CN112480258B

CN112480258B - anti-CD 123 nano antibody and application thereof

Info

Publication number: CN112480258B
Application number: CN202011458886.6A
Authority: CN
Inventors: 狄升蒙; 侯莉; 石磊; 刘芳; 茅健; 余学军
Original assignee: Huadao Shanghai Biopharma Co ltd
Current assignee: Huadao Shanghai Biopharma Co ltd
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2022-07-29
Anticipated expiration: 2040-12-11
Also published as: CN112480258A

Abstract

The invention provides an anti-CD 123 nano antibody and application thereof, wherein the nano antibody comprises a heavy chain variable region; the heavy chain variable region includes CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3. The antibody screened from the camel VHH immune library has the CDR regions shown in SEQ ID NO 1-3, and the nano antibody formed by combining with different framework region FRs has strong affinity with CD123, so that the constructed chimeric antigen receptor and the chimeric antigen receptor immune cell have obvious cytotoxicity to CD123 positive cells, and have wide application prospect in the field of tumor treatment.

Description

anti-CD 123 nano antibody and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and relates to an anti-CD 123 nano antibody 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 significant 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 CD123 CAR-T, so that the CD123 CAR-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 an anti-CD 123 nano antibody and application thereof, the anti-CD 123 nano antibody with high affinity 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-T cell, and the CAR-T 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 present invention provides a nanobody against CD123, comprising a heavy chain variable region;

the heavy chain variable region comprises CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;

SEQ ID NO:1：GFTFSSYD；

SEQ ID NO:2：IKPGPVGT；

SEQ ID NO:3：ARGSRYSWNLDGPL。

in the invention, an antibody with high affinity is screened from a CD123 immune camel VHH immune library, and the result shows that when the CDR region of the heavy chain variable region of the antibody is the amino acid sequence shown in SEQ ID NO. 1-3, the antibody which is specifically combined with CD123 and has strong affinity can be obtained by combining with different framework region FRs, so that the CDR region shown in SEQ ID NO. 1-3 has a decisive effect on the affinity of the antibody and CD 123.

Preferably, the heavy chain variable region of the nanobody comprises the amino acid sequence shown in SEQ ID NO. 4 or SEQ ID NO. 5;

SEQ ID NO:4：

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSHIKPGPVGTDYTDSVKGRFTISRDNAKNTLYLQMDSLKTEDTAVYYCARGSRYSWNLDGPLRGQGTQVTVSS；

SEQ ID NO:5：

EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSHIKPGPVGTDYTDSVKGRFTISRDNAKNTLYLQMDSLKTEDTAVYYCARGSRYSWNLDGPLRGQGTQVTVSS。

in the invention, a phage display technology is utilized to screen a CD123 immune camel VHH library to obtain anti-CD 123 nano antibodies CD123-9(SEQ ID NO:4) and CD123-12(SEQ ID NO:5) only containing a heavy chain variable region, wherein the two nano antibodies have the same CDR region SEQ ID NO: 1-3, have high affinity to CD123 and have important application prospect in the aspect of constructing a chimeric antigen receptor targeting CD 123.

In a second aspect, the present invention provides a nucleic acid molecule comprising a DNA fragment encoding the nanobody of the first aspect.

Preferably, the nucleic acid molecule comprises a nucleic acid sequence shown as SEQ ID NO. 6 or SEQ ID NO. 7, wherein SEQ ID NO. 6 is a coding sequence of CD123-9, and SEQ ID NO. 7 is a coding sequence of CD 123-12;

SEQ ID NO:6：

GAGGTGCAGCTGGTGGAGAGCGGAGGAGGCCTGGTGCAGCCTGGCGGCAGCCTGAGACTGTCTTGTGCCGCCTCTGGCTTTACATTTTCTTCTTATGATATGAGCTGGGTGAGACAGGCTCCAGGAAAAGGACTGGAGTGGGTGTCTCATATCAAACCTGGACCAGTGGGCACCGATTATACAGATAGCGTGAAAGGCAGATTCACAATTTCTAGAGATAATGCCAAAAATACACTGTATCTGCAGATGGACAGCCTGAAGACCGAGGACACCGCCGTGTACTACTGCGCCAGAGGCAGCAGATACAGCTGGAACCTGGACGGCCCCCTGAGAGGCCAGGGCACCCAGGTGACCGTGAGCAGC；

SEQ ID NO:7：

GAGGTGCAGCTGGTGGAGTCTGGCGGCGATCTGGTGCAGCCTGGCGGATCTCTGAGACTGTCTTGCGCTGCCTCCGGATTCACATTTTCTAGCTATGATATGTCTTGGGTGAGACAGGCCCCAGGAAAGGGACTGGAATGGGTGTCTCACATCAAGCCTGGACCTGTGGGAACAGATTATACAGATAGCGTGAAAGGAAGGTTTACAATTTCTAGAGATAATGCCAAAAATACACTGTATCTGCAGATGGATTCTCTGAAAACAGAGGATACCGCCGTGTATTATTGTGCCAGAGGATCTAGATATTCTTGGAATCTGGATGGACCTCTGAGAGGCCAGGGCACACAGGTGACCGTGAGCTCT。

in a third 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 a nanobody according to the first aspect.

In the invention, the nano antibody of the first aspect is used as an extracellular region of the chimeric antigen receptor and 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 is referred to NM _001145873, the sequence of the CD28 transmembrane region is referred to NM _006139 and the sequence of the DAP10 transmembrane region is referred to NM _ 014266.

Preferably, the signaling domain comprises CD3 ζ and the sequence can be referred 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 ζ sequentially from N-terminus to C-terminus.

Preferably, the chimeric antigen receptor comprises the nanobody of the first aspect, a CD8 α transmembrane region, 4-1BB, and CD3 ζ in sequence from N-terminus to C-terminus.

Preferably, the chimeric antigen receptor comprises the nanobody of the first aspect, a CD28 transmembrane region, a CD28 intracellular region and a CD3 ζ in sequence from N-terminus to C-terminus.

Preferably, the chimeric antigen receptor comprises the nanobody of the first aspect, a CD8 α transmembrane region, OX40 and CD3 ζ in sequence from N-terminus to C-terminus.

Preferably, the chimeric antigen receptor comprises the nanobody of the first aspect, a CD8 α transmembrane region, ICOS, and CD3 ζ in sequence from N-terminus to C-terminus.

Preferably, the chimeric antigen receptor comprises the nanobody of the first aspect, a CD28 transmembrane region, a CD28 intracellular region, OX40 and CD3 ζ in sequence from N-terminus to C-terminus.

Preferably, the chimeric antigen receptor comprises an amino acid sequence shown as SEQ ID NO. 8 or SEQ ID NO. 9;

8(CD 8. alpha. signal peptide-CD 123-9-CD 8. alpha. hinge-TM-41BB-CD3 ζ):

MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSHIKPGPVGTDYTDSVKGRFTISRDNAKNTLYLQMDSLKTEDTAVYYCARGSRYSWNLDGPLRGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；

9(CD 8. alpha. signal peptide-CD 123-12-CD 8. alpha. hinge-TM-41BB-CD3 ζ):

MALPVTALLLPLALLLHAARPEVQLVESGGDLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSHIKPGPVGTDYTDSVKGRFTISRDNAKNTLYLQMDSLKTEDTAVYYCARGSRYSWNLDGPLRGQGTQVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。

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

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

In a fifth 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 fourth aspect.

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

Preferably, the chimeric antigen receptor immune cell comprises the expression vector of the fourth aspect and/or the recombinant lentivirus of the fifth 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 a seventh aspect, the present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cell of the sixth 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 an eighth aspect, the present invention provides a nanobody of the first aspect, a nucleic acid molecule of the second aspect, a chimeric antigen receptor of the third aspect, an expression vector of the fourth aspect, a recombinant lentivirus of the fifth aspect, a chimeric antigen receptor immune cell of the sixth aspect, or a pharmaceutical composition of the seventh 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 nano antibody library, and screen out nano antibodies which can specifically combine CD123 antigen with CD123-9 and CD 123-12;

(2) the invention further utilizes the screened nano 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. 1A shows the affinity of Biacore for detecting CD123-9 antibody, and FIG. 1B shows the affinity of Biacore for detecting CD123-12 antibody;

FIG. 2 shows FACS detection of CD123-9 and CD123-12 nanobodies recognizing CD123 antigen on the cell surface;

FIG. 3 is a pRRL. EF1 α -CD123 CAR-WPRE lentiviral vector plasmid map;

FIG. 4 is a schematic diagram of the structure of a chimeric antigen receptor expressing CD 123;

FIG. 5 shows the chimeric antigen receptor expression rate of T lymphocytes;

FIG. 6 is the killing effect of CD123 CAR-T cells on CD123 positive tumor cells;

FIG. 7 is the level of IL-2 secretion by CD123 CAR-T cells;

FIG. 8 is the level of TNF α secretion by CD123 CAR-T cells;

FIG. 9 is the level of IFN γ secretion by CD123 CAR-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 Nanobody library, panning and ELISA Primary screening

(1) Construction of phage Nanobody 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

Reverse transcriptase synthesis of first strand cDNA followed by amplification of VHH gene 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 volume of 50% glycerol, and mixingSubpackaging, and storing at-80 deg.C to obtain a product with a storage capacity of greater than 10 ⁹ The phage display camelid VHH immune library of (a).

(2) Panning of phage Nanobody 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 × 10 ¹¹ pfu/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 eluate 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 rest bacterial liquid, resuspending the rest bacterial liquid by using 2 XYT culture solution with proper volume, and smearing the 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 of 50% glycerol, split-filled, 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 helper phage M13K07 (1.7X 10) ¹³ PFU/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

In the step (2), 3 rounds of panning are carried out on the phage library by adopting the CD123 antigen, 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 a VHH antibody sequence of a monoclonal strain is 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 10 ⁶ Per mL;

adding 2X 10 to each well of a V-bottom 96-well plate ⁵ Centrifuging at 300g for 5min to remove supernatant, adding VHH antibody crude extract to resuspend cells, and incubating at 4 ℃ for 1 h;

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 Nanobodies

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

the gene encoding the CD 123-resistant heavy chain variable region is amplified by PCR, wherein an upstream primer of CD123-9(SEQ ID NO:6) is HD-F, a downstream primer is HD-B12-R2, an upstream primer of CD123-12(SEQ ID NO:7) is HD-F, a downstream primer is HD-CD-12-R, 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, elongation at 72 ℃ for 10s, 30 cycles, elongation 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-4 pCP.Stuffer-mCg2a-FC	～5μg
10 Xdigestion Buffer (10 Xreaction Buffer)	5μL
		FspA I	2μL
PfI 23II	2μL
		ddH ₂ O	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 a VHH expressing vector C-4 pcp. stuffer VHH-mCg2a-Fc with a mouse Fc tag.

TABLE 7

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

Approximately 24h before plasmid transfection, 293E cells were subcultured to a cell density of approximately 0.6X 10 ⁶ Per mL; when the cell density is (1.0-1.2) × 10 ⁶ unit/mL, rate of activity>At 95%, 0.15. mu. g C-4 pCP.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% CO ₂ Culturing 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 ^TM SuRe ^TM Concentrating 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 antibodies CD123-9(SEQ ID NO:4), CD123-12(SEQ ID NO:5) were subjected to affinity assay using Biacore, a bioanalytical sensing technique developed based on Surface Plasmon Resonance (SPR), which can detect the binding and dissociation changes of molecules in the tracking solution to the chip surface molecules, record them in sensorgram form, 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, fig. 1A and fig. 1B.

TABLE 8

Fixability of	Mobile phase	Ka(1/Ms)	Kd(1/s)	KD(M)
					0.5μg/mL CD123-9	Human IL-3R alpha/CD 123 protein	3.05E+05	1.42E-05	4.66E-11
0.5μg/mL CD123-12	Human IL-3R alpha/CD 123 protein	2.95E+05	1.62E-05	5.51E-11

Example 4 flow assay of anti-CD 123 VHH Nanobodies

After mixing K562(CD123-), KG-1 alpha (CD123+), THP-1(CD123+) tumor cells with purified recombinant anti-CD 123-9/21 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, which indicates that the nanobody can recognize the CD123 antigen on the cell surface.

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

This example constructs lentiviral vectors expressing a CAR molecule with an EFS-anti-CD123 VHH CAR-WPRE map as shown in FIG. 3 and a schematic representation of the CAR molecule as shown in FIG. 4, comprising the CD8 α signal peptide (SEQ ID NO:13), anti CD123 VHH (SEQ ID NO:4/SEQ ID NO:5), CD8 α hinge and transmembrane regions (SEQ ID NO:14), 4-1BB (SEQ ID NO:15), CD3 ζ (SEQ ID NO:16) with an amino acid sequence as shown in SEQ ID NO:8 or SEQ ID NO: 9;

SEQ ID NO:13：

MALPVTALLLPLALLLHAARP；

SEQ ID NO:14：

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC；

SEQ ID NO:15：

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；

SEQ ID NO:16：

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

Reagent	Volume (μ L)
		10×buffer	5
2mM dNTP	5
		25mM MgSO ₄	3
10 μ M upstream primer	1
		10 μ M upstream primer	1
Template DNA (cDNA clone)	1
		PCR grade water	33
KOD-Plus-Neo	1

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:22)：accacgacgccagcgccgcgac；

Vector-R(SEQ ID NO:23)：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 10 ⁷ 293T cells were plated in 15cm dishes in DMEM containing 10% Fetal Bovine Serum (FBS) at 37 ℃ with 5% CO ₂ Culturing 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) ⁵ Individual 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% CO ₂ Culturing overnight, and replacing fresh culture medium;

after 72h of infection, 400g was centrifuged for 5min to collect cells, 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 on ice was carried out 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) as number of cells (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 10 ⁶ mL, then inoculated into CD3 and CD28 antibody coated well plates, activated for 48 h;

collecting activated T cells, adjusting cell density to 1 × 10 ⁶ Per 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 ℃ ₂ After 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 taken ⁵ Centrifuging 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 lentivirus infection efficiency was measured by flow cytometry and the results are shown in FIG. 5.

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 10 ⁵ /mL，Inoculating 100 μ L of the target cells in a 96-well plate; adding CD123 CAR-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 of ⁵¹ Cr 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 6, where CD123 CAR-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 the liquid, adding 300 mu L of washing liquid into each hole to wash the plate for 6 times, and patting the enzyme label plate on absorbent paper after washing the plate each time;

adding 100 mu L of horseradish peroxidase labeled streptavidin diluent (diluted 1: 100) into each well, sealing the wells with a new sealing plate, and incubating at room temperature at 300rpm/min under oscillation 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 a dark place, then adding 100 mu L of stop solution into each hole, changing the color from blue to yellow, measuring the OD value under 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 after calibration.

The secretion results of IL-2, TNF-alpha and IFN-gamma factors are respectively shown in figure 7, figure 8 and figure 9, 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, and obtains anti-CD 123 nano antibodies with high affinity; the anti-CD 123 nano 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 gene 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> anti-CD 123 nanobody and application thereof

<130> 20201210

<160> 23

<170> SIPOSequenceListing 1.0

<210> 1

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<212> PRT

<213> Artificial sequence ()

<400> 1

Gly Phe Thr Phe Ser Ser Tyr Asp

1 5

<210> 2

<211> 8

<212> PRT

<213> Artificial sequence ()

<400> 2

Ile Lys Pro Gly Pro Val Gly Thr

1 5

<210> 3

<211> 14

<212> PRT

<213> Artificial sequence ()

<400> 3

Ala Arg Gly Ser Arg Tyr Ser Trp Asn Leu Asp Gly Pro Leu

1 5 10

<210> 4

<211> 121

<212> PRT

<213> Artificial sequence ()

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ser His Ile Lys Pro Gly Pro Val Gly Thr Asp Tyr Thr Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ser Arg Tyr Ser Trp Asn Leu Asp Gly Pro Leu Arg Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 5

<211> 121

<212> PRT

<213> Artificial sequence ()

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ser His Ile Lys Pro Gly Pro Val Gly Thr Asp Tyr Thr Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gly Ser Arg Tyr Ser Trp Asn Leu Asp Gly Pro Leu Arg Gly

100 105 110

Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 6

<211> 363

<212> DNA

<213> Artificial sequence ()

<400> 6

gaggtgcagc tggtggagag cggaggaggc ctggtgcagc ctggcggcag cctgagactg 60

tcttgtgccg cctctggctt tacattttct tcttatgata tgagctgggt gagacaggct 120

ccaggaaaag gactggagtg ggtgtctcat atcaaacctg gaccagtggg caccgattat 180

acagatagcg tgaaaggcag attcacaatt tctagagata atgccaaaaa tacactgtat 240

ctgcagatgg acagcctgaa gaccgaggac accgccgtgt actactgcgc cagaggcagc 300

agatacagct ggaacctgga cggccccctg agaggccagg gcacccaggt gaccgtgagc 360

agc 363

<210> 7

<211> 363

<212> DNA

<213> Artificial sequence ()

<400> 7

gaggtgcagc tggtggagtc tggcggcgat ctggtgcagc ctggcggatc tctgagactg 60

tcttgcgctg cctccggatt cacattttct agctatgata tgtcttgggt gagacaggcc 120

ccaggaaagg gactggaatg ggtgtctcac atcaagcctg gacctgtggg aacagattat 180

acagatagcg tgaaaggaag gtttacaatt tctagagata atgccaaaaa tacactgtat 240

ctgcagatgg attctctgaa aacagaggat accgccgtgt attattgtgc cagaggatct 300

agatattctt ggaatctgga tggacctctg agaggccagg gcacacaggt gaccgtgagc 360

tct 363

<210> 8

<211> 365

<212> PRT

<213> Artificial sequence ()

<400> 8

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

1 5 10 15

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

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser His Ile Lys Pro Gly Pro Val Gly Thr Asp

65 70 75 80

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

85 90 95

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

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Gly Ser Arg Tyr Ser Trp Asn Leu Asp

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

<210> 9

<211> 365

<212> PRT

<213> Artificial sequence ()

<400> 9

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

1 5 10 15

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

20 25 30

Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe

35 40 45

Thr Phe Ser Ser Tyr Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys

50 55 60

Gly Leu Glu Trp Val Ser His Ile Lys Pro Gly Pro Val Gly Thr Asp

65 70 75 80

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

85 90 95

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

100 105 110

Ala Val Tyr Tyr Cys Ala Arg Gly Ser Arg Tyr Ser Trp Asn Leu Asp

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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<212> DNA

<213> Artificial sequence ()

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cgcgattctt aagggtgtcc agtgcgaggt gcagctggtg ga 42

<210> 11

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 11

gcatggagga cagggcttga ttgtggggct gctcacggtc acctg 45

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 12

gcatggagga cagggcttga ttgtgggaga gctcacggtc acctg 45

<210> 13

<211> 21

<212> PRT

<213> Artificial sequence ()

<400> 13

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

1 5 10 15

His Ala Ala Arg Pro

20

<210> 14

<211> 69

<212> PRT

<213> Artificial sequence ()

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 15

<211> 42

<212> PRT

<213> Artificial sequence ()

<400> 15

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

1 5 10 15

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

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 16

<211> 112

<212> PRT

<213> Artificial sequence ()

<400> 16

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 17

<211> 67

<212> DNA

<213> Artificial sequence ()

<400> 17

ctgccgctgg ccttgctgct ccacgccgcc aggccggagg tgcagctggt ggagagcgga 60

ggaggcc 67

<210> 18

<211> 32

<212> DNA

<213> Artificial sequence ()

<400> 18

gcgctggcgt cgtggtgctg ctcacggtca cc 32

<210> 19

<211> 67

<212> DNA

<213> Artificial sequence ()

<400> 19

ctgccgctgg ccttgctgct ccacgccgcc aggccggagg tgcagctggt ggagtctggc 60

ggcgatc 67

<210> 20

<211> 32

<212> DNA

<213> Artificial sequence ()

<400> 20

gcgctggcgt cgtggtagag ctcacggtca cc 32

<210> 21

<211> 75

<212> DNA

<213> Artificial sequence ()

<400> 21

gctgcaggtc gactctagag gatcccgcca ccatggcctt accagtgacc gccttgctcc 60

tgccgctggc cttgc 75

<210> 22

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 22

accacgacgc cagcgccgcg ac 22

<210> 23

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 23

tcgataagct tgatatcg 18

Claims

1. A nanobody against CD123, consisting of a heavy chain variable region;

the heavy chain variable region includes CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3.

2. The nanobody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the nanobody comprises the amino acid sequence shown in SEQ ID No. 4 or SEQ ID No. 5.

3. A nucleic acid molecule comprising a DNA fragment encoding the nanobody of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein the nucleotide sequence of the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO. 6 or SEQ ID NO. 7.

5. 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 a nanobody according to claim 1 or 2.

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

the hinge region comprises a CD8 a hinge region;

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

the signaling domain comprises CD3 ζ;

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

7. The chimeric antigen receptor according to claim 6, wherein the chimeric antigen receptor comprises the nanobody of claim 1 or 2, CD8 a transmembrane region, 4-1BB, and CD3 ζ.

8. The chimeric antigen receptor according to claim 6, wherein the chimeric antigen receptor comprises the nanobody of claim 1 or 2, the CD28 transmembrane region, the CD28 intracellular region, and CD3 ζ.

9. The chimeric antigen receptor according to claim 6, wherein the chimeric antigen receptor comprises the nanobody of claim 1 or 2, a CD8 a transmembrane region, OX40, and CD3 ζ.

10. The chimeric antigen receptor according to claim 6, wherein the chimeric antigen receptor comprises the nanobody of claim 1 or 2, a CD8 a transmembrane region, ICOS and CD3 ζ.

11. The chimeric antigen receptor according to claim 6, wherein the chimeric antigen receptor comprises the nanobody of claim 1 or 2, the CD28 transmembrane region, the CD28 intracellular region, OX40, and CD3 ζ.

12. The chimeric antigen receptor according to claim 6, wherein the amino acid sequence of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO. 8 or SEQ ID NO. 9.

13. An expression vector comprising a gene encoding the chimeric antigen receptor of any one of claims 5-12.

14. The expression vector of claim 13, wherein the expression vector is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector comprising the gene encoding the chimeric antigen receptor of any one of claims 5 to 12.

15. The expression vector of claim 14, wherein the expression vector is a lentiviral vector.

16. A recombinant lentivirus prepared from a mammalian cell transfected with the expression vector of claim 15 and a helper plasmid.

17. A chimeric antigen receptor immune cell, wherein said chimeric antigen receptor immune cell expresses the chimeric antigen receptor of any one of claims 5-12.

18. The chimeric antigen receptor immune cell according to claim 17, characterized in that it comprises the expression vector of any one of claims 13-15 and/or the recombinant lentivirus of claim 16;

the immune cells include any one of T cells, B cells, NK cells, mast cells or macrophages or a combination of at least two of the same.

19. A pharmaceutical composition comprising the chimeric antigen receptor immune cell of claim 17 or 18;

the pharmaceutical composition further comprises any one or a combination of at least two of pharmaceutically acceptable carriers, excipients or diluents.

20. Use of the nanobody of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the chimeric antigen receptor of any one of claims 5 to 12, the expression vector of any one of claims 13 to 15, the recombinant lentivirus of claim 16, the chimeric antigen receptor immune cell of claim 17 or 18, or the pharmaceutical composition of claim 19 for the preparation of a medicament for the treatment of a malignant tumor;

the malignant tumor is leukemia.