CN116063569A

CN116063569A - EPHA2 chimeric antigen receptor and uses thereof

Info

Publication number: CN116063569A
Application number: CN202211354863.XA
Authority: CN
Inventors: 孙卫平; 张丹丹
Original assignee: Jinan Taihe Pharmaceutical Technology Co ltd
Current assignee: Bonentai Shandong Biomedical Technology Group Co ltd
Priority date: 2022-11-01
Filing date: 2022-11-01
Publication date: 2023-05-05
Anticipated expiration: 2042-11-01
Also published as: CN116063569B

Abstract

The invention belongs to the technical fields of immunology and molecular biology, and particularly relates to an EPHA2 chimeric antigen receptor and application thereof. According to the invention, a series of novel anti-EPHA 2 single-domain antibodies are obtained and subjected to humanized design, so that the novel anti-EPHA 2 single-domain antibodies can be combined with an EPHA2 antigen, and an EPHA2 chimeric antigen receptor is further designed and targeted, and experiments prove that the CAR-T cells for expressing the EPHA2 chimeric antigen receptor have excellent expression rate and durability, and the cells show good lasting stability and anti-tumor, especially anti-brain glioma effects, and meanwhile, the experiment proves that under the condition of lower application dosage, obvious curative effects can be obtained, so that the safety is higher, and the novel EPHA2 chimeric antigen receptor has good practical application value.

Description

EPHA2 chimeric antigen receptor and uses thereof

Technical Field

The invention belongs to the technical fields of immunology and molecular biology, and particularly relates to an EPHA2 chimeric antigen receptor and application thereof.

Background

The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Brain gliomas are the most common and most invasive advanced primary adult brain tumors. Current standard of care treatments, including surgical excision, radiation therapy, and chemotherapy, can extend the patient's lifetime to 14-15 months. Survival rate is about 10% in 5 years, and final mortality rate is close to 100%. The brain glioma is virtually incurable due to heterogeneity, immune escape, infiltration and protection of the blood brain barrier. Therefore, there is an urgent need to develop new therapeutic methods to eliminate brain gliomas.

T cells expressing chimeric antigen receptor show remarkable curative effect in treating malignant hematopathy, and provide wide prospect for tumor treatment. Such immunotherapy may involve the modification of T cells by viral or plasmid vectors to express a Chimeric Antigen Receptor (CAR), to recognize tumor-associated antigens (TAAs) or antigens over-expressed by cancer cells, and to utilize the ability of T cells to kill malignant cells. The CAR molecule consists of an extracellular antigen recognition region, a transmembrane region and an intracellular signal activation region, and T cells modified by the CAR molecule can directly attack tumors expressing surface antigens.

ephrin type a receptor 2 (EPHA 2) is a transmembrane tyrosine kinase receptor, belonging to one of the superfamily of tyrosine kinase Receptor (RTKs). Like other tyrosine kinases, the EPHA2 molecule can be divided into three domains, and an extracellular ligand-binding region, an intracellular tyrosine kinase active functional region, and a transmembrane region. EPHA2 is involved in the development of the nervous system and vascular system, and EPHA2 is significantly over-expressed in malignant gliomas compared to benign tumors and is significantly correlated with a poorer prognosis, further demonstrating that EPHA2 contributes to malignant transformation of tumors. The EphA2/Ephrin A1 system can be targeted for cancer treatment by at least two mechanisms. First, the oncogenic function of EphA2 may be inhibited, such as decreasing EphA2 expression, promoting EphA2 degradation, and blocking activation of endogenous EphA 2. Alternatively, ephA2 receptors can be used to deliver therapeutic agents (exogenous agents or endogenous immune cells) to cancer cells and related blood vessels. EphA2 targeted therapy has emerged in clinical trials of various types and stages of malignancy. Strategies for clinically inhibiting EphA2 include EphA 2-targeted antibody-cytotoxic drug conjugates (ADCs) or peptide-drug conjugates (PDCs); tyrosine Kinase Inhibitors (TKIs), such as dasatinib, which has been approved for the market; CAR-T cells that recognize and target EphA2 antigen; and nanocarriers designed to deliver EphA 2-targeted siRNAs to tumor cells. Future strategies for potential inhibition of non-canonical signals may also include EphA2 agonists, such as soluble EphA2 agonists (A1-Fc), or other small molecule inhibitors, to block EphA2 phosphorylation at S897 (Akti/rski/PKAi).

There are studies showing that EPHA2 plays an important role in brain glioma development, including invasion, metastasis and angiogenesis. There are also researchers that have successfully demonstrated the anti-tumor activity of EPHA2 as a target for CAR-T treatment glioma xenograft models; however, data on duration of remission is limited and most remain in the research and experimental stages. EPHA2 is overexpressed on almost all brain gliomas, but is minimally or not expressed at all in normal brain tissue. Thus, EPHA2 is considered a suitable target for brain glioma for developing CAR-T cell immunotherapy.

Disclosure of Invention

The present invention provides EPHA2 chimeric antigen receptors and uses thereof. It is an object of the present invention to provide CAR-T cells, CAR expression vectors and CAR treatment platforms having significant therapeutic advantages in tumors (such as brain gliomas) with high expression of EPHA2 compared to known CAR-T cells.

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

in a first aspect of the invention there is provided an EPHA2 chimeric antigen receptor comprising at least an antigen binding domain, a transmembrane domain, a costimulatory domain, and a signaling domain, wherein the antigen binding domain is an anti-EPHA 2 single domain antibody;

Wherein the amino acid sequence of the anti-EPHA 2 single domain antibody is selected from the group consisting of:

(a) Amino acid sequences as shown in SEQ ID NO.5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39; or alternatively, the first and second heat exchangers may be,

(b) Amino acid sequences as shown in SEQ ID No.5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 are formed by substitution, addition or deletion of one or more amino acids, can be specifically bound to chimeric antigen receptor, and have variants which bind to EPHA2 and induce T cell signaling.

The EPHA2 chimeric antigen receptor comprises a CD8 alpha signal peptide, the antigen binding domain which binds to the EPHA2 antigen, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain and a CD3 zeta signaling domain which are connected in series.

In a second aspect of the invention there is provided a nucleic acid sequence capable of encoding an EPHA2 chimeric antigen receptor according to the first aspect.

In a third aspect of the invention there is provided a CAR expression vector comprising a nucleotide according to the second aspect.

In a fourth aspect of the present invention, there is provided a CAR-T cell capable of expressing the chimeric antigen receptor of EPHA2 described above.

Specifically, it is prepared by introducing the above CAR expression vector.

In a fifth aspect of the invention, there is provided an anti-tumour agent comprising at least a CAR-T cell as described above.

In a sixth aspect of the invention, there is provided a method of treating glioma, the method comprising: administering to the subject a therapeutically effective dose of the CAR-T cell or anti-tumor drug described above.

The beneficial technical effects of one or more of the technical schemes are as follows:

according to the technical scheme, a series of novel anti-EPHA 2 single-domain antibodies are obtained and subjected to humanized design, so that the novel anti-EPHA 2 single-domain antibodies can be combined with an EPHA2 antigen, and an EPHA2 chimeric antigen receptor is further designed and targeted, and experiments prove that the CAR-T cells for expressing the EPHA2 chimeric antigen receptor have excellent expression rate and durability, and the cells show good lasting stability and anti-tumor, especially anti-glioma effects, and meanwhile, the experiment proves that the novel EPHA2 chimeric antigen receptor can achieve remarkable curative effects under the condition of low application dosage, so that the novel EPHA2 chimeric antigen receptor has higher safety, and has good practical application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a plasmid map of an example of the present invention;

FIG. 2 is a SDS-PAGE diagram showing an example of the present invention, wherein M is marker protein and Lane 1 is 2. Mu.g EPHA2-His-ek-huFc protein;

FIG. 3 is a graph showing the activity of EPHA2 protein according to the present invention;

FIG. 4 is a diagram showing a sequence analysis using Vector NTI in an embodiment of the present invention;

FIG. 5 is a diagram showing the binding between clones A5, A2, C10, A12 and A10 and the target protein in the examples of the present invention;

FIG. 6 is a diagram showing the binding of antibodies A5, A2, C10, A12, A10-VHH-huFc fusion antibodies to CHO-K1/CHO-K1-EPHA2 cells in the examples of the present invention;

FIG. 7 is a graph showing activation of Jurkat-NFAT-Luc-GFP cell lines by EPHA2 endogenously expressed tumor cells in the examples of the present invention;

FIG. 8 is a graph of in vitro antitumor activity of EPHA2 CAR-T cell targeting in examples of the present invention;

FIG. 9 is a graph showing the change in tumor volume of a tumor-transplanted (U87) mouse treated with humanized EPHA2 CAR-T cells according to the embodiment of the present invention;

FIG. 10 is a graph showing survival of mice with engrafted tumor (U87) after treatment with humanized EPHA2 CAR-T cells according to the examples of the present invention;

FIG. 11 is a graph showing tumor volume change in tumor cells of mice transplanted with tumor cells (U87) after treatment with different doses of humanized EPHA2 CAR-T cells in the examples of the present invention;

FIG. 12 is a graph showing survival of mice transplanted with tumor (U87) after the use of different doses of humanized EPHA2 CAR-T cells in the examples of the present invention.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology within the skill of the art, and are fully explained in the literature. See, e.g., sambrook et al, molecular cloning: the techniques and conditions described in the handbook, or as recommended by the manufacturer.

The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.

In one exemplary embodiment of the invention, there is provided an EPHA2 chimeric antigen receptor comprising at least an antigen binding domain, a transmembrane domain, a costimulatory domain, and a signaling domain, wherein the antigen binding domain is an anti-EPHA 2 single domain antibody;

(b) The amino acid sequences shown in SEQ ID No.5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39 are formed by substitution, addition or deletion of one or more amino acids, can be specifically bound to chimeric antigen receptor, and have the functions of binding to EPHA2 and inducing T cell signaling.

According to the invention, a large number of EPHA2 antibodies are screened, so that the anti-EPHA 2 single domain antibodies are obtained, and the therapeutic effect of the chimeric antigen receptor is improved; experiments prove that the CAR-T cells prepared from the anti-EPHA 2 single domain antibodies with the amino acid sequences shown in SEQ ID No.25, 29 and 37 have remarkable therapeutic effects on resisting cerebral glioma under the condition of low dosage administration, and are remarkably superior to the products in the prior art.

In one or more embodiments of the present invention, the amino acid sequence as shown in SEQ ID No.5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 is at least 90% identical, including 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical, to the amino acid sequence shown in SEQ ID No.5, 7, 9, 11, 13, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, by substitution, addition or deletion of one or more amino acids, and still has the ability to specifically bind to chimeric antigen receptor, having the ability to bind EPHA2 and induce T cell signaling.

In one or more embodiments of the invention, the nucleotide sequence of the anti-EPHA 2 single domain antibody comprises a sequence having the sequence set forth in SEQ ID No.6, 8, 10, 12, 14, 16, 18, 26, 28, 30, 32, 34, 36, 38, 40.

In one or more embodiments of the invention, the nucleotide sequence of the anti-EPHA 2 single domain antibody comprises a variant having at least 80%, including but not limited to 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to the sequence shown in SEQ ID No.6, 8, 10, 12, 14, 16, 18, 26, 28, 30, 32, 34, 36, 38, 40, and which expresses a protein having the ability to specifically bind to a chimeric antigen receptor, having the ability to bind EPHA2 and induce T cell signaling.

In one or more embodiments of the invention, the chimeric antigen receptor further comprises a signal peptide that is a CD8 a signal peptide; the nucleotide sequence of the CD8 alpha signal peptide is shown as SEQ ID NO. 41.

In one or more embodiments of the invention, the antigen binding domain and the transmembrane domain are linked by a hinge region, which may comprise a CD8 hinge region having a nucleotide sequence as set forth in SEQ ID NO. 42.

In one or more embodiments of the invention, the transmembrane domain is a CD8 transmembrane domain, and the nucleotide sequence of the CD8 transmembrane domain is shown in SEQ ID NO. 43.

In one or more embodiments of the invention, the co-stimulatory domain comprises any one or more of a 4-1BB domain, a CD27 domain, a CD40 domain or an OX40 domain, preferably a 4-1BB domain; the nucleotide sequence of the human 4-1BB domain is shown as SEQ ID NO. 44.

The signal transduction domain is CD3 zeta signal transduction domain, and the nucleotide sequence of the signal transduction domain is shown as SEQ ID NO. 45.

In one or more embodiments of the invention, the chimeric antigen receptor comprises a CD8 alpha signal peptide, the antigen binding domain described above that binds to the EPHA2 antigen, a CD8 hinge region, a CD8 transmembrane domain, a human 4-1BB costimulatory domain, and a CD3 zeta signaling domain in tandem.

In one or more embodiments of the invention, a nucleic acid sequence is provided that is capable of encoding the EPHA2 chimeric antigen receptor of the first aspect.

In one or more embodiments of the invention, there is provided a CAR expression vector comprising a nucleotide according to the second aspect.

In one or more embodiments of the invention, the CAR expression vector may be a viral vector, including but not limited to a lentiviral vector, a retroviral vector, an adenoviral vector, a plasmid, or the like, with lentiviral vectors being preferred in view of current technical maturity.

The lentiviral vector comprises the nucleotide sequence of the chimeric antigen receptor targeted to EPHA2 described above.

In one or more embodiments of the invention, a CAR-T cell is provided that is capable of expressing the chimeric antigen receptor of EPHA2 described above.

Specifically, it is prepared by introducing the above CAR expression vector. In the art, this can be accomplished by well known techniques, such as by transfecting the EPHA2 chimeric antigen receptor through its coding nucleotide sequence into T cells for expression, and by transfecting the chimeric antigen receptor into T cells with a CAR expression vector.

In one or more embodiments of the invention, an anti-tumor agent is provided that comprises at least the CAR-T cell described above.

Wherein the tumor is a tumor-associated disease associated with the overexpression of EPHA2, in particular brain glioma.

The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a buffer, an emulsifier, a suspending agent, a stabilizer, a preservative, an excipient, a filler, a coagulant and a blending agent, a surfactant, a dispersing agent, or an antifoaming agent.

The medicament may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a microcapsule, liposome, nanoparticle, or polymer, and any combination thereof. The delivery vehicle for the pharmaceutically acceptable carrier may be a liposome, biocompatible polymer (including natural and synthetic polymers), lipoprotein, multi-skin, polysaccharide, lipopolysaccharide, artificial viral envelope, inorganic (including metallic) particles, as well as bacterial or viral (e.g., baculovirus, adenovirus and retrovirus), phage, cosmid or plasmid vectors.

The medicament may also be used in combination with other medicaments for the prevention and/or treatment of recurrent abortion, and other prophylactic and/or therapeutic compounds may be administered simultaneously with the main active ingredient, even in the same composition.

The medicament may also be administered alone in separate compositions or in a dosage form different from the primary active ingredient, with other prophylactic and/or therapeutic compounds. A partial dose of the principal component may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the medicament of the invention may be adjusted during the course of treatment according to the severity of the symptoms, the frequency of recurrence and the physiological response of the treatment regimen.

The medicament of the invention may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.

In one or more embodiments of the present invention, there is provided a method of brain glioma treatment, the method comprising: administering to the subject a therapeutically effective dose of the CAR-T cell or anti-tumor drug described above.

The subject is an animal, preferably a human, who has been the subject of treatment, observation or experiment. By "therapeutically effective amount" is meant that amount of active compound or pharmaceutical agent, including a compound of the present invention, which causes a biological or medical response in a tissue system, animal or human that is sought by a researcher, veterinarian, medical doctor or other medical personnel, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and spacing of the active ingredients of the present invention is determined by its nature and external conditions such as the form, route and site of administration and the particular mammal being treated, and that such optimal dosage may be determined by conventional techniques. It must also be appreciated that the optimal course of treatment, i.e. the daily dosage of the simultaneous compounds over the nominal time period, can be determined by methods well known in the art. In particular, the CAR-T cells of the present invention can achieve satisfactory therapeutic effects at low doses, thereby providing a safer and more effective therapeutic approach to glioma.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are noted, and are generally conducted under conventional conditions.

Examples

Preparation of EPHA2 recombinant proteins

(1) Construction of EPHA 2-huFc expression plasmid

The extracellular segment Ala24-Val343 (SEQ ID NO: 1) gene (SEQ ID NO: 2) of human EPHA2 is synthesized in vitro, inserted into eukaryotic expression plasmid containing Fc segment Asp104-Lys330 of human IgG1 heavy chain constant region, and connected with 6 times His-enterokinase site (ek) to form fusion expression protein EPH_huFc (shown as SEQ ID NO: 3) and corresponding gene sequence (shown as SEQ ID NO: 4).

(2) EPHA2 huFc virus packaging

a. Day before transfection, inoculation 3X 10 ⁵ HEK293T cells/ml in 10cm dishes.

b. On the day of transfection, HEK293T cells reached about 70% confluency, the original medium was aspirated off, washed once with PBS, and incompletely cultured with 9ml DMEM at 37℃with 5% CO ₂ Incubator for standby

C. Preparing PEI-DNA complex: 1mL of DMEM is taken into a 1.5mL sterile centrifuge tube, 7.5 mug of EPHA 2-huFc plasmid, 5.7 mug of pGP and 3.75 mug of pVSVG are respectively added, the mixture is fully and uniformly mixed up and down by a pipette, 50.75 mug/mug PEI is added, immediately and uniformly mixed up and down by a pipette, and the mixture is kept stand for 10-15min at room temperature.

d. Transfection: the DNA-PEI complex is added into a 10cm culture dish drop by drop, the culture dish is gently rocked in a shape of a Chinese character 'mi', and the mixture is fully and uniformly mixed. The dishes were placed at 37℃in 5% CO ₂ After culturing in the incubator for 6 to 8 hours, the medium containing the transfection reagent was removed and replaced with fresh complete medium (DMEM+10% FBS). Will be cultivatedThe culture dish is placed at 37 ℃ and 5 percent CO ₂ Incubator, culture for 48h.

e. First time detoxification: the culture medium in the petri dish was collected into a 50mL sterile centrifuge tube and placed at 4 ℃ for use. 10mL fresh complete medium [ DMEM+10% FBS ] was carefully added along the edge of the dish]The dishes were placed at 37℃with 5% CO ₂ The incubator continues to cultivate for 24 hours. And (5) collecting virus liquid.

d. Secondary detoxification: the culture broth in the petri dish was collected into a sterile 50mL centrifuge tube for the first time and centrifuged at 6000x g overnight at 4 ℃ for 16h.

f. The supernatant after centrifugation was aspirated off, and resuspended in 300ul PBS to form the EPHA 2. HuFc virus solution.

(3) Viral infection expresses epha2_hufc.

a. CHO-S cell density was adjusted to 1X 10 with CHO GROW CD1 ⁶ Per ml, inoculated in 6-well plates.

b. Dripping EPHA 2-huFc virus solution into 6-hole plate dropwise, mixing, standing at 37deg.C and 5% CO ₂ Culturing in a shaking incubator at 130 rpm.

After 24h, the cell culture solution in the six-well plate is collected and centrifuged at 200Xg and 25 ℃ for 5min.

d. After centrifugation, the supernatant was aspirated, 2mL of CHO GROW CD1 containing puromycin was added, resuspended and transferred to a 6-well plate, and placed at 37℃in 5% CO ₂ Screening was performed by culturing in an incubator on a 130rpm shaker (note that a blank CHO-S cell control group was set). Maintenance of cell Density during screening 1X 10 ⁶ /ml。

f. Cells within the control wells were essentially dead and the screen ended. The CHO-S-EPHA2-His-ek-Fc2-Puro cell line was grown in an expanded culture during which H410KJ CellTurbo feed1 was added to enhance protein expression. Expression was continued for 4 days, and the culture expression supernatant was collected and filtered with a 0.45 μm pinhole filter to form a filtered sample.

(4) Affinity purification using protein A packing pre-packed column

a. Balance: the pre-packed column was washed with PBS to UV curve equilibrium.

b. Loading: and (5) placing the sample injection pipe into a filtered sample for sample loading.

c. Washing: after loading, PBS is used for flushing until the curve is balanced;

d. eluting: the protein solution was collected by eluting with glycine buffer (pH 3.0) and immediately neutralized with an appropriate amount of Tris-HCl (pH 9.0) solution.

e. Protein eluent was concentrated using a millipore ultrafiltration tube with an cutoff of 10KD and protein concentration was detected using an ultra-trace nucleic acid protein detector OD280 module.

f. 2ug was run on SDS-PAGE and the results are shown in FIG. 2.

(5) EPHA2 protein Activity assay

Constructing a pCDH-EF1a-4H5-CAR-EGFRt plasmid of an anti-EPHA2 positive antibody 4H5, and transfecting HEK293T cells. And incubating the transfected HEK293T-4H5 cells with purified EPHA2-His-ek-Fc protein, then incubating anti-human FC-PE, and taking the HEK293T cells as negative cells to compare with FACS analysis results to show that the EPHA2-ek-Fc protein is functional and can be used for the next step of immunization and detection. (FIG. 3)

2. Screening of VHH antibodies specific for EPHA2 using phage display library

(1) The method comprises the steps of immunizing alpaca by using an EPHA2 protein (cutting huFc tag), performing subcutaneous multipoint immunization, taking peripheral blood to measure antibody titer, combining immune serum with the EPHA2 recombinant protein, and performing gradient dilution along with the immune serum, wherein OD value changes in a gradient manner, meets the requirement of library establishment, and arranging and constructing a phage display library.

(2) Collecting peripheral blood of immune alpaca, extracting RNA, preparing cDNA sample, PCR cloning VHH antibody coding gene, constructing phage display library (reservoir capacity 2x 10) ⁹ The empty rate is 0%, 16 monoclone are selected randomly for sequencing, and the sequence analysis is carried out by using Vector NTI, so that the result shows that each monoclone has large sequence difference and better library diversity. (FIG. 4)

(3) Solid phase panning was performed using EPHA2 recombinant protein antigen, 2-4 rounds of Phage panning experiments were performed, input and output values were calculated after the end of each round, and Phage ELISA was performed on the acquired monoclonal Phage beginning at round 2.

a. The EPHA2 recombinant protein antigen was coated with CBS at a concentration of 1. Mu.g/mL overnight at 4 ℃.

b. The antigen was discarded and blocked with 3% MPBS for 2h at room temperature, which is a Sample plate; at the same time, a blank ELISA plate, which is a Negative control plate, was blocked with MPBS.

c. MPBS was discarded and washed 4 times with 0.05% PBST; the monoclonal phage supernatant was diluted with 0.01% PBST 1:1, 100. Mu.L per well, incubated for 1h at 4 ℃.

d. Discarding the primary antibody and washing with 0.05% pbst 5 times; anti-M13 anti-HRP was diluted 1:3000 with 0.05% PBST and incubated at 4℃for 1h at 100. Mu.L per well.

f. Discarding the secondary antibody, and washing the PBST for 5 times; TMB was developed at 37℃for 15min, sulfuric acid was used to terminate the reaction, and OD450 was read. g. S/N values were calculated and samples with larger ratios were selected for Sanger sequencing.

h. And obtaining nucleic acid and amino acid sequence information of the candidate single domain antibody, performing sequence comparison, and selecting candidate antibodies with different CDR region amino acid sequences. CMV promoter, signal peptide and hu IgG1 Fc tag are introduced at the N end and the C end of the candidate antibody sequence through overlay PCR respectively, the purified PCR product is transiently transfected into 293F cells, and the culture medium supernatant is collected.

I. Resuscitation 1 x 10 ⁶ The CHO-K1-EPHA2 recombinant cell strain in logarithmic growth phase was centrifuged at 400Xg at 4℃for 5min. CHO-K1 cell line served as a negative control. After the supernatant was discarded, the 100uL of the culture medium supernatant was added, after incubation at 4℃for 30min, the cells were washed 3 times with 1mL of PBS, then resuspended with 100uL of PBS, 5uL of PE-labeled Anti-human IgG antibody was added, after incubation at room temperature for 30min in the absence of light, washed three times with 1mL of PBS, and finally resuspended with 500uL of PBS. And preliminarily determining the binding condition of the candidate antibody and the target protein through a flow cytometer. The results showed that CHO-K1 did not bind to the EphA2 antibody, but did bind specifically to CHO-K1-EphA2, which was an EPHA2 specific antibody. Clone numbers A5, A2, C10, a12, a10, respectively. (FIG. 5)

The VHH amino acid sequence of A5 is shown as (SEQ ID NO: 5), and the gene sequence is shown as (SEQ ID NO: 6); the VHH amino acid sequence of A2 is shown as (SEQ ID NO: 7), and the gene sequence is shown as (SEQ ID NO: 8); the VHH amino acid sequence of C10 is shown as (SEQ ID NO: 9), and the gene sequence is shown as (SEQ ID NO: 10); the VHH amino acid sequence of A12 is shown as (SEQ ID NO: 11), and the gene sequence is shown as (SEQ ID NO: 12); the VHH amino acid sequence of A10 is shown as (SEQ ID NO: 13), and the gene sequence is shown as (SEQ ID NO: 14).

3. Construction of anti-EPHA 2-VHH_huFc fusion antibody and transient expression purification and activity identification in eukaryotic cells thereof

a. Cloning the single domain antibody coding region gene sequence into pFUSE-huIgG1-Fc2 vector to construct eukaryotic expression vector. 293F cells in the logarithmic growth phase were transiently transfected. Culture supernatants were collected 5-7 days after transfection and affinity purified by Protein A.

b. The CHO-K1-EPHA2 cells were resuscitated, and the purified candidate single domain antibodies were incubated with them after gradient dilution, with CHO-K1 as a negative control group. PE-labeled Anti-human IgG antibodies were then added separately for incubation. Detection analysis was performed by cell flow and curves were drawn with MFI values and antibody concentrations. The results showed that antibodies A5, A2, C10, A12, A10-VHH-huFc fusion antibodies and

CHO-K1/CHO-K1-EPHA2 cells. The five antibodies E2-A2 gradually shift in binding with target cells with gradient dilution, but antibodies A5, A2 showed lower non-specific binding at high concentrations, that is, C10, A12, A10 specifically bound to EPHA 2-expressing CHO-K1-EPHA2 cells, and not negative cells CHO-K1, at all dilution gradients and dilution gradients of A5, A2 below 2ug/ml (FIG. 6). Further performing activation experiments to perform sequence screening.

c. Cloning the gene sequence of the single domain antibody coding region into the Lenti-EF1a-pCAR-puro vector to construct a eukaryotic expression vector. HEK293T cells were transiently transfected, and virus-transduced Jurkat-NFAT-Luc-GFP cells, and non-transduced Jurkat-NFAT-Luc-GFP cells, were used as negative controls. And 5-7 days of screening by puromycin, and screening to obtain the CAR-Jurkat-NFAT-Luc-GFP cell strain after the cells of the negative control group basically die.

d. And (3) performing co-culture on brain glioma cells U87 and U251 which endogenously express the EPHA2 and are in a logarithmic growth phase respectively with Jurkat-NFAT-Luc-GFP cell strains according to a certain proportion, and detecting activation conditions of the Jurkat-NFAT-Luc-GFP cell strains by the tumor cells endogenously expressing the EPHA2 after the co-culture. The results showed that the fold activation of C10 was highest and A10 times under the same co-culture (FIG. 7). C10 and a10 were selected for further humanization and their function was verified.

d. The EPHA2 antigen was coupled to the BiaCore T200 chip using an amino coupling protocol, and affinity assays were performed using the C10/A10-VHH antibody as a mobile relative single domain antibody, all with affinities on the order of nM.

4. Single domain antibody humanization design

(21) Based on the single domain antibody identification results, C10 and a10 were humanized. The CDR1 amino acid sequence of the antibody C10-VHH is shown in (SEQ ID NO: 15); the CDR2 amino acid sequence is shown in (SEQ ID NO: 16); the CDR3 amino acid sequence is shown in (SEQ ID NO: 17); the CDR1 amino acid sequence of antibody A10-VHH is shown in (SEQ ID NO: 18); the CDR2 amino acid sequence is shown in (SEQ ID NO: 19); the CDR3 amino acid sequence is shown in (SEQ ID NO: 20). C10 and A10 were humanised using CDR & SDR Grafting and Back mutation. The amino acid sequence after C10-HM1 humanization is shown as (SEQ ID NO: 21), and the nucleic acid sequence after C10-HM1 humanization is shown as (SEQ ID NO: 22); the amino acid sequence after C10-HM2 humanization is shown as (SEQ ID NO: 23), and the nucleic acid sequence after C10-HM2 humanization is shown as (SEQ ID NO: 24); the amino acid sequence after C10-HM3 humanization is shown as (SEQ ID NO: 25), and the nucleic acid sequence after C10-HM3 humanization is shown as (SEQ ID NO: 26); the amino acid sequence after C10-HM4 humanization is shown as (SEQ ID NO: 27), and the nucleic acid sequence after C10-HM4 humanization is shown as (SEQ ID NO: 28); the amino acid sequence after C10-HM5 humanization is shown as (SEQ ID NO: 29), and the nucleic acid sequence after C10-HM5 humanization is shown as (SEQ ID NO: 30). The amino acid sequence after A10-HM1 humanization is shown as (SEQ ID NO: 31), and the nucleic acid sequence after A10-HM1 humanization is shown as (SEQ ID NO: 32); the amino acid sequence after A10-HM2 humanization is shown as (SEQ ID NO: 33), and the nucleic acid sequence after A10-HM2 humanization is shown as (SEQ ID NO: 34); the amino acid sequence after A10-HM3 humanization is shown as (SEQ ID NO: 35), and the nucleic acid sequence after A10-HM3 humanization is shown as (SEQ ID NO: 36); the amino acid sequence after A10-HM4 humanization is shown as (SEQ ID NO: 37), and the nucleic acid sequence after A10-HM4 humanization is shown as (SEQ ID NO: 38); the amino acid sequence after A10-HM5 humanization is shown as (SEQ ID NO: 39), and the nucleic acid sequence after A10-HM5 humanization is shown as (SEQ ID NO: 40).

Preparation of CAR-T cells and anti-tumor Activity study

C10 and a10 and the respective humanized sequences were selected for CAR-T cell preparation and anti-tumor activity studies.

(1) Construction of chimeric antigen receptor lentiviral expression vector targeting EPHA2

The pCDH-EF1a plasmid is used as a vector to construct a lentiviral plasmid for expressing the second-generation chimeric antigen receptor of the EPHA2 antibody. Including pCDH-EF1a-C10-BBZ, pCDH-EF1a-C10-HM1-BBZ, pCDH-EF1a-C10-HM2-BBZ, pCDH-EF1a-C10-HM3-BBZ, pCDH-EF1a-C10-HM4-BBZ, pCDH-EF1a-C10-HM5-BBZ, pCDH-EF1a-A10-HM1-BBZ, pCDH-EF1a-A10-HM3-BBZ, pCDH-EF1a-A10-HM4-BBZ, pCDH-EF1a-A10-HM5-BBZ.

The C10-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 10), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 ζ (SEQ ID NO: 45) of CD 3.

The C10-HM1-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 22), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

The C10-HM2-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 24), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

The C10-HM3-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 26), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

The C10-HM4-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 28), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

The C10-HM5-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 30), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

A10-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 14), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 ζ (SEQ ID NO: 45) of CD 3.

The A10-HM1-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 32), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

The A10-HM2-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 34), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

The A10-HM3-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 36), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

The A10-HM4-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 38), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

The A10-HM5-BBZ nucleic acid sequence consists of the CD8 alpha signal peptide (SEQ ID NO: 41), the C10-VHH (SEQ ID NO: 40), the CD8 hinge region (SEQ ID NO: 42), the CD8 transmembrane region (SEQ ID NO: 43) and the intracellular signaling domain 4-1BB (SEQ ID NO: 44) and the intracellular segment CD3 zeta (SEQ ID NO: 45) of CD 3.

After all plasmids were sequenced correctly, plasmids were extracted and purified using Qiagen's plasmid purification kit to obtain transfection grade plasmids for HEK293F suspension cell lentiviral packaging experiments.

(2) EPHA 2-targeted chimeric antigen receptor lentivirus preparation

a. HEK293F cell density was adjusted to 4.5X10 with FreeStyle 293 Medium ⁶ cells/mL, volume 90% of the package volume. High capacity 50 amplitude CO at temp. of 37.0 deg.C ₂ And culturing by a superimposed constant-temperature oscillator for standby.

b. Preparing plasmid/PEI complex: 2 sterile centrifuge tubes were prepared and 5% of the packaging volume of FreeStyle 293 medium was added, respectively. To the first tube, pLP1, pLP2, pLP/VSVG and pCDH-EF1a-CAR plasmids were added in sequence in a certain proportion, the total amount of plasmids was 180. Mu.g/100 mL of packaging volume, gently mixed, and incubated at room temperature for 5min. To the other tube was added PEI solution, PEI dose (μg) =total plasmid amount (μg) 3, gently mixed and incubated for 5min at room temperature.

c. And adding the incubated PEI diluent into the plasmid diluent, quickly and fully mixing, and incubating for about 12min in a vertical laminar flow clean workbench.

d. Transfection: taking out the prepared HEK293F cells, adding the prepared plasmid/PEI complex while shaking, mixing, and mixing at 37.0deg.C, 130rpm, amplitude of 50.0mm, and 5.0% CO ₂ Culturing overnight.

e. About 16-18h post transfection. Adding OPM-CHO PFF06 with volume of 10% of transfection system, mixing well at 37.0deg.C, 130rpm, amplitude of 50.0mm, 5.0% CO ₂ Culturing overnight.

f. About 48h after transfection. Centrifugation was performed at 3000 Xg at 22.0deg.C for 15min, and the supernatant virus was collected and filtered through a 0.65 μm syringe filter.

g. Nuclease digestion. The Super nucleic solution is added into the virus liquid according to 100U/mL, the mixture is inverted for 5 to 8 times, and the mixture is treated for about 16 hours at 4 ℃.

Centrifugation was performed at 6000 Xg overnight at 4℃for about 16h, and the supernatant was discarded. Resuspension was performed with 1% packaging volume DPBS (containing 10% human serum albumin). Taking 50ul virus liquid to detect infection titer, and storing at-80 ℃ after the rest of virus liquid is packaged.

i. The 293T cells were infected with different dilutions of the virus solution, and after 48h, the positive cell rate of the infection was detected by flow cytometry and converted to the infection titer of the virus solution for subsequent transduction of T cells.

(3) Preparation of CART cells

A host cell comprising the recombinant expression vector. The host cells we use are human peripheral blood T cells or T cell-containing cell populations.

a. Peripheral blood T cell isolation

5mL of the anticoagulated blood sample was transferred to a 15mL sterile centrifuge tube, the centrifugal force was 800 Xg, the centrifugal deceleration was minimized, and the blood sample was centrifuged at room temperature for 20min. The serum layer was removed, and an equal volume of physiological saline was added to the lower red peripheral blood cell layer and mixed well.

5mL of lymphocyte separation liquid is added into a new 15mL centrifuge tube, then the blood sample diluted by normal saline is slowly added into the upper layer of lymphocyte separation reagent along the tube wall, the centrifugal force is 800 Xg, the rotating speed is set to be the lowest, and the centrifugation is carried out for 30min at room temperature. The white mononuclear cell layer was aspirated into a new 15mL sterile centrifuge tube, and an equal volume of physiological saline was added and mixed well. Centrifugal force is 800 Xg, centrifugal force is 5min. Absorbing and discarding the supernatant, adding 5mL of physiological saline, uniformly mixing, taking part of cell suspension for counting, and detecting CD3 by using flow cytometry ⁺ T cell ratio.

According to Dynabeads and CD3 ⁺ T cell number ratio of 1:1, T cells were isolated and part of the cell suspension was counted.

b.T cell activation

T cell growth medium (containing X-Vivo 15 medium, 300IU/mL interleukin 2, 10ng/mL interleukin 7, 5ng/mL interleukin 15, 5ng/mL interleukin 21) was added to adjust the T cell density to 1E6/mL. The cells were exposed to 5% CO at 37 ℃ ₂ Culturing in an incubator for 40h.

c.T cell culture transduction

The lentivirus was removed from the-80℃refrigerator and thawed on ice.

The activated T cells were removed from the incubator, polybrene was added to the culture vessel to a final concentration of 6. Mu.g/mL, virus solution (MOI about 50) was added, and the vessel was sealed with a sealing film and centrifuged at 800 Xg at room temperature for 1 hour.

After centrifugation, the culture vessel was centrifuged at 37℃and 5% CO ₂ In the incubator of (2), the culture was continued for 24 hours.

Centrifugation at 400 Xg for 10min, the virus-containing medium supernatant was discarded, the cell pellet was resuspended in fresh T-cell growth medium, and the cells were transferred to a new culture vessel and cultured continuously. Maintaining cell density at 1-2x10 ⁶ /mL。

After 4 days of culture, a fraction of the cells were taken and examined for expression of T cell surface CAR molecules using a flow cytometer. Centrifuging to collect the prepared CAR-T cells and NC-T cells (control group), washing the supernatant once with PBS, adding EPHA2-huFc protein, and incubating for 30min; washing twice with PBS, adding PE anti-human IgGFc antibody, and incubating for 30min in dark; the supernatant was washed twice with PBS, resuspended, and finally the flow cytometer detected the proportion of T cells positive for CAR. The expression efficiency of the CAR molecule was about 40%.

(4) In vitro antitumor Activity targeting EPHA2 CAR-T cells

The antitumor activity of the EPHA2 CART cells is judged by a cytotoxicity experiment. In comparing the in vitro killing activity of UTD, C10-BBZ, C10-HM1-BBZ, C10-HM2-BBZ, C10-HM3-BBZ, C10-HM4-BBZ, C10-HM5-BBZ, A10-HM1-BBZ, A10-HM2-BBZ, C10-HM3-BBZ, C10-HM4-BBZ, C10-HM5-BBZ CAR-T cells, the infection positive rates of twelve CAR T cells were 27.8%, 22.6%, 21.8%, 27.8%, 27.0%, 27.8%, 31.2%, 28.7%, 30.9%, 25.1%, 30.7% and 28.2%, respectively.

a. U251-Luc, U87-Luc cells endogenously expressing EPHA2 were used as target cells, and the density was adjusted to 5X 10 with RPMI1640 complete medium ⁵ mu.L/mL, then 100. Mu.L/well, 96 well plate, i.e. 5X 10 ⁴ And/or holes.

car-T cells and UTD as effector cells, while RPMI1640 complete medium was adjusted to appropriate densities, respectively, experimental groups were following effector cells: target cells = 2.5:1,5:1, 10:1 ratio was added to target cell wells, 100 μl/well, respectively. And setting the control group as a group without effector cells, namely the maximum fluorescent release hole of the target cells. Each proportion is provided with 2 compound holes

c. Placing at 37deg.C 5% CO ₂ Culturing in an incubator for 18h.

d. According to the plate layout, 100uL of cell suspension is transferred to a full white enzyme label plate per hole, 100uL of D-luciferin is added into each hole, the mixture is uniformly mixed, the whole process is operated in a dark place, and the operation is rapid, and the mixture is kept stand for 5min. And detecting by using a multifunctional enzyme-labeled instrument bioluminescence signal detection system. The cytotoxicity calculation formula is: % cytotoxicity= [ (control-experimental)/control ]. Times.100. The results showed significant antitumor activity, with more than 85% of tumor cells (better than CN 113980138A) lysed at a medium E: T ratio (5:1). Taken together, the anti-tumor activity of C10-HM3/C10-HM5/A10-HM4 was better than that of C10, A10 and other humanized sequences before humanization, FIG. 8. And evaluating the anti-tumor effect in the animal body.

IFN-gamma secretion detection after CART and target cell co-culture

U251-Luc, U87-Luc cells as target cells (containing EPHA2 target protein) were conditioned to a density of 5X 10 with RPMI1640 complete medium ⁵ Per mL, then 100 μl/well, 96-well plate, i.e. 5×10 ⁴ And/or holes.

CAR-T cells and UTD (control) were effector cells, adjusted to 2.5X10 respectively with RPMI1640 complete medium ⁶ Per mL, 100. Mu.L/well, 3 multiplex wells were placed in the target cell well.

c. Placing at 37deg.C 5% CO ₂ Culturing in an incubator for 18h.

d. ELISA plates were prepared, coated and blocked.

Coating a capture anti-body: the capture anti-ibody was diluted to 2. Mu.g/mL using PBS, 96-well ELISA plate, 100. Mu.L/well. Incubate overnight at 4 ℃.

Closing: plates were washed three times with PBST and blocked with 1% BSA-PBS at room temperature for 1h, 300. Mu.L/well.

e. The 96-well cell plate was placed in a centrifuge, centrifuged at 800 Xg at room temperature for 5min, and the culture supernatant was collected.

f. Adding a sample: plates were washed three times with PBST, and the collected culture supernatants and standards [ 9.38pg/mL-600pg/mL ] were added, 100. Mu.L/well, and incubated for 2h at room temperature.

g. Add detection antibody: plates were washed three times with PBST and then diluted detection antibody to 125ng/mL with PBS, 100. Mu.L/well, and incubated for 2h at room temperature.

h. Adding strepitavidin-HRP B: plates were washed three times with PBST and then diluted 40-fold with PBS, 100. Mu.L/well, incubated at room temperature for 20min, taking care of protection from light.

i. Adding a color development liquid: the plates were washed three times with PBST, then with the addition of the chromogenic solution, 100. Mu.L/well, incubated at room temperature for 20min, and noted protected from light.

g. Terminating the reaction: add 2M H ₂ SO ₄ 50. Mu.L/well, care was taken to avoid light.

k. And (3) detection: the OD450 was read with a multifunctional microplate reader with a reference wavelength of 540nm.

Analysis of the data, the CAR-T group was significantly higher than the UTD group, demonstrated that CAR-T was able to secrete significant amounts of IFN- γ upon stimulation of EPHA 2-expressing tumor cells.

IL-2 release after CART co-culture with target cells

U251-Luc, U87-Luc cells as target cells (containing EPHA2 target protein) were adjusted to a density of 5X 10 with RPMI1640 complete medium ⁵ Per mL, then 100 μl/well, 96-well plate, i.e. 5×104/well.

CAR-T cells and UTD (control) were effector cells, adjusted to 2.5X10 respectively with RPMI1640 complete medium ⁶ Per mL, 100. Mu.L/well, 3 multiplex wells per ratio.

c. Placing at 37deg.C 5% CO ₂ Culturing in an incubator for 18h.

d. ELISA plates were prepared, coated and blocked.

Coating a capture anti-body: the capture anti-bodies were diluted to 4. Mu.g/mL in PBS, 96-well ELISA plates, 100. Mu.L/well. Incubate overnight at 4 ℃.

f. Adding a sample: plates were washed three times with PBST, and the collected culture supernatants and standards [ 15.6pg/mL-1000pg/mL ] were added, 100. Mu.L/well, and incubated for 2h at room temperature.

g. Add detection antibody: plates were washed three times with PBST and then diluted detection antibody to 100ng/mL with PBS, 100. Mu.L/well, and incubated for 2h at room temperature.

Analysis of the data, the CAR-T group was significantly higher than the UTD group, demonstrated that CAR-T was able to secrete significant amounts of IL-2 upon stimulation of EPHA 2-expressing tumor cells.

(5) Antitumor Activity in vivo in animals targeting EPHA2 CAR-T cells

NSG mice knock out the Il2rg gene on the basis of NOD-SCID, are severely immunodeficiency mice, lack mature T, B, NK cells, and are important vectors for humanized mice, xenografts and immune reconstruction; has important significance for researching human hematopoietic stem cells, tumorigenesis, treatment, immunodeficiency diseases and in vivo immune mechanism. Is a tool mouse which is internationally accepted at present and has higher immunodeficiency degree and is more suitable for transplanting human cells or tissues.

a. Harvesting U87 cells in logarithmic growth phase, re-suspending the cells with PBS, and adjusting cell density to 1.5X10 ⁷ Per mL, it was mixed with Matrigel 1:1 (v: v). 200 ul/volume was inoculated subcutaneously in the right anterior axilla of 6-8 week old female NSG mice to establish glioma xenograft mouse models.

b. The health condition and the tumorigenesis condition of the mice are observed daily, and the tumor volume reaches 100-300mm after about 14 days ³ 。

c. Mice were equally divided into 6 groups of 8C 10-HM5/C10-HM3/A10-HM4/UTD/Temozolomide (a clinically conventional small molecule drug)/PBS. Intravenous injection of 1X 10 per mouse tail ⁷ Single treatment (lower than CN113980138A dose) was performed with single CART cells.

d. Following dosing, mice were observed daily for general symptoms for a total of 40 days, and the mice were euthanized at the end of the experiment and dissected. The size of the U87 engrafted tumor volume was measured every 3-4 days, the change in tumor volume and survival of each group of mice were recorded, and the growth curve of tumor volume with time and the survival curve of mice were plotted. The results showed that both C10-HM5/C10-HM3/A10-HM4 CAR-T cells were effective in inhibiting U87 cell proliferation, FIG. 9. And when the UTD/Temozolomide/PBS mice in the control group are all dead, only the C10-HM5/C10-HM3 CAR-T treated group mice are all alive for more than 40d, the anti-tumor activity of the targeted EPHA2 CAR-T cells in animals is remarkable (the transplanted tumor of the mice in the experimental group is almost completely inhibited in 12-16 days, the tumor volume is almost not measured, and the result is better than that of CN 113980138A). Fig. 10. The CDR1 amino acid sequence of antibody C10-HM3 is shown in (SEQ ID NO: 46); the CDR2 amino acid sequence is shown in (SEQ ID NO: 47); the CDR3 amino acid sequence is shown in (SEQ ID NO: 48); the CDR1 amino acid sequence of antibody C10-HM5 is shown in (SEQ ID NO: 49); the CDR2 amino acid sequence is shown as (SEQ ID NO: 50); the CDR3 amino acid sequence is shown in (SEQ ID NO: 51); the CDR1 amino acid sequence of antibody A10-HM4 is shown as (SEQ ID NO: 52); the CDR2 amino acid sequence is shown in (SEQ ID NO: 53); the CDR3 amino acid sequence is shown in (SEQ ID NO: 54). Taking C10-HM3 CAR-T as an example, the therapeutic doses of CAR-T cells were further explored.

(6) Antitumor active dose of targeting EPHA2 CAR-T cells in animals

To further examine the dose of anti-tumor activity of EPHA2-CAR T cells in vivo, we will have U87 cells

Is injected into 6-8 week old female NSG mice subcutaneously, and 1.5X10 s subcutaneously in the left armpit ⁶ And U87 cells, and establishing a glioma xenograft mouse model. The volume of the U87 transplanted tumor reaches 100-300mm ³ When mice were divided into 5 groups of 8 mice per group, C10-HM3-1E6/C10-HM3-2E6/C10-HM3-4E 6/UTD/PBS. Intravenous injection of 1X 10 per mouse tail ⁶ /2×10 ⁶ /4×10 ⁶ Single treatment was performed with CART cells. Following dosing, the general symptoms of the mice were closely observed, and the mice were measured for U87 shift 2-3 times per weekTumor volume size, tumor volume change and survival of each group of mice were recorded, and tumor volume growth curves and mouse survival curves were plotted over time, and mice were euthanized at the end of the experiment and dissected. The results showed that all of the C10-HM3-1E6/C10-HM3-2E6/C10-HM3-4E6 CAR-T cells were effective in inhibiting U87 cell proliferation, FIG. 11. And when the control UTD/PBS mice were all dead, the three different doses of CAR-T treated mice survived for more than 40d, i.e., the lowest therapeutic dose was 1X 10 ⁶ The anti-tumor activity of the targeted EPHA2 CAR-T cells in animals was also shown to be significant (at day 12, the experimental group mice transplanted tumors had been completely inhibited, tumor volumes had not been measured), fig. 12. After the CART treatment dosage is reduced, the animal treatment efficacy result is still good, which indicates that the efficacy is strong, and the cost is low and the safety is high when the product ratio is equal to the future product ratio. Blood collection in D3/D7/D14/D28 can detect CART cells, which shows that the EPHA2 CAR-T cells have good persistence in animals. Laying a good foundation for the next clinical trial research.

Amino acid and nucleotide sequences referred to in the examples

(1) EPHA2 protein sequence

AQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAV

(2) EPHA2 nucleic acid sequences

GCTCAGGGAAAAGAAGTGGTGCTGCTGGACTTTGCTGCCGCTGGCGGAGAACTTGGATGGCTGACACACCCTTACGGCAAAGGCTGGGACCTGATGCAGAACATCATGAACGACATGCCCATCTACATGTACAGCGTGTGCAACGTGATGAGCGGCGACCAGGACAATTGGCTGAGGACCAACTGGGTGTACAGAGGCGAGGCCGAGCGGATCTTCATCGAGCTGAAGTTCACCGTGCGGGACTGCAACAGCTTTCCTGGCGGAGCCAGCAGCTGCAAAGAGACATTCAACCTGTACTACGCCGAGAGCGACCTGGACTACGGCACCAACTTCCAGAAGCGGCTGTTCACCAAGATCGACACAATCGCCCCTGACGAGATCACCGTGTCCAGCGATTTTGAGGCCCGGCACGTGAAGCTGAACGTGGAAGAAAGAAGCGTGGGCCCTCTGACCAGAAAGGGCTTCTACCTGGCCTTCCAGGATATCGGAGCCTGTGTGGCACTGCTGTCTGTGCGGGTGTACTACAAGAAGTGCCCCGAGCTGCTGCAAGGCCTGGCTCACTTTCCTGAGACAATCGCCGGAAGCGACGCCCCATCTCTGGCTACAGTTGCCGGCACATGTGTGGATCATGCCGTGGTTCCACCTGGCGGCGAGGAACCTAGAATGCACTGTGCTGTGGATGGCGAGTGGCTGGTGCCTATCGGACAGTGTCTGTGTCAGGCCGGCTACGAGAAGGTGGAAGATGCCTGTCAGGCCTGCTCTCCCGGCTTCTTCAAGTTTGAGGCCAGCGAGAGCCCCTGCCTGGAATGTCCTGAACACACCCTGCCATCTCCAGAGGGCGCCACATCTTGCGAGTGCGAGGAAGGCTTCTTTCGGGCCCCTCAGGATCCAGCCAGCATGCCTTGTACCAGACCTCCAAGCGCTCCCCACTATCTGACAGCCGTTGGAATGGGCGCCAAAGTGGAACTGAGATGGACCCCTCCACAGGACAGCGGCGGCAGAGAAGATATCGTGTACTCCGTGACCTGCGAGCAGTGCTGGCCTGAGTCTGGCGAATGTGGACCTTGTGAAGCCAGCGTGCGGTACTCTGAACCTCCTCACGGACTGACAAGAACCAGCGTGACCGTGTCCGACCTGGAACCTCACATGAACTACACCTTCACCGTGGAAGCCCGGAATGGCGTGTCCGGACTGGTCACCAGCAGAAGCTTTAGAACCGCCAGCGTGTCCATCAACCAGACCGAGCCTCCAAAAGTGCGGCTGGAAGGCAGAAGCACCACAAGCCTGTCCGTGTCTTGGAGCATCCCTCCACCTCAGCAGAGCAGAGTGTGGAAGTACGAAGTGACCTACCGGAAGAAGGGCGACAGCAACAGCTACAACGTGCGGAGAACCGAGGGCTTTAGCGTGACCCTGGATGATCTGGCCCCTGACACCACATACCTGGTGCAGGTTCAGGCCCTGACACAAGAAGGACAAGGCGCCGGATCTAAGGTGCACGAGTTCCAGACACTGAGCCCTGAAGGCAGCGGAAACCTGGCTGTG

(3) EPHA 2-huFc protein sequence

AQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVHHHHHHDDDDKMDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(4) EPHA2 huFc nucleic acid sequences

GCTCAGGGAAAAGAAGTGGTGCTGCTGGACTTTGCTGCCGCTGGCGGAGAACTTGGATGGCTGACACACCCTTACGGCAAAGGCTGGGACCTGATGCAGAACATCATGAACGACATGCCCATCTACATGTACAGCGTGTGCAACGTGATGAGCGGCGACCAGGACAATTGGCTGAGGACCAACTGGGTGTACAGAGGCGAGGCCGAGCGGATCTTCATCGAGCTGAAGTTCACCGTGCGGGACTGCAACAGCTTTCCTGGCGGAGCCAGCAGCTGCAAAGAGACATTCAACCTGTACTACGCCGAGAGCGACCTGGACTACGGCACCAACTTCCAGAAGCGGCTGTTCACCAAGATCGACACAATCGCCCCTGACGAGATCACCGTGTCCAGCGATTTTGAGGCCCGGCACGTGAAGCTGAACGTGGAAGAAAGAAGCGTGGGCCCTCTGACCAGAAAGGGCTTCTACCTGGCCTTCCAGGATATCGGAGCCTGTGTGGCACTGCTGTCTGTGCGGGTGTACTACAAGAAGTGCCCCGAGCTGCTGCAAGGCCTGGCTCACTTTCCTGAGACAATCGCCGGAAGCGACGCCCCATCTCTGGCTACAGTTGCCGGCACATGTGTGGATCATGCCGTGGTTCCACCTGGCGGCGAGGAACCTAGAATGCACTGTGCTGTGGATGGCGAGTGGCTGGTGCCTATCGGACAGTGTCTGTGTCAGGCCGGCTACGAGAAGGTGGAAGATGCCTGTCAGGCCTGCTCTCCCGGCTTCTTCAAGTTTGAGGCCAGCGAGAGCCCCTGCCTGGAATGTCCTGAACACACCCTGCCATCTCCAGAGGGCGCCACATCTTGCGAGTGCGAGGAAGGCTTCTTTCGGGCCCCTCAGGATCCAGCCAGCATGCCTTGTACCAGACCTCCAAGCGCTCCCCACTATCTGACAGCCGTTGGAATGGGCGCCAAAGTGGAACTGAGATGGACCCCTCCACAGGACAGCGGCGGCAGAGAAGATATCGTGTACTCCGTGACCTGCGAGCAGTGCTGGCCTGAGTCTGGCGAATGTGGACCTTGTGAAGCCAGCGTGCGGTACTCTGAACCTCCTCACGGACTGACAAGAACCAGCGTGACCGTGTCCGACCTGGAACCTCACATGAACTACACCTTCACCGTGGAAGCCCGGAATGGCGTGTCCGGACTGGTCACCAGCAGAAGCTTTAGAACCGCCAGCGTGTCCATCAACCAGACCGAGCCTCCAAAAGTGCGGCTGGAAGGCAGAAGCACCACAAGCCTGTCCGTGTCTTGGAGCATCCCTCCACCTCAGCAGAGCAGAGTGTGGAAGTACGAAGTGACCTACCGGAAGAAGGGCGACAGCAACAGCTACAACGTGCGGAGAACCGAGGGCTTTAGCGTGACCCTGGATGATCTGGCCCCTGACACCACATACCTGGTGCAGGTTCAGGCCCTGACACAAGAAGGACAAGGCGCCGGATCTAAGGTGCACGAGTTCCAGACACTGAGCCCTGAAGGCAGCGGAAACCTGGCTGTGCACCATCACCATCACCATGATGACGATGACAAGATGGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCACGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

(5) A5-VHH amino acid sequence

QLKVVESGGGLVQPGGSLKLSCAASQSIFDFNAMDWYRQAPGKQRELVARIENGGNTSYYNSVQGRFAISRDNVKNTVYLQMNELKPEDTAVYFCCALRTKAWGPDEYYWGQGTQVTVSS

(6) A5-VHH nucleic acid sequences

CAGCTGAAGGTTGTGGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAAGCTGTCTTGTGCCGCCAGCCAGAGCATCTTCGACTTCAACGCCATGGACTGGTACAGACAGGCCCCTGGCAAACAGAGAGAGCTGGTCGCCAGAATCGAGAACGGCGGCAACACCAGCTACTACAACAGCGTGCAGGGCAGATTCGCCATCAGCCGGGACAACGTGAAGAACACCGTGTACCTGCAGATGAACGAGCTGAAGCCCGAGGATACCGCCGTGTACTTCTGTTGTGCCCTGCGGACAAAAGCCTGGGGACCCGATGAGTACTATTGGGGCCAGGGCACCCAAGTGACCGTGTCATCT

(7) A2-VHH amino acid sequence

QVQLVESGGGLVQPGGSLRLSCAASTSIFNFNAMDWYRQAPGKERELVARIENGGNTSYTNSVQGRFTISRDSVKNTVYLQMNALKPEDTAVYFCCALRTKAWGPDEFHWGQGAQVTVSS

(8) A2-VHH nucleic acid sequences

CAGGTTCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCACCAGCATCTTCAACTTCAACGCCATGGACTGGTACAGACAGGCCCCTGGCAAAGAGAGAGAGCTGGTCGCCAGAATCGAGAACGGCGGCAATACCAGCTACACCAACAGCGTGCAGGGCAGATTCACCATCAGCCGGGACAGCGTGAAGAACACCGTGTACCTGCAGATGAACGCCCTGAAGCCTGAGGATACCGCCGTGTACTTCTGCTGTGCCCTGAGGACAAAAGCCTGGGGACCCGATGAGTTTCACTGGGGACAAGGCGCCCAAGTGACCGTGTCATCT

(9) C10-VHH amino acid sequence

AVQLVESGGGLVQPGGSLRLACVASGRTQSTYNTGWFRQAPGKEREFVASITWSSDNWYHADSVRGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCATSTAWSTLATRYDNWGQGAQVTVSS

(10) C10-VHH nucleic acid sequences

GCTGTTCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGGCCTGTGTGGCCTCTGGCAGAACCCAGAGCACATACAACACCGGCTGGTTCAGACAGGCCCCTGGCAAAGAGAGAGAGTTCGTCGCCAGCATCACCTGGTCCAGCGACAACTGGTATCACGCCGATTCTGTGCGGGGCAGATTCACCATCAGCCGGGACAATGCCAAGAACGCCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAGGACACAGCCGTGTACTACTGTGCCACCAGCACCGCCTGGTCTACCCTGGCCACCAGATACGATAATTGGGGCCAGGGCGCCCAAGTGACCGTTTCTTCT

(11) A12-VHH amino acid sequence

MAQVQLVESGGGLVQPGGSLRLSCAASGVIFSRHDMAWYRQAAGKQREVVATIGTGPIITYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYIPRAWGSDYWGQGTQVTVSS

(12) A12-VHH nucleic acid sequences

ATGGCTCAGGTGCAGCTTGTTGAATCTGGCGGCGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCTGCCAGCGGCGTGATCTTCAGCAGACACGACATGGCCTGGTACAGACAGGCCGCTGGAAAGCAGAGAGAGGTGGTCGCCACAATCGGCACAGGCCCCATCATCACATACGCCGACTCTGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCTGAGGACACCGCCGTGTACTACTGCTACATCCCTAGAGCCTGGGGCAGCGATTATTGGGGCCAGGGAACACAAGTGACCGTGTCCTCT

(13) A10-VHH amino acid sequence

EVQLAESGGGLVQPGGSLRLSCAASGIDFSISDMAWYRQAPGKQREVVAFVSSGNLIQYSESAKGRFTISRDNAKNSVHLEMNNVKPEDTGVYLCYARTYSNGLRIHWGQGTQVTVST

(14) A10-VHH nucleic acid sequences

GAAGTTCAGCTGGCTGAATCTGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGTCTTGTGCCGCCTCTGGCATCGACTTCAGCATCAGCGACATGGCCTGGTACAGACAGGCCCCTGGCAAGCAGAGAGAAGTGGTCGCCTTTGTGTCCAGCGGCAACCTGATCCAGTACAGCGAGTCTGCCAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCGTGCACCTGGAAATGAACAACGTGAAGCCCGAGGACACCGGCGTGTACCTGTGTTACGCCAGAACCTACAGCAACGGCCTGAGAATCCACTGGGGCCAGGGAACCCAAGTGACCGTGTCTACA

(15) C10-CDR1 amino acid sequence

GRTQSTYN

(16) C10-CDR2 amino acid sequence

ITWSSDNW

(17) C10-CDR3 amino acid sequence

ATSTAWSTLATRYDN

(18) A10-CDR1 amino acid sequence

GIDFSISD

(19) A10-CDR2 amino acid sequence

VSSGNL

(20) A10-CDR3 amino acid sequence

YARTYSNGLRIH

(21) C10-HM1 amino acid sequence

EVQLVESGGGLVKPGGSLRLSCAASGRTQSTYNTGWFRQAPGKGREFVASITWSSDNWYHADSVKGRFTISRDNAKNAVYLQMNSLRAEDTAVYYCATSTAWSTLATRYDNWGQGTTVTVSS

(22) C10-HM1 nucleic acid sequences

GAGGTGCAGCTGGTTGAATCTGGCGGCGGACTTGTGAAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCTCTGGCAGAACCCAGAGCACATACAATACCGGCTGGTTCAGACAGGCCCCTGGCAAGGGCAGAGAATTTGTGGCCAGCATCACCTGGTCCAGCGACAACTGGTATCACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACGCCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAGCCGTGTACTACTGTGCCACAAGCACCGCCTGGTCTACCCTGGCCACCAGATACGATAATTGGGGCCAGGGCACCACCGTGACCGTTTCTTCT

(23) C10-HM2 amino acid sequence

AVQLVESGGGLVKPGGSLRLSCVASGRTQSTYNTGWFRQAPGKGREFVASITWSSDNWYHADSVKGRFTISRDNAKNAVYLQMNSLRAEDTAVYYCATSTAWSTLATRYDNWGQGTTVTVSS

(24) C10-HM2 nucleic acid sequences

GCTGTGCAGCTGGTTGAATCTGGCGGCGGACTTGTGAAGCCTGGCGGATCTCTGAGACTGAGCTGTGTGGCCTCTGGCAGAACCCAGAGCACCTACAATACCGGCTGGTTCAGACAGGCCCCTGGCAAGGGCAGAGAATTTGTGGCCAGCATCACCTGGTCCAGCGACAACTGGTATCACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACGCCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAGCCGTGTACTACTGTGCCACAAGCACCGCCTGGTCTACCCTGGCCACCAGATACGATAATTGGGGCCAGGGCACCACCGTGACCGTTTCTTCT

(25) C10-HM3 amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGRTQSTYNTGWFRQAPGKGREFVASITWSSDNWYHADSVKGRFTISRDNAKNAVYLQMNSLRAEDTAVYYCATSTAWSTLATRYDNWGQGTTVTVSS

(26) C10-HM3 nucleic acid sequences

GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCTCTGGCAGAACCCAGAGCACCTATAATACCGGCTGGTTCAGACAGGCCCCTGGCAAGGGCAGAGAATTTGTGGCCAGCATCACCTGGTCCAGCGACAACTGGTATCACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACGCCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAGCCGTGTACTACTGTGCCACAAGCACCGCCTGGTCTACCCTGGCCACCAGATACGATAATTGGGGCCAGGGCACCACCGTGACCGTTTCTTCT

(27) C10-HM4 amino acid sequence

AVQLVESGGGLVQPGGSLRLSCVASGRTQSTYNTGWFRQAPGKGREFVASITWSSDNWYHADSVKGRFTISRDNAKNAVYLQMNSLRAEDTAVYYCATSTAWSTLATRYDNWGQGTTVTVSS

(28) C10-HM4 nucleic acid sequences

GCTGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGAGCTGTGTGGCCTCTGGCAGAACCCAGAGCACCTACAATACCGGCTGGTTCAGACAGGCCCCTGGCAAGGGCAGAGAATTTGTGGCCAGCATCACCTGGTCCAGCGACAACTGGTATCACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACGCCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAGCCGTGTACTACTGTGCCACAAGCACCGCCTGGTCTACCCTGGCCACCAGATACGATAATTGGGGCCAGGGCACCACCGTGACCGTTTCTTCT

(29) C10-HM5 amino acid sequence

EVQLVESGGGLVKPGGSLRLSCAASGRTQSTYNTGWFRQAPGKGREFVASITWSSDNWYHADSVRGRFTISRDNAKNAVYLQMNSLRAEDTAVYYCATSTAWSTLATRYDNWGQGTTVTVSS

(30) C10-HM5 nucleic acid sequences

GAGGTGCAGCTGGTTGAATCTGGCGGCGGACTTGTGAAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCTCTGGCAGAACCCAGAGCACATACAATACCGGCTGGTTCAGACAGGCCCCTGGCAAGGGCAGAGAATTTGTGGCCAGCATCACCTGGTCCAGCGACAACTGGTATCACGCCGATTCTGTGCGGGGCAGATTCACCATCAGCCGGGACAATGCCAAGAACGCCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACAGCCGTGTACTACTGTGCCACAAGCACCGCCTGGTCTACCCTGGCCACCAGATACGATAATTGGGGCCAGGGCACCACCGTGACCGTTTCTTCT

(31) A10-HM1 amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGIDFSISDMAWYRQAPGKGREWVAYVSSGNLIQYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCYARTYSNGLRIHWGQGTLVTVSS

(32) A10-HM1 nucleic acid sequences

GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCATCGACTTCAGCATCTCTGACATGGCCTGGTACAGACAGGCCCCTGGCAAGGGCAGAGAATGGGTCGCATATGTGTCCAGCGGCAACCTGATCCAGTACGCCGATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTTACGCCCGGACCTACAGCAACGGCCTGAGAATCCATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT

(33) A10-HM2 amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGIDFSISDMAWYRQAPGKGREVVAFVSSGNLIQYADSVKGRFTISRDNAKNSVHLQMNSLRAEDTAVYLCYARTYSNGLRIHWGQGTLVTVSS

(34) A10-HM2 nucleic acid sequences

GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCATCGACTTCAGCATCTCTGACATGGCCTGGTACAGACAGGCCCCTGGCAAGGGAAGAGAAGTGGTGGCCTTTGTGTCCAGCGGCAACCTGATCCAGTACGCCGATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACTCCGTGCACCTCCAGATGAACAGCCTGAGAGCCGAGGATACCGCCGTGTACCTGTGTTACGCCCGGACCTACAGCAACGGCCTGAGAATCCATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT

(35) A10-HM3 amino acid sequence

EVQLVESGGGLVKPGGSLRLSCAASGIDFSISDMAWYRQAPGKGREWVSFVSSGNLIQYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYLCYARTYSNGLRIHWGQGTLVTVSS

(36) A10-HM3 nucleic acid sequences

GAGGTGCAGCTGGTTGAATCTGGCGGCGGACTTGTGAAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCTCTGGCATCGACTTCAGCATCAGCGACATGGCCTGGTACAGACAGGCCCCTGGCAAGGGCAGAGAATGGGTGTCCTTTGTGTCCAGCGGCAACCTGATCCAGTACGCCGATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACAGCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTATCTGTGTTACGCCCGGACCTACAGCAACGGCCTGAGAATCCATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT

(37) A10-HM4 amino acid sequence

EVQLVESGGGLVKPGGSLRLSCAASGIDFSISDMAWYRQAPGKGREVVAFVSSGNLIQYADSVKGRFTISRDNAKNSVHLQMNSLRAEDTAVYLCYARTYSNGLRIHWGQGTLVTVSS

(38) A10-HM4 nucleic acid sequences

GAGGTGCAGCTGGTTGAATCTGGCGGCGGACTTGTGAAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCTCTGGCATCGACTTCAGCATCAGCGACATGGCCTGGTACAGACAGGCCCCTGGCAAGGGAAGAGAAGTGGTGGCCTTTGTGTCCAGCGGCAACCTGATCCAGTACGCCGATAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACTCCGTGCACCTCCAGATGAACAGCCTGAGAGCCGAGGATACCGCCGTGTACCTGTGTTACGCCCGGACCTACAGCAACGGCCTGAGAATCCATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT

(39) A10-HM5 amino acid sequence

EVQLVESGGGLVQPGGSLRLSCAASGIDFSISDMAWYRQAPGKGREVVAFVSSGNLIQYADSAKGRFTISRDNAKNSVHLQMNSLRAEDTAVYLCYARTYSNGLRIHWGQGTLVTVSS

(40) A10-HM5 nucleic acid sequences

GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCATCGACTTCAGCATCTCTGACATGGCCTGGTACAGACAGGCCCCTGGCAAGGGAAGAGAAGTGGTGGCCTTTGTGTCCAGCGGCAACCTGATCCAGTACGCCGATTCTGCCAAGGGCAGATTCACCATCAGCCGGGACAACGCCAAGAACTCCGTGCACCTCCAGATGAACAGCCTGAGAGCCGAGGATACCGCCGTGTACCTGTGTTACGCCCGGACCTACAGCAACGGCCTGAGAATCCATTGGGGCCAGGGCACACTGGTCACCGTTTCTTCT

(41) CD8 alpha signal peptide

ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCC

(42) CD8 hinge region

ACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCGCCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATACCCGGGGTCTTGACTTCGCCTGCGAT

(43) CD8 transmembrane region

ATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTCCTGCTGCTTTCACTCGTGATCACTCTTTACTGT

(44)4-1BB

AAGCGCGGTCGGAAGAAGCTGCTGTACATCTTTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTTCCCAGAGGAGGAGGAAGGCGGCTGCGAACTG

(45)CD3ξ

CGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG

(46) CD10-HM3-CDR1 amino acid sequence

GRTQSTYN

(47) C10-HM3-CDR2 amino acid sequence

ITWSSDNW

(48) C10-HM3-CDR3 amino acid sequence

ATSTAWSTLATRYDN

(49) C10-HM5-CDR1 amino acid sequence

GRTQSTYN

(50) C10-HM5-CDR2 amino acid sequence

ITWSSDNW

(51) C10-HM5-CDR3 amino acid sequence

ATSTAWSTLATRYDN

(52) A10-HM4-CDR1 amino acid sequence

GIDFSISD

(53) A10-HM4-CDR2 amino acid sequence

VSSGNL

(54) A10-HM4-CDR3 amino acid sequence

YARTYSNGLRIH

Claims

1. An EPHA2 chimeric antigen receptor comprising at least an antigen binding domain, a transmembrane domain, a costimulatory domain, and a signaling domain, wherein the antigen binding domain is an anti-EPHA 2 single domain antibody;

2. The EPHA2 chimeric antigen receptor according to claim 1, wherein the amino acid sequence of said anti-EPHA 2 single domain antibody is selected from the amino acid sequences shown in SEQ ID No.25, 29 or 37.

3. The EPHA2 chimeric antigen receptor according to claim 1, wherein said chimeric antigen receptor further comprises a signal peptide, said signal peptide being a CD8 a signal peptide; the nucleotide sequence of the CD8 alpha signal peptide is shown as SEQ ID NO. 41.

4. The EPHA2 chimeric antigen receptor according to claim 1, wherein the antigen binding domain and the transmembrane domain are linked by a hinge region comprising a CD8 hinge region having the nucleotide sequence shown in SEQ ID No. 42.

5. The EPHA2 chimeric antigen receptor according to claim 1, wherein the transmembrane domain is a CD8 transmembrane domain, and the nucleotide sequence of the CD8 transmembrane domain is shown in SEQ ID No. 43.

6. The EPHA2 chimeric antigen receptor according to claim 1, wherein the costimulatory domain comprises the 4-1BB domain; the nucleotide sequence of the human 4-1BB domain is shown as SEQ ID NO. 44;

7. A nucleic acid sequence capable of encoding the EPHA2 chimeric antigen receptor of the first aspect.

8. A CAR expression vector comprising the nucleic acid sequence of claim 7.

9. A CAR-T cell expressing the chimeric antigen receptor of EPHA2 of claim 1 or 2.

10. An anti-tumor drug comprising at least the CAR-T cell of claim 9;

the tumor is a tumor-associated disease associated with the overexpression of EPHA2, including brain glioma.