 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 Leu Ala 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 hinges

<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 hinges

<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 regions

<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 regions

<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 costimulatory 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 hinges

<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 hinges

<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 regions

<400> 16

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210>17

<211> 87

<212> DNA

<213>

<220>

<223>CD28 transmembrane regions

<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 costimulatory 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. identifying the polypeptide of CD123 antigens, which is characterized in that the amino acid sequence of the polypeptide such as SEQ ID NO:Shown in 1.

2. polypeptide described in claim 1 is in the application for the single-chain antibody for preparing anti-CD123 antigens.

3. the Chimeric antigen receptor of the anti-CD123 antigens containing polypeptide described in claim 1, which is characterized in that by identifying CD123 The polypeptide of antigen, hinge area, transmembrane region and intracellular signal domain are sequentially connected composition；The ammonia of the polypeptide of the identification CD123 antigens Base acid sequence contains polypeptide described in claim 1.

4. Chimeric antigen receptor according to claim 3, which is characterized in that the amino acid sequence of hinge area such as SEQ ID NO:24 or SEQ ID NO:Shown in 25.

5. Chimeric antigen receptor according to claim 3, which is characterized in that the amino acid sequence of the transmembrane region such as SEQ ID NO:3 or SEQ ID NO:Shown in 4.

6. Chimeric antigen receptor according to claim 3, which is characterized in that the intracellular signal domain be CD28 and/or CD137 and/or CD3 ζ chains；The amino acid sequence of the CD28 such as SEQ ID NO:The amino acid sequence such as SEQ of 7, the CD137 ID NO:8, the amino acid sequence such as SEQ ID NO of the CD3 ζ chains:9.

7. the preparation method of the slow virus carrier of the Chimeric antigen receptor described in claim 3, which is characterized in that specifically include with Lower step：

1) gene order of the Chimeric antigen receptor of the Chimeric antigen receptor of anti-CD123 antigens is synthesized：Synthesis successively contain leader peptide, Different single-chain antibody (ScFv), hinge area, transmembrane region and the intracellular signal domains of anti-human anti-CD123 antigens；The hinge area core Acid sequence such as SEQ ID NO:14 or SEQ ID NO:15, transmembrane region nucleic acid sequence such as SEQ ID NO:16 or 17, intracellular signal Domain nucleic acid sequence such as SEQ ID NO:18、SEQ ID NO:19 and SEQ ID NO:20；

2) slow virus carrier of structure expression Chimeric antigen receptor：Design primer, the nucleotide sequence such as SEQ ID of forward primer NO:Shown in 21, the nucleotide sequence such as SEQ ID NO of reverse primer:Shown in 22, using the sequence of the Chimeric antigen receptor as mould Plate carries out PCR amplification, obtains DNA fragmentation；

By the gene order of DNA fragmentation restriction enzyme NheI and SalI double digestion, while using restriction enzyme NheI and SalI digestion Lentiviral pCDH-EF1, the target fragment after then cutting plum and Lentiviral piece Section is attached by T4 ligases, obtains the slow virus carrier of expression Chimeric antigen receptor.

8. the slow virus carrier obtained by preparation method described in claim 7.

9. the T cell of slow virus carrier infection according to any one of claims 8.

10. application of the T cell in the drug for being used to prepare treatment malignant hematologic disease described in claim 9.

11. application according to claim 10, which is characterized in that the cell or tissue of the malignant hematologic disease can express CD123。

12. application of the polypeptide described in claim 1 in the drug for being used to prepare treatment malignant hematologic disease.

13. application according to claim 12, which is characterized in that the cell of the malignant hematologic disease can express CD123。

14. polypeptide described in claim 1 can express the carriers of CD123 Hematologic Malignancy Cells being used to prepare precisely capture In application.

15. such as SEQ ID NO:The application of antigen recognition domain of the amino acid sequence shown in 1 in preparing CAR-T skeletons.