CN112521506A

CN112521506A - CD123 antigen binding fragment and application thereof

Info

Publication number: CN112521506A
Application number: CN202011461164.6A
Authority: CN
Inventors: 狄升蒙; 童东阳; 周建国; 冯冬歌; 余学军
Original assignee: Huadao Shanghai Biological Medicine Co ltd
Current assignee: Huadao Shanghai Biological Medicine Co ltd
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-03-19
Anticipated expiration: 2040-12-11
Also published as: CN112521506B

Abstract

The invention provides a CD123 antigen binding fragment and application thereof, wherein the CD123 antigen binding fragment comprises a heavy chain CDR1 shown in SEQ ID NO. 1, a heavy chain CDR2 shown in SEQ ID NO. 2 and a heavy chain CDR3 shown in SEQ ID NO. 3. The invention screens and obtains CD123 antigen binding fragments with CDR regions shown in SEQ ID NO 1-3 from a camel VHH immune library, and an antibody formed by combining the fragment and a frame region FR has strong affinity to CD123, so that the constructed chimeric antigen receptor and chimeric antigen receptor immune cells have obvious cytotoxicity to CD123 positive cells, and the application prospect in the field of tumor treatment is wide.

Description

CD123 antigen binding fragment and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and relates to a CD123 antigen binding fragment and application thereof.

Background

Over 30 years ago, the "3 + 7" regimen (3 days for daunorubicin chemotherapy combined with 7 days for cytarabine chemotherapy) alleviated the disease in about 60% of AML patients, becoming the standard induction regimen for the treatment of acute leukemia in children and adults. In the last 90 s of the century, clinical experts began to focus on post-remission treatment regimens and their benefits and conducted a great deal of research including high dose cytarabine chemotherapy or Hematopoietic Stem Cell Transplantation (HSCT). Although remission rates and overall survival rates for childhood acute leukemia currently reach greater than 90% and 60%, respectively, existing treatment regimens are still limited to anthracycline-based drugs, nucleoside analogs, and intensive post-remission therapy. In order to improve the prognosis of AML patients, protocols including alternatives to mitoxantrone and daunorubicin, cytarabine-based intensive therapy, etc. were used clinically, however, most clinical studies showed no significant difference in outcome for each treatment group.

The curative effect of stem cell transplantation on leukemia shows that the antitumor immunity can effectively eliminate and prevent leukemia recurrence. In fact, many studies have found that the recurrence rate of hematopoietic stem cell transplantation is significantly reduced compared to chemotherapy. However, hematopoietic stem cell transplantation in remission is controversial due to the high mortality rate of hematopoietic stem cell transplantation. In recent years, with improvements in supportive care, comprehensive HLA and NK typing contributes to reduction of side effects of hematopoietic stem cell transplantation therapy. Tyrosine Kinase Inhibitors (TKIs) have become one of the important chemotherapeutic regimens. For example, the FLT3 gene (FLT3ITD) can elicit AML in about 15% of children and 30% of adults, and individuals with a higher FLT 3-ITD/wild-type FLT3 ratio exhibit a more pronounced poor prognosis, associated with poor outcome. Sorafenib (sorafenib), sunitinib and other FLT3 inhibitors can effectively inhibit FLT3 mutation, but the drug resistance phenomenon appears after long-term use of the drugs, which is mainly related to D835 or F691 kinase region point mutation. The novel tyrosine kinase inhibitor crenolanib has a good therapeutic effect on Sorafenib (Sorafenib) resistant AML mouse models, and the inhibitor can prolong clinical benefit. Although TKIs offer different leukemia treatment regimens, they are still in the development stage and more treatment strategies are clinically needed.

The concept of Chimeric antigen receptor modified T lymphocytes (CAR-T) was developed as early as 1989, but the ideal effect has not been achieved in clinical trials. Over the next two decades scientists continually optimized this technique until 2011, CAR-T cells targeted to CD19(B lymphocyte antigen CD19, CD19) achieved surprising efficacy in treating relapsed/refractory chronic B-lymphocyte leukemia, and the use of CAR-T cells in tumor therapy opened new chapters.

The chimeric antigen receptor is an artificially synthesized fusion protein with the function similar to that of a T cell receptor, and mainly comprises a signal peptide, an antigen recognition region, a hinge region, a transmembrane region and an intracellular signal region. Upon binding to the target antigen, the chimeric antigen receptor forms a dimer, activates T cells via intracellular signaling molecules, secretes perforin and granzyme B, and effects killing of the target cells. CAR-T cells recognize target cells independent of Major Histocompatibility Complex (MHC), avoiding immune escape due to down-regulation of tumor cell MHC molecules. In recent years, chimeric antigen modified T lymphocytes (CAR-T) have developed rapidly, and at present, two products are approved by FDA in the united states and are on the market, and many products are approved by clinical trials of drugs in China.

CD123, also known as CD3 receptor alpha chain, is expressed on leukemic cells and leukemic stem cells, normal hematopoietic stem cells are not or weakly expressed, and are also expressed on endothelial cells and monocytes, plasma cell DCs, but CD123 positive monocytes and plasma cells account for a small proportion of reactive lymph nodes. High expression of CD123 promotes proliferation of tumor cells. CD123 is currently the most studied AML antigen, and several monoclonal antibody-based drugs targeting CD123 have been developed, such as 7G3, CSL360 and CSL362, in preclinical and clinical trials. In addition, CAR-T therapies targeting CD123 have also completed preclinical studies and showed significant anti-tumor effects.

The anti-CD 123 chimeric antigen receptor gene is introduced into T cells through a genetic engineering method to prepare CD123CAR-T, so that the CD123CAR-T cells specifically recognize CD 123-expressing acute myeloid leukemia cells and eliminate tumor cells, thereby realizing the anti-tumor effect of the CD123 chimeric antigen receptor gene. Therefore, immunotherapeutic approaches targeting CD123 have great clinical value.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides a CD123 antigen binding fragment and application thereof, a high-affinity anti-CD 123 antibody containing the CD123 antigen binding fragment is screened by utilizing a phage display technology and is used as an antigen binding domain of a chimeric antigen receptor molecule to construct a CAR immune cell, and the CAR immune cell can specifically recognize and kill CD123 positive cells and has important application prospect in the field of tumor treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a CD123 antigen-binding fragment, the CD123 antigen-binding fragment comprising the heavy chain CDR1 of SEQ ID NO. 1, the heavy chain CDR2 of SEQ ID NO. 2, and the heavy chain CDR3 of SEQ ID NO. 3;

SEQ ID NO:1：ENVYC；

SEQ ID NO:2：VSRVGWT；

SEQ ID NO:3：AADSRVCGLRSGRKYTD。

in the invention, an antibody with high affinity is screened from a CD123 immune camel VHH immune library, and the fact that a fragment with heavy chain CDRs shown in SEQ ID NO. 1-3 has specific high binding capacity to a CD123 antigen is found, and the CDR regions shown in SEQ ID NO. 1-3 have decisive effect on the affinity of the antigen binding fragment or the antibody with the CD 123.

In a second aspect, the present invention provides an anti-CD 123 antibody comprising a CD123 antigen-binding fragment according to the first aspect.

Preferably, the anti-CD 123 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO 4; 4, SEQ ID NO:

EVQLVESGGGSVQAGGSLKLSCSASENVYCMGWFRQAPGKEREGVATVSRVGWTSYADSVKGRFTISRDNAKNALYLQMNSLKPEDTAMYYCAADSRVCGLRSGRKYTDWGQGTQVTVSS。

in the invention, a phage display technology is utilized to screen a CD123 immune camel VHH library to obtain an anti-CD 123 antibody CD123-35(SEQ ID NO:4) only containing a heavy chain variable region, wherein the anti-CD 123 antibody has CDR regions shown in SEQ ID NO: 1-3, has high affinity to CD123 and has important application prospect in the aspect of constructing a chimeric antigen receptor of a target CD 123.

In a third aspect, the present invention provides a nucleic acid molecule comprising a DNA fragment encoding the CD123 antigen-binding fragment of the first aspect.

Preferably, the nucleic acid molecule comprises a DNA fragment encoding the anti-CD 123 antibody of the second aspect.

Preferably, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO. 5, which is the coding sequence of the anti-CD 123 antibody CD 123-35;

SEQ ID NO:5：

GAAGTGCAGCTGGTGGAGAGCGGCGGCGGATCTGTGCAGGCCGGAGGCTCTCTGAAACTGTCTTGTTCTGCCAGCGAGAATGTGTATTGTATGGGATGGTTTAGACAGGCCCCAGGCAAAGAGAGAGAGGGAGTGGCTACAGTGTCTAGAGTGGGATGGACATCTTATGCCGATTCTGTGAAAGGAAGATTCACAATTTCTAGAGATAATGCCAAAAATGCCCTGTATCTGCAGATGAATAGCCTGAAGCCTGAGGATACAGCCATGTATTATTGTGCCGCCGATTCTAGAGTGTGTGGACTGAGATCTGGCAGAAAATATACAGATTGGGGCCAGGGAACACAGGTGACAGTGTCTAGC。

in a fourth aspect, the present invention provides a chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, and a signaling domain;

the antigen binding domain is an anti-CD 123 antibody according to the second aspect.

In the invention, the anti-CD 123 antibody of the second aspect is used as an extracellular region of the chimeric antigen receptor and is combined with CD123 in a targeted manner, and the constructed chimeric antigen receptor and immune cells expressing the chimeric antigen receptor have a high-efficiency targeting effect on CD123 positive cells and have important significance in the field of CD123 positive tumor treatment.

Preferably, the signal peptide comprises a CD8 a signal peptide.

Preferably, the hinge region comprises a CD8 a hinge region.

Preferably, the transmembrane domain comprises any one or a combination of at least two of a CD8 a transmembrane region, a CD28 transmembrane region or a DAP10 transmembrane region, the sequence of the CD8 a transmembrane region being referred to NM _001145873, the sequence of the CD28 transmembrane region being referred to NM _006139 and the sequence of the DAP10 transmembrane region being referred to NM _ 014266.

Preferably, the signaling domain comprises CD3 ζ, the sequence of which can be referenced to NM _ 198053.

Preferably, the signal transduction domain further comprises any one or a combination of at least two of the intracellular domains of 4-1BB, CD28, OX40, ICOS or DAP10, the sequence of 4-1BB is referred to NM _001561, the sequence of CD28 is referred to NM _006139, the sequence of OX40 is referred to NM _003327, the sequence of ICOS is referred to NM _012092, and the sequence of DAP10 is referred to NM _ 014266.

In a specific embodiment of the invention, the signaling domain comprises 4-1BB and CD3 zeta in order from N-terminus to C-terminus.

Preferably, the chimeric antigen receptor comprises, from N-terminus to C-terminus, the anti-CD 123 antibody of the second aspect, the CD8 a transmembrane region, 4-1BB and CD3 ζ in that order.

Preferably, the chimeric antigen receptor comprises, from N-terminus to C-terminus, the anti-CD 123 antibody, CD28 transmembrane region, CD28 intracellular region and CD3 ζ of the second aspect in that order.

Preferably, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, the anti-CD 123 antibody of the second aspect, a CD8 a transmembrane region, OX40, and CD3 ζ.

Preferably, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, the anti-CD 123 antibody of the second aspect, a CD8 a transmembrane region, ICOS, and CD3 ζ.

Preferably, the chimeric antigen receptor comprises, from N-terminus to C-terminus, the anti-CD 123 antibody of the second aspect, the CD28 transmembrane region, the CD28 intracellular region, OX40 and CD3 ζ in that order.

Preferably, the chimeric antigen receptor comprises an amino acid sequence shown as SEQ ID NO. 6;

6(CD 8. alpha. signal peptide-CD 123-35-CD 8. alpha. hinge-TM-41BB-CD 3. zeta):

MALPVTALLLPLALLLHAARPEVQLVESGGGSVQAGGSLKLSCSASENVYCMGWFRQAPGKEREGVATVSRVGWTSYADSVKGRFTISRDNAKNALYLQMNSLKPEDTAMYYCAADSRVCGLRSGRKYTDWGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

in a fifth aspect, the present invention provides an expression vector comprising the gene encoding the chimeric antigen receptor of the fourth aspect.

Preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to the fourth aspect, preferably a lentiviral vector.

In the invention, a virus vector, especially a lentivirus vector, containing the encoding gene of the chimeric antigen receptor of the fourth aspect is constructed, and an anti-CD 123CAR-T cell is constructed by using a genetic engineering method, so that an anti-tumor effect is realized.

In a sixth aspect, the present invention provides a recombinant lentivirus prepared from a mammalian cell transfected with an expression vector and a helper plasmid according to the fifth aspect.

In a seventh aspect, the present invention provides a chimeric antigen receptor immune cell expressing the chimeric antigen receptor of the fourth aspect.

Preferably, the chimeric antigen receptor immune cell comprises the expression vector of the fifth aspect and/or the recombinant lentivirus of the sixth aspect.

Preferably, the immune cells comprise any one of T cells, B cells, NK cells, mast cells or macrophages or a combination of at least two thereof.

In an eighth aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cell of the seventh aspect.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In a ninth aspect, the present invention provides a CD123 antigen-binding fragment according to the first aspect, an anti-CD 123 antibody according to the second aspect, a nucleic acid molecule according to the third aspect, a chimeric antigen receptor according to the fourth aspect, an expression vector according to the fifth aspect, a recombinant lentivirus according to the sixth aspect, a chimeric antigen receptor immune cell according to the seventh aspect, or a pharmaceutical composition according to the eighth aspect, for use in the preparation of a medicament for the treatment of a malignant tumor.

Preferably, the malignancy comprises a hematological tumor.

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

(1) the invention utilizes a transient transfection mode to introduce plasmids containing CD123 protein extracellular region gene segments into eukaryotic cells, express recombinant CD123 protein for immunizing bactrian camel, construct a phage display antibody library, and screen CD123-35 antibodies which can be specifically combined with CD123 antigen;

(2) the invention further utilizes the screened antibody to transform into a chimeric antigen receptor, and utilizes a genetic engineering method to express the chimeric antigen receptor in immune cells, so that the constructed immune cells expressing the anti-CD 123 chimeric antigen receptor have a cytotoxic effect on CD123 positive tumor cells, efficiently secrete cell factors IL-2, TNF-alpha and IFN-gamma after being co-cultured with the CD123 positive cells, and have important application prospects in the aspect of treating CD123 positive acute myeloid leukemia.

Drawings

FIG. 1 is Biacore for detecting the affinity of CD123-35 antibody;

FIG. 2 shows FACS detection of CD123-35 antibody recognizing CD123 antigen on the cell surface;

FIG. 3 is a schematic diagram of the structure of a chimeric antigen receptor expressing CD 123;

FIG. 4 shows the chimeric antigen receptor expression rate of T lymphocytes;

FIG. 5 is the killing effect of CD123CAR-T cells on CD123 positive tumor cells;

FIG. 6 is the level of IL-2 secretion by CD123CAR-T cells;

FIG. 7 is the level of TNF α secretion by CD123CAR-T cells;

FIG. 8 is the level of IFN γ secretion by CD123CAR-T cells.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 construction of phage antibody library, panning and ELISA Primary screening

(1) Construction of phage antibody libraries

In the embodiment, firstly, CD123 antigen extracellular region is adopted to immunize bactrian camel, and 200mL of peripheral blood is extracted after the titer is verified by ELISA; separating lymphocytes from peripheral blood to obtain peripheral blood mononuclear lymphocyte sediment, and extracting RNA;

using the extracted RNA as a template

III reverse transcriptase synthesizing first strand cDNA, and subsequently amplifying VHH gene by using nested PCR; inserting the amplified VHH gene into a pMECS phage display carrier, electrically converting TG1 competent cells, taking a proper amount of bacterial liquid for library identification, and uniformly coating all the rest cultures on an LB/AMPGLU flat plate;

collecting thallus Porphyrae after bacteria growth, adding 1/3 vol 50% glycerol, mixing, packaging, and storing at-80 deg.C to obtain product with storage capacity greater than 10⁹The phage display camelid VHH immune library of (a).

(2) Panning of phage antibody libraries

Performing 3 rounds of solid phase screening on the immune library, and enriching to obtain phage clones with binding activity; after prokaryotic induction expression is carried out on the monoclonal phage, phage clones which can be combined with the extracellular region of the CD123 antigen are further screened by ELISA;

diluting the purified CD123 recombinant protein to 4 mu g/mL by using PBS buffer solution, taking a 96-well enzyme label plate, selecting 3 wells, adding 100 mu L (400 ng/well) of CD123 recombinant protein diluent into each well, coating overnight at 4 ℃, and setting a PBS negative control group; discarding the coating solution, adding 150 mu L of 2% skimmed milk powder into each hole, sealing at room temperature for 1h, and preparing an ELISA plate coated with the CD123 recombinant protein;

washing enzyme labeling plate with PBST for 4 times, diluting the prepared phage solution with 2% skimmed milk powder to 5 × 10¹¹pfu/mL, then adding 100 mu L/hole into the enzyme label plate, and incubating for 2h at room temperature; discarding a phage sample, washing with PBST for 10 times, then washing with PBS for 5 times, adding 100 μ L of freshly prepared 0.1M triethylamine into each well, and standing at room temperature for 10 min; the eluate was immediately neutralized with an equal volume of 1M Tris-HCl (pH 7.4) by aspiration;

taking part of the eluent to determine the titer of the phage; infecting 4mL of log-phase TG1 bacterial solution (OD600 about 0.6) with 400 μ L of eluate, and incubating at 37 deg.C for 30 min; then adding 16mL of 2 XYT/ampicillin/glucose (2 XYT/AMP-GLU), and continuing to culture at 37 ℃ and 200r/min until OD600 reaches 0.6-0.8;

taking 100 mu L of bacterial suspension, performing gradient dilution, uniformly smearing the bacterial suspension on a 2 XYT/ampicillin/glucose agar plate, and performing library capacity and diversity determination; inoculating 100 mu L of bacterial suspension into a 2 XYT/AMP-GLU culture medium, culturing to a logarithmic phase, adding an auxiliary phage, performing library rescue, obtaining phage particles, performing phage titer detection, and concentrating and purifying to obtain phage particles for the next round of screening; the screening operation was repeated 3 times;

after centrifuging the residual bacterial liquid, re-suspending the residual bacterial liquid by using 2 XYT culture solution with proper volume, and smearing the re-suspended bacterial liquid on a flat plate with screening resistance for overnight culture; the bacteria were scraped from the plate using the appropriate amount of liquid culture medium, resuspended in 2 XYT medium containing 1/3 vol 50% glycerol, aliquoted, and stored at-80 ℃.

(3) Phage packaging

Adding 100 mu L of the bacterium solution elutriated in the step (2) into 100mL of 2 XYT/AMPGL culture solution, and carrying out shaking culture at 37 ℃ and 200rpm until the logarithmic phase (the OD600 value is 0.6-0.8); add 90. mu.L of the helper phage M13K07 (1.7X 10)¹³PFU/mL), standing at 37 ℃ for 30min, centrifuging at 2800g for 10min to collect the thallus, resuspending with 200mL 2 XYT/AMP-KAN culture medium, and culturing at 37 ℃ for 12h under shaking at 200 rpm;

centrifuging at 4 deg.C and 3800g for 30min, collecting supernatant, adding 1/5 volume of precooled PEG/NaCl, mixing, and precipitating bacteriophage for 2 h; centrifuging at 4 ℃ and 3800g for 30min, collecting phage, resuspending the thallus with PBS solution with the final volume of 2mL, and transferring to a 15mL centrifuge tube; centrifuging at 4 ℃ and 12000g for 15min, collecting supernatant, adding 1/5 volumes of precooled PEG/NaCl solution, turning upside down and mixing uniformly, and standing on ice for 2 h; centrifuging at 4 deg.C and 10000g for 10min, discarding supernatant, resuspending phage precipitate with 1mL PBS, shaking and incubating overnight at 4 deg.C to dissolve phage particles completely, mixing phage solution with equal volume of 60% glycerol, packaging into 1.5mL EP tube, and storing at-80 deg.C.

(4) ELISA Primary screening

As 3 rounds of panning are carried out on the phage library by adopting the CD123 antigen in the step (2), in order to avoid losing sequence diversity, ELISA primary screening is carried out on panning products of the 2 nd round and the 3 rd round, positive clones are randomly selected from the panning products and are induced to express, an expression supernatant is a crude extraction VHH antibody, and the sequence SEQ ID NO. 4 and SEQ ID NO. 5 of the VHH antibody CD123-35 sequence of a monoclonal strain are determined by sequencing.

Example 2 FACS screening of candidate clones

This example performs cell culture according to standard cell culture protocols:

digesting the cells with pancreatin to prepare a suspension of CD 123-positive cells or CD 123-negative cells, centrifuging at 300g for 5min to remove the culture medium, and resuspending the cells with a Flow Buffer to a cell concentration of 2X 10⁶Per mL;

adding 2X 10 to each well of a V-bottom 96-well plate⁵Each cell was centrifuged at 300g for 5min to remove the supernatant, and VHH antibody crude was addedThe extract is used for resuspending the cells and is incubated for 1h at 4 ℃;

centrifuging at 300g for 5min to remove supernatant, resuspending cells with Flow Buffer, adding 100. mu.L of Flow Buffer diluted APC anti-his antibody (2. mu.g/mL), and incubating at 4 ℃ for 1 h;

after the cells were washed 3 times with the Flow Buffer, the cells were resuspended using 200. mu.L of the Flow Buffer and subjected to Flow assay.

Example 3 expression, purification and affinity determination of VHH-mIgG2a Fc antibodies

To further identify the antibodies screened, the example constructed vector C-4pcp. stuffer VHH-mCg2a-FC (with mouse FC tag) expressing VHH, as follows:

the anti-CD 123 heavy chain variable region encoding gene is amplified by PCR, wherein an upstream primer of CD123-35(SEQ ID NO:5) is HD-CD 35-F, a downstream primer is HD-B8-R1, the sequence is shown in Table 2, a PCR reaction system is shown in Table 3, the reaction conditions are pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 10s, extension at 72 ℃ for 10s, 30 cycles, extension at 72 ℃ for 5min and storage at 4 ℃.

TABLE 2

TABLE 3

Subjecting the empty vector to enzyme digestion treatment at 37 deg.C for 6h, wherein the system is shown in Table 4, and the enzyme digested vector is used

The PCR purification kit was recovered and purified, and dissolved in 45. mu.L of water to detect the DNA concentration.

TABLE 4

Reagent	Dosage of
		C-4pCP.Stuffer-mCg2a-FC	～5μg
10 Xdigestion Buffer (10 Xreaction Buffer)	5μL
		FspA I	2μL
PfI 23II	2μL
		ddH₂O	Make up to 50 μ L

The PCR amplification product was ligated into the enzyme-digested linearized vector by homologous recombination, as shown in Table 5, in a 37 ℃ water bath for 30 min.

TABLE 5

Reagent	Volume (μ L)
		Exnase II	1
2×Exnase II buffer	2
		Linearized vector	4
Amplification product (Insert fragment)	3

The entire homologous recombination reaction was added to DH 5. alpha. competent cells under the transformation conditions shown in Table 6.

TABLE 6

Procedure	Temperature (. degree.C.)	Time
			Ice bath
	0	5min
			Heating of	42	1min
Ice bath
		0	3min
Adding 500. mu.L LB medium, shaking at 220rpm				37	1.5h
	Pipette
200. mu.L of the solution and spread on LB/Amp plates		37	Overnight (16 to 18h)

Selecting a single clone from a transformation plate to carry out PCR pre-identification, wherein the system is shown in Table 7, the conditions are pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, extension at 72 ℃ for 5min, storage at 4 ℃, and the amplification product is sent to a sequencing company for sequencing identification. Sequencing results were found to be in line with expectations, indicating the successful construction of expression vector C-4pCP. Stuffer VHH-mCg2a-FC with mouse Fc-tagged VHH (scFV-mIgG 1).

TABLE 7

Reagent	Volume (μ L)
		pEF1A	1
PSV40	1
		2×Fast Taq Mix	15
ddH₂O	Is supplemented to 30

Approximately 24h before plasmid transfection, 293E cells were subcultured to fineThe cell density was about 0.6X 10⁶Per mL; when the cell density is (1.0-1.2) × 10⁶unit/mL, rate of activity>At 95%, 0.15 μ g C-4pCP.Stuffer VHH-mCg2a-FC was transfected into 100mL 293E cells with PEI, the ratio of plasmid DNA to PEI being 1: 2;

293E cells transfected with plasmids at 37 ℃ at 130rpm in 8% CO₂Culturing in a shaking table for 5-7 days, centrifuging at 3000rpm for 30min, collecting supernatant, sterile filtering with Millex-GP Filter Unit 0.45 μm, and selecting with MabSelect^TMSuRe^TMConcentrating by centrifugation, washing the column with 1 × PBS, eluting the protein with 0.1M Gly-HCl, and neutralizing with 1/10 volumes of Tris-HCl at pH 8.5 to obtain antibody protein; the resulting antibody protein was dialyzed overnight at 4 ℃ and the antibody concentration was quantitatively determined by NanoDrop 2000 and the antibody purity was determined by SEC-HPLC.

The purified anti-CD 123 VHH antibody CD123-35(SEQ ID NO:4) was subjected to affinity assay using Biacore, a bioanalytical sensing technique developed based on Surface Plasmon Resonance (SPR), which can detect binding of molecules in a tracking solution to molecules on the chip surface, the course of dissociation changes, recorded as sensorgrams, and provide kinetic and affinity data. In the measurement process, the antibody is immobilized on the surface of the chip, and the mobile phase is a solution containing the antigen. The measurement results are shown in table 8 and fig. 1.

TABLE 8

Fixability of	Mobile phase	Ka(1/Ms)	Kd(1/s)	KD(M)
					0.5μg/mL CD123-35	Human IL-3R alpha/CD 123 protein	1.68E+05	2.76E-04	1.64E-09

Example 4 flow assay of anti-CD 123 VHH antibodies

After mixing K562(CD123-), KG-1 alpha (CD123+), THP-1(CD123+) tumor cells with purified recombinant CD123-35 antibody, ice-cooling for 30min, then adding APC labeled goat anti-mouse IgG antibody, incubating for 30min, and detecting with flow cytometry.

The results are shown in FIG. 2, indicating that the anti-CD 123-35 antibody recognizes CD123 antigen on the cell surface.

EXAMPLE 5 design and construction of expression vectors for chimeric antigen receptors

This example constructs a lentiviral vector expressing a CAR molecule, the CAR molecule is schematically depicted in FIG. 3 and comprises a CD8 α signal peptide (SEQ ID NO:9), anti CD123 VHH (SEQ ID NO:4), a CD8 α hinge and transmembrane region (SEQ ID NO:10), 4-1BB (SEQ ID NO:11), CD3 ζ (SEQ ID NO:12), and an amino acid sequence depicted in SEQ ID NO: 6;

SEQ ID NO:9：

MALPVTALLLPLALLLHAARP；

SEQ ID NO:10：

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC；

SEQ ID NO:11：

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；

SEQ ID NO:12：

RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

(1) the gene coding for the anti CD123 VHH is amplified by PCR, the primers are shown in Table 9, the system is shown in Table 10 (the reagents are from TOYOBO Inc.), and the PCR program is shown in Table 11;

TABLE 9

Watch 10

TABLE 11

(2) The CD8 α signal peptide was added to the scFv fragment by PCR, the primers are shown in Table 12, the PCR reaction system is shown in Table 13, and the PCR reaction was carried out according to the PCR program shown in Table 11; after the reaction is finished, detecting the PCR product by using 1% agarose gel electrophoresis, recovering fragments of about 460bp, and quantitatively detecting by using an ultraviolet absorption method;

TABLE 12

Watch 13

(3) The CD8 α hinge region-transmembrane region-41 BB-CD3 ζ (CD8 α hinge-TM-41BB-CD3Z) was amplified by PCR using the following primers:

CD8αH-F(SEQ ID NO:16)：accacgacgccagcgccgcgac；

Vector-R(SEQ ID NO:17)：tcgataagcttgatatcg；

the PCR reaction system is shown in table 14 (reagents derived from TOYOBO Inc.) and PCR reactions were performed according to the PCR program in table 11; after the reaction is finished, detecting the PCR product by using 1% agarose gel electrophoresis, recovering fragments of about 780bp, and quantitatively detecting by using an ultraviolet absorption method;

TABLE 14

(4) Mu.g of HD CD19 CAR plasmid was digested with BamHI and EcoRI in a water bath at 37 ℃ for 2h to recover the vector.

The fragments were ligated with recombinase in 37 ℃ water bath for 0.5h in the reaction system shown in Table 15, and the ligation products were transformed into E.coli stbl3 competent cells according to the conventional method. And selecting a single clone from a solid culture medium for overnight culture, performing PCR identification, wherein the system is shown in table 16, the program is shown in table 17, selecting a positive clone after PCR is finished, further performing sequencing identification, and the sequencing result is in line with expectation.

Watch 15

Reagent	Dosage of
		HD CD19 CAR	184.54ng
CD8αsignal CD123 VHH	31.32ng
		CD8αhinge-TM-41BB-CD3Z	29.72ng
5×CE buffer	2μL
		Exnase^TM II	1μL
PCR grade water	Make up to 10 mu L

TABLE 16

Reagent	Volume (μ L)
		Taq PCR Master Mix	10
10μM F Seq-trEF1a-F	1
		10μM R Vector-R	1
Template DNA bacterial liquid	1
		PCR grade water	7

TABLE 17

Example 6 Lentiviral packaging, concentration and Titer assay

(1) Lentiviral packaging

Mixing 1.6X 10⁷293T cells were plated in 15cm dishes in DMEM containing 10% Fetal Bovine Serum (FBS) at 37 ℃ with 5% CO₂Culturing overnight; adding 30 μ g pRRL. EF1 α -CD123 CAR-WPRE lentiviral vector, 12.5 μ g gag/pol helper plasmid and 10 μ g VSVg envelope plasmid into 2000 μ L serum-free DMEM medium, and mixing; mu.g PEI (1. mu.g/. mu.L) was dissolved in 2000. mu.L serum free DMEM medium, gently mixed (or vortexed at 1000rpm for 5 seconds) and incubated at room temperature for 5 min;

adding the PEI mixed solution into the DNA mixed solution, immediately mixing by vortex or gently mixing uniformly, and incubating for 20min at room temperature; dripping 4mL of the transfection compound into 293T cells, and replacing a fresh culture medium after 4-5 h; after 48h, the viral supernatant was collected.

(2) Lentiviral concentration

Filtering the virus supernatant with a 0.45-micron filter membrane, collecting the filtrate in a 50mL centrifuge tube, adding 1/4-volume PEG-NaCl virus concentrated solution, turning upside down, mixing uniformly, and standing overnight at 4 ℃; centrifuging at 4 deg.C and 3500rpm for 30min, removing supernatant, adding appropriate amount of RPMI 1640 culture medium (containing 10% FBS), and resuspending virus precipitate; the concentrated lentivirus suspension was divided into 50 μ L portions and stored at-80 ℃.

(3) Lentiviral titer detection

500 μ L K562 cells (1X 10)⁵Individual cells) were inoculated in a 24-well cell culture plate, 1. mu.L, 0.2. mu.L and 0.04. mu.L of the concentrated lentivirus, respectively, and polybrene was added to a final concentration of 5. mu.g/mL, 37 ℃ and 5% CO₂Culturing overnight, and replacing fresh culture medium;

after 72h of infection, 400g of the cells were collected by centrifugation for 5min, 100. mu.L of PBS + 2% FBS was added to resuspend the cells, 1. mu.g of hCD123-EcD-Fc antibody was added, and incubation was performed on ice for 30 min; after washing with PBS + 2% FBS for 1 time, adding 100. mu.L PBS + 2% FBS for resuspension of cells, adding APCanti-human IgG Fc antibody, and incubating on ice for 30 min; after washing with PBS + 2% FBS for 2 times, adding 300 μ L of PBS + 2% FBS for resuspension of cells, detecting infection efficiency by using a flow cytometer, preferably taking a cell sample with a positive rate of 5-20%, and calculating the lentivirus titer according to the following formula.

Titer (TU/mL) is cell number (10)⁵) X Positive Rate/Virus volume (mL)

Example 7 Lentivirally transduced T lymphocytes

Diluting anti-human CD3 antibody and anti-human CD28 antibody with PBS to final concentration of 1 μ g/mL and 0.5 μ g/mL respectively, coating the well plate, and standing overnight at 4 deg.C; discarding the antibody coating solution in the pore plate, and washing twice with 1mL of PBS;

X-VIVO + 10% FBS + IL-2(300U/mL) T cell culture medium was added to human PBMC to adjust the cell density to 1X 10⁶mL, then inoculated into CD3 and CD28 antibody coated well plates, activated for 48 h;

collecting activated T cells, adjusting cell density to 1 × 10⁶Per mL, lentivirus was added at a multiplicity of infection (MOI) of 10, polybrene was added to a final concentration of 5. mu.g/mL, and 5% CO was added at 37 ℃₂After overnight culture in the environment, replacing a fresh culture medium, and carrying out passage every 2-3 days;

after 5 days of T cell infection with lentivirus, 3X 10 cells were taken⁵Centrifuging T cells at 4 ℃ for 5min at 400g, discarding the supernatant, and washing the cells once with PBS and 2% FBS; adding 100 μ L PBS + 2% FBS to resuspend the cells, adding 0.25 μ g Human CD123 protein antibody, incubating on ice for 30min, and washing with PBS + 2% FBS for 1 time; adding 100 μ L PBS + 2% FBS to resuspend cells, adding APC anti-human IgG Fc antibody, and incubating on ice for 30 min; after washing 2 times with PBS + 2% FBS, 300. mu.L of PBS + 2% FBS was added to resuspend the cells.

The lentiviral infection efficiency was measured by flow cytometry, and the results are shown in fig. 4, where the expression efficiency of the CAR molecule was 79.2%.

Example 8 in vitro toxicity assay of CAR-T cells

293T (CD123-), 293T-CD123(CD123+), BxPC3-CD123(CD123+) were adjusted to a cell concentration of 1X 10⁵mL, 100. mu.L of the target cells were inoculatedSeeding in 96-well plates; adding CD123CAR-T cells and control T cells to a 96-well plate at an effective target ratio of 0.3:1, 1:1, and 3: 1; each set was provided with 3 multiple wells. The experimental and control groups were as follows:

experimental groups: each target cell + CAR-T;

control group 1: maximal release of LDH by target cells;

control group 2: target cells spontaneously release LDH;

control group 3: the effector cells spontaneously release LDH;

after the effector cells and the target cells are co-cultured for 18h, the killing effect of the effector cells on the target cells is detected by using a CytoTox 96 nonradioactive cytotoxicity detection kit (Promega). The method is based on a colorimetric method, and reflects the cracking degree of cells by detecting the content of Lactate Dehydrogenase (LDH). LDH is a stable cytosolic enzyme that is released after cell lysis in a manner similar to that of⁵¹Cr is released in the radioactive assay in essentially the same manner, and the released LDH can be detected by a coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan (formazan), with the amount of red product produced being proportional to the number of cells lysed. Reference is made in particular to the instructions of the CytoTox 96 nonradioactive cytotoxicity detection kit.

The killing ability of effector cells to target cells was calculated according to the following cytotoxicity calculation formula:

percent cytotoxicity ═ 100% (experimental-control 2-control 3)/(control 1-control 2) ×

The results are shown in figure 5, where CD123CAR-T cells have killing activity against CD123 positive tumor cells, but no killing effect on CD123 negative cells.

Example 9 CAR-T cytokine secretion

Centrifuging 400g of a cell culture with an effective target ratio of 1:1 for 10min, removing precipitates, placing 100 mu L of supernatant into a sample hole of an enzyme label plate, adding 50 mu L of detection antibody diluent (1:100 dilution), and performing shaking incubation for 2 hours at room temperature at 300rpm/min by using a plate sealing membrane sealing plate; discarding liquid, adding 300 mu L of washing liquid into each hole to wash the plate for 6 times, and drying the ELISA plate on absorbent paper after washing the plate each time;

adding 100 μ L of horse radish peroxidase labeled streptavidin diluent (1:100 dilution) into each well, sealing with a new sealing plate membrane, and incubating at room temperature at 300rpm/min under shaking for 45 min; discarding liquid, adding 300 mu L of washing liquid into each hole to wash the plate for 6 times, and drying the ELISA plate on absorbent paper after washing the plate each time;

adding 100 mu L of chromogenic substrate TMB into each hole, incubating for 5-30 min at room temperature in the dark, then adding 100 mu L of stop solution into each hole, changing the color from blue to yellow, measuring the OD value at the maximum absorption wavelength of 450nm and the reference wavelength of 570nm or 630nm by using an enzyme labeling instrument, and subtracting the measured value of 570nm or 630nm from the measured value of 450nm by using the calibrated OD value.

The secretion results of IL-2, TNF-alpha and IFN-gamma factors are respectively shown in figure 6, figure 7 and figure 8, and the constructed CAR-T cells release cytokines after being incubated with CD123 positive tumor cells, but have no obvious cytokine secretion after being incubated with CD123 negative cells.

In conclusion, the invention utilizes phage display technology to screen camel VHH immune libraries immunized by CD123 to obtain anti-CD 123 antibody with high affinity; the anti-CD 123 antibody is used as an antigen binding structural domain to construct a chimeric antigen receptor, a chimeric antigen receptor gene is introduced into immune cells through a genetic engineering method, and the prepared CD123 chimeric antigen receptor immune cells can specifically recognize and kill tumor cells expressing CD123, so that the anti-tumor effect is realized.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

<110> Huadao (Shanghai) biopharmaceutical Co., Ltd

<120> CD123 antigen binding fragment and application thereof

<130> 20201210

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Val Ser Arg Val Gly Trp Thr

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Ala Ala Asp Ser Arg Val Cys Gly Leu Arg Ser Gly Arg Lys Tyr Thr

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Asp

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Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Thr Val

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

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Thr Ile Ser Arg Asp Asn Ala Lys Asn Ala Leu Tyr Leu Gln Met Asn

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Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Asp Ser

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Arg Val Cys Gly Leu Arg Ser Gly Arg Lys Tyr Thr Asp Trp Gly Gln

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Gly Thr Gln Val Thr Val Ser Ser

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser

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Val Gln Ala Gly Gly Ser Leu Lys Leu Ser Cys Ser Ala Ser Glu Asn

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Val Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu

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

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

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Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr

100 105 110

Cys Ala Ala Asp Ser Arg Val Cys Gly Leu Arg Ser Gly Arg Lys Tyr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe

245 250 255

Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu

260 265 270

Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp

275 280 285

Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys

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Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala

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

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

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

355 360

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gcatggagga cagggcttga ttgtggggct agacactgtc acctg 45

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Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

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His Ala Ala Arg Pro

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Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

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Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

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Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

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Ile Thr Leu Tyr Cys

65

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

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

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Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

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

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

100 105 110

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ctgccgctgg ccttgctgct ccacgccgcc aggccggaag tgcagctggt ggagagcggc 60

ggcggat 67

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gcgctggcgt cgtggtgcta gacactgtca cc 32

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<213> Artificial sequence ()

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gctgcaggtc gactctagag gatcccgcca ccatggcctt accagtgacc gccttgctcc 60

tgccgctggc cttgc 75

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<213> Artificial sequence ()

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accacgacgc cagcgccgcg ac 22

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tcgataagct tgatatcg 18

Claims

1. A CD123 antigen binding fragment, wherein said CD123 antigen binding fragment comprises the heavy chain CDR1 of SEQ ID NO. 1, the heavy chain CDR2 of SEQ ID NO. 2, and the heavy chain CDR3 of SEQ ID NO. 3.

2. An anti-CD 123 antibody, wherein said anti-CD 123 antibody comprises the CD123 antigen-binding fragment of claim 1;

preferably, the anti-CD 123 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 4.

3. A nucleic acid molecule comprising a DNA segment encoding the CD123 antigen-binding fragment of claim 1;

preferably, the nucleic acid molecule comprises a DNA fragment encoding the anti-CD 123 antibody of claim 2;

preferably, the nucleic acid molecule comprises the nucleic acid sequence shown in SEQ ID NO. 5.

4. A chimeric antigen receptor comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, and a signaling domain;

the antigen binding domain is the anti-CD 123 antibody of claim 2.

5. The chimeric antigen receptor according to claim 4, wherein the signal peptide comprises a CD8 a signal peptide;

preferably, the hinge region comprises a CD8 a hinge region;

preferably, the transmembrane domain comprises any one or a combination of at least two of the CD8 a transmembrane region, CD28 transmembrane region, or DAP10 transmembrane region;

preferably, the signaling domain comprises CD3 ζ;

preferably, the signaling domain further comprises any one of or a combination of at least two of the 4-1BB, CD28 intracellular domains, OX40, ICOS or DAP10 intracellular domains;

preferably, the chimeric antigen receptor comprises the anti-CD 123 antibody of claim 2, a CD8 a transmembrane region, 4-1BB, and CD3 ζ;

preferably, the chimeric antigen receptor comprises the anti-CD 123 antibody of claim 2, a CD28 transmembrane region, a CD28 intracellular region, and a CD3 ζ;

preferably, the chimeric antigen receptor comprises the anti-CD 123 antibody of claim 2, a CD8 a transmembrane region, OX40, and CD3 ζ;

preferably, the chimeric antigen receptor comprises the anti-CD 123 antibody of claim 2, a CD8 a transmembrane region, ICOS, and CD3 ζ;

preferably, the chimeric antigen receptor comprises the anti-CD 123 antibody of claim 2, a CD28 transmembrane region, a CD28 intracellular region, OX40, and CD3 ζ;

preferably, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 6.

6. An expression vector comprising a gene encoding the chimeric antigen receptor of claim 4 or 5;

preferably, the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector containing the gene encoding the chimeric antigen receptor according to claim 4 or 5, preferably a lentiviral vector.

7. A recombinant lentivirus prepared from a mammalian cell transfected with the expression vector of claim 6 and a helper plasmid.

8. A chimeric antigen receptor immune cell, wherein said chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 4 or 5;

preferably, the chimeric antigen receptor immune cell comprises the expression vector of claim 6 and/or the recombinant lentivirus of claim 7;

9. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of claim 8;

10. Use of the CD123 antigen-binding fragment of claim 1, the anti-CD 123 antibody of claim 2, the nucleic acid molecule of claim 3, the chimeric antigen receptor of claim 4 or 5, the expression vector of claim 6, the recombinant lentivirus of claim 7, the chimeric antigen receptor immune cell of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for the treatment of a malignant tumor;

preferably, the malignancy comprises a hematological tumor.