A nanobody against human EphA2 and its preparation method and application

Abstract

The present invention discloses a nanobody against human EphA2, a preparation method and application thereof. The nanobody specifically binds to human EphA2 to block the activity of EphA2. The nanobody is nanobody NbEphA2#1 or nanobody NbEphA2#6; the amino acid sequence of NbEphA2#1 is shown in SEQ ID NO.1; the amino acid sequence of NbEphA2#6 is shown in SEQ ID NO.2. The present invention successfully constructed an artificial synthesis library of nanobodies and screened out the two most effective nanobody sequences against EphA2. The nanobody can effectively inhibit the proliferation and stemness maintenance of human breast cancer cells and promote their apoptosis, and therefore has therapeutic prospects for targeting EphA2-positive tumor cells. In addition, the nanobody can significantly inhibit the growth of breast cancer in situ in mice without affecting the weight of mice, further confirming its potential and safety in tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of nano antibodies, and in particular to an anti-human EphA2 nano antibody and a preparation method and application thereof. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women worldwide. Triple-negative breast cancer (TNBC) has a poor prognosis and relatively limited treatment options due to the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2).

[0003] EphA2 (erythropoietin type A ephrin receptor-2) is a tyrosine kinase receptor that is highly expressed in a variety of tumor cells, including triple-negative breast cancer cells. It plays an important role in the proliferation, migration and invasion of tumor cells. Therefore, targeted therapy for EphA2 has become a potential treatment strategy. At present, the target research on EphA2 has made the following progress in the preclinical stage: In terms of antibody drugs, DS-8895a, as an antibody against EphA2, can significantly enhance the therapeutic effect on colorectal cancer when used in combination with cetuximab, and can reverse the secondary resistance to cetuximab. In addition, the new EphA2-targeted liposome (EphA2-ILs-DTXp) showed synergistic effects with PD-1 inhibitors in preclinical tumor models, which can significantly improve the therapeutic effect and increase CD8 + T cell infiltration in tumor tissues. In terms of peptide drug conjugates (PDCs), BT5528, as a PDC targeting EphA2, has shown significant anti-tumor activity in a variety of EphA2-positive tumor models by linking a cyclic peptide to an MMAE payload. In addition, EphA2 siRNA, as a nanoliposomal EphA2-targeted therapy, has been evaluated in a Phase I clinical trial (NCT01591356) for patients with advanced or recurrent solid tumors. These studies provide a variety of potential therapeutic strategies for EphA2-targeted therapy. Although there are not many successful clinical results at present, these studies provide new directions and hope for future tumor treatment.

[0004] Nanobodies (Nb) are ideal for many therapeutic and biotechnology applications due to their unique physicochemical properties, such as small size (approximately 15 kDa), high stability, high solubility, strong tissue penetration, low immunogenicity, and short production cycle, as well as the ability to bind to epitopes that are inaccessible to conventional antibodies. Currently, a large number of biologics based on multivalent, multispecific nanobodies and their modified structures have entered different stages of clinical research in the fields of immune diseases, tumors, neurological diseases, blood diseases, and infectious diseases. For example, Caplacizumab is the first Nb-based drug used to treat a rare coagulation disorder in adults with acquired thrombotic thrombocytopenic purpura, laying the foundation for the medical application of Nb; KN035 Envoli monoclonal antibody is the world's first nanobody for immunotherapy and is currently the only approved subcutaneous PD-(L)1 antibody for the treatment of advanced colorectal cancer. Although no other drugs based on Nb structures have been approved for cancer treatment. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the present invention provides an anti-human EphA2 nanobody, its preparation method, and application. The nanobody can specifically bind to human EphA2 and block its activity, thereby inhibiting the proliferation and stemness maintenance of cancer cells and promoting their apoptosis, providing a new means for the treatment of EphA2-positive tumor cells.

[0006] To solve the above technical problems, the technical solution of the present invention is as follows: a nanobody against human EphA2, wherein the nanobody is nanobody NbEphA2#1 or nanobody NbEphA2#6; the amino acid sequence of NbEphA2#1 is as shown in SEQ ID NO.1; the amino acid sequence of NbEphA2#6 is as shown in SEQ ID NO.2.

[0007] The present invention also provides a multivalent nanobody against human EphA2, wherein the multivalent nanobody is constructed by connecting one or both of the nanobody NbEphA2#1 and the nanobody NbEphA2#6.

[0008] Preferably, the nanobody NbEphA2#1 or nanobody NbEphA2#6 comprises four framework regions FR1, FR2, FR3, FR4 and three complementarity determining regions CDR1, CDR2 and CDR3;

[0009] Among them, in the nano antibodies NbEphA2#1 and NbEphA2#6, the amino acid sequence of FR1 is shown as SEQ ID NO.3; the amino acid sequence of FR2 is shown as SEQ ID NO.4; the amino acid sequence of FR3 is shown as SEQ ID NO.5; the amino acid sequence of FR4 is shown as SEQ ID NO.6; the amino acid sequence of CDR1 is shown as SEQ ID NO.7, and the amino acid sequence of CDR2 is shown as SEQ ID NO.8.

[0010] As further preferred, the amino acid sequence of the complementarity determining region CDR3 of the nanobody NbEphA2#1 is shown in SEQ ID NO.9;

[0011] The amino acid sequence of the complementarity determining region CDR3 of the nanobody NbEphA2#6 is shown in SEQ ID NO.10.

[0012] The present invention also provides a method for preparing the anti-human EphA2 nanobody, which comprises the following steps:

[0013] Step 1: Design and synthesize a CDR3 region DNA fragment library, then construct a plasmid DNA vector containing a nanobody backbone, and finally connect the CDR3 region DNA library into the nanobody backbone vector to form the final nanobody artificial synthesis library;

[0014] Step 2: Clone the gene fragments of the artificial synthesis library of nanobodies into an expression vector with a Flag tag, clone the EphA2 cDNA into an expression vector with HA, co-transfect HEK293T cells, and screen for nanobody gene sequences with higher abundance;

[0015] Step 3: Expression and purification of nanobodies;

[0016] Step 4: Verification of affinity and activity of nanobodies.

[0017] As a further description of the above scheme: Step 1 detailed operations are as follows:

[0018] Synthetic CDR3 DNA fragment library: Design and synthesize a CDR3 DNA fragment library containing 15-21 random amino acid sequences. Each amino acid is encoded by three bases. By randomly combining bases, a diverse CDR3 DNA fragment library is generated to ensure that a variety of possible amino acid sequence combinations are covered.

[0019] Construction of a Nanobody Backbone Plasmid DNA Vector: Design and construct a plasmid DNA vector containing the Nanobody backbone. The vector should contain the Nanobody framework region, CDR1 region, CDR2 region, and necessary expression regulatory elements. In addition, a 3×Flag tag should be fused to the C-terminus of the vector to facilitate subsequent detection and purification.

[0020] Connect the CDR3 region DNA library to the nanobody backbone vector: Use restriction enzymes or homologous recombination technology to connect the CDR3 region DNA library to the corresponding position of the nanobody backbone vector to form the final nanobody artificial synthetic library.

[0021] As a further description of the above scheme: Step 2 detailed operations are as follows:

[0022] The gene fragments of the artificial synthesis library of nanobodies were cloned into an expression vector with a 3×Flag tag to construct a nanobody expression plasmid; the EphA2 cDNA was cloned into an expression vector with an HA tag to construct an EphA2 expression plasmid;

[0023] The constructed nanobody expression plasmid and EphA2 expression plasmid were co-transfected into HEK293T cells. 24-60 hours after transfection, the cells were fixed, an isPLA experiment was performed, and positive cells were sorted using a flow cytometer. The plasmids in the sorted positive cells were PCR amplified, the amplified DNA fragments were recovered, and then high-throughput second-generation sequencing was performed. The CDR3 region DNA fragment sequences and abundances of the nanobody candidate factors binding to EphA2 were obtained. The top 6-10 nanobodies with the highest abundance were recombined into a protein expression vector and affinity tested, ultimately obtaining nanobodies against EphA2 with high affinity and specificity.

[0024] As a further description of the above scheme: Step 3 detailed operations are as follows:

[0025] Cloning the Nanobody gene sequence: Clone the Nanobody gene sequence screened in step 2 into an expression vector to construct an expression vector containing the following elements: 8 His tags, a TEV protease cleavage site, a transmembrane peptide TAT domain, a Nanobody sequence that recognizes EphA2, and a 3×Flag tag; transform the constructed expression vector into Escherichia coli BL21 competent cells and express the Nanobody protein under IPTG induction;

[0026] Purification and enzyme digestion

[0027] Nickel bead affinity purification: harvest the bacterial culture after expression, disrupt the cells by ultrasonication, and collect the supernatant;

[0028] Purification was performed using a nickel bead affinity chromatography column, which specifically bound the nanobody protein by utilizing the high affinity of the His8 tag to the nickel beads;

[0029] TEV protease cleavage: The purified protein solution is mixed with TEV protease in a certain proportion for enzymatic cleavage. After cleavage, the C-terminus of the nanobody protein will be carrying a TAT transmembrane peptide domain and a 3×Flag tag;

[0030] Repurification: Use affinity chromatography columns to further purify the enzymatically cleaved nanobody protein to remove uncleaved proteins and TEV protease.

[0031] The present invention also provides the use of the nanobody in the preparation of an anti-tumor drug. Preferably, the tumor is breast cancer.

[0032] The present invention successfully constructed a synthetic library of nanobodies and screened for two of the most effective anti-EphA2 nanobody sequences. These nanobodies can effectively inhibit the proliferation and stemness of human breast cancer cells and promote their apoptosis, thus showing promise for therapeutic applications targeting EphA2-positive tumor cells. Furthermore, animal experiments demonstrated that these nanobodies significantly inhibited the growth of triple-negative breast carcinoma in situ in mice without affecting their body weight, further confirming their potential and safety in tumor treatment.

[0033] Compared with existing technologies, the present invention has the following advantages: the nanobodies provided by the present invention can be used to prepare drugs for treating EphA2-positive tumors, particularly breast cancer. The nanobodies specifically bind to EphA2, blocking its activity, inhibiting tumor cell proliferation and stemness maintenance, and promoting tumor cell apoptosis, thereby exerting anti-tumor effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a map of the plasmid DNA vector containing the nanobody backbone;

[0035] Figure 2 The amino acid sequence and connection relationship corresponding to the above-mentioned nanocarrier backbone base sequence;

[0036] Figure 3 is a map of the expression vector with HA;

[0037] Figure 4 Figure 1 is the screening of EphA2 nanoantibodies; A is the flow chart of EphA2 nanoantibody screening; B is the isPLA of 293T cells followed by flow cytometry sorting of positive cells; C is the observation of positive signals of sorted positive cells under a confocal microscope, scale bar, 50 μm; D is the amplification of DNA fragments in the CDR3 region by PCR and subsequent second-generation sequencing;

[0038] Figure 5 This is a map of the pET-28a-SUMO expression vector;

[0039] Figure 6 Figure 1 is the affinity test of EphA2 nanobody; AB is the GST pull-down assay to detect antigen-antibody binding; C is the SPR assay to detect the affinity of EphA2 nanobody; D is the molecular docking prediction of the domain of EphA2 nanobody binding to EphA2 protein;

[0040] Figure 7 EphA2 nanobodies kill triple-negative breast cancer cells; A is a schematic diagram of the TAT transmembrane peptide fusion expression of nanobodies, as well as the seven expressed EphA2 nanobodies; B is the IF experiment to detect the localization and amount of nanobodies in HCC1806 and HCC1937 cells after the nanobodies were added to treat them, with a scale bar of 200 μm; C is the WB experiment to detect the amount of nanobodies in HCC1806 cells after the nanobodies were added to treat them;

[0041] Figure 8 EphA2 nanobody significantly inhibits the proliferation of triple-negative breast cancer cells. A shows that different concentrations of EphA2 nanobody were added to HCC1806 and MDA-MB-231 cells and treated for 48 hours, followed by a CCK8 assay to measure the number of viable cells. B shows that different concentrations of EphA2 nanobody were added to HCC1806 and MDA-MB-231 cells and subjected to a colony formation assay. The ability of tumor colony formation was then assessed by crystal violet staining. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005.

[0042] Figure 9 EphA2 nanobody significantly promoted apoptosis of triple-negative breast cancer cells. EphA2#1 and Nb EphA2#6 HCC1806 and MDA-MB-231 cells were treated with nanoantibodies, and tumor cell apoptosis was detected by Annexin V and PI staining. Scale bar, 100 μm. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005;

[0043] Figure 10 EphA2 nanobody inhibits tumor cell stemness; A is the use of 2μg / ml Nb E ph A2#1 and Nb EphA2#6HCC1806 and MDA-MB-231 cells were treated with nanoantibodies, and the proportion of ALDH-positive cell population was detected by flow cytometry; B is the treatment with 2 μg / ml Nb EphA2#1 and Nb EphA2#6 Nanobodies were used to treat HCC1806 and MDA-MB-231 cells, and the stemness of tumor cells was detected by mamosphere assay. Scale bar, 200 μm. C shows the effect of different concentrations of Nb EphA2#1 and Nb EphA2#6 HCC1806 and MDA-MB-231 cells were treated with nanoantibodies, and the expression of tumor cell stemness markers was detected by Western blotting. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005;

[0044] Figure 11 EphA2 nanobody inhibits tumor malignancy through the JAK-STAT3 pathway; AB are nanobody Nb with different concentrations EphA2#1 and Nb EphA2#6 After treating HCC1806 and MDA-MB-231 cells for 48 h, the expression of related proteins was detected by WB assay;

[0045] Figure 12 Bivalent nanobody BiNb EphA2#1 and BiNb EphA2#6 Inhibits the growth of mouse breast carcinoma in situ; A is a bivalent nanobody BiNb EphA2#1 and BiNb EphA2#6 SDS-PAGE staining of prokaryotic expression and purification; B is a schematic diagram of the mouse experiment; CH are HCC1806 and MDA-MB-231 cell lines inoculated into the mammary fat pad of mice, followed by administration of the bivalent nanobody BiNb EphA2#1 and BiNb EphA2#6 After treatment with 5 mg / kg of leukemia virus (LEV), the body weight, tumor volume, and tumor weight of mice were monitored. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the technical solution of the present invention is not limited thereto.

[0047] Example 1 Preparation of Nanobodies

[0048] 1. Construction of artificial synthetic library of nanoantibodies

[0049] First, a CDR3 region DNA fragment library was synthesized by merging bases, with the number of combined bases reaching 20 amino acids. Next, a plasmid DNA vector containing the Nanobody backbone was constructed. Finally, the CDR3 region DNA library was ligated into the Nanobody backbone vector to form the final synthetic Nanobody library. This synthetic library encompasses a large number of Nanobodies with diverse sequences, providing a rich set of candidate sequences for screening Nanobodies against EphA2.

[0050] Detailed steps

[0051] Synthetic CDR3 DNA Fragment Library: A CDR3 DNA fragment library containing 20 random amino acid sequences was designed and synthesized. Each amino acid is encoded by three bases, so the CDR3 DNA fragments are 60 bases long. By randomly combining bases, a diverse CDR3 DNA fragment library was generated, ensuring coverage of a wide range of possible amino acid sequence combinations.

[0052] Construction of nanobody backbone plasmid DNA vector: Design and construct a plasmid DNA vector containing the nanobody backbone. The vector should contain the framework region of the nanobody (FR1, FR2, FR3, FR4, CDR1, CDR2) and necessary expression regulatory elements, such as promoters, terminators, etc. In addition, the C-terminus of the vector should be fused with a 3×Flag tag for subsequent detection and purification. The vector map is as follows: Figure 1 shown.

[0053] Connect the CDR3 region DNA library to the Nanobody backbone vector: Use appropriate restriction enzymes or homologous recombination technology to connect the CDR3 region DNA library to the corresponding position of the Nanobody backbone vector. Ensure that the connected plasmid DNA vector contains the complete Nanobody sequence, including FR1, FR2, FR3, FR4 and CDR3 regions, as well as the 3×Flag tag.

[0054] The base sequence corresponding to the nanobody backbone is shown in SEQ ID NO. 11. The amino acid sequence corresponding to the base sequence of the nanocarrier backbone is shown in Figure 2 shown. Figure 2 In the present invention, the amino acid sequence of FR1 is shown in SEQ ID NO.3; the amino acid sequence of FR2 is shown in SEQ ID NO.4; the amino acid sequence of FR3 is shown in SEQ ID NO.5; the amino acid sequence of FR4 is shown in SEQ ID NO.6; the amino acid sequence of CDR1 is shown in SEQ ID NO.7 and the amino acid sequence of CDR2 is shown in SEQ ID NO.8.

[0055] The experimental conditions for homologous recombination of the nanobody backbone fragment into the pCDNA3.1 vector to construct the pCDNA3.1-Nb-3 X flag vector are as follows:

[0056] 1. Linearization of Nanobody Backbone Fragments and Vectors

[0057] A nanobody backbone fragment with 15-30 bp homology arms (synthesized by Qingke) was taken and dissolved in 20 μL of nuclease-free water. The fragment size was verified by 1% agarose gel electrophoresis (the expected length must match the target insertion region) and set aside.

[0058] pCDNA3.1 vector double enzyme digestion

[0059] Reaction system (50 μL):

[0060] pCDNA3.1 plasmid: 5 μg;

[0061] BamHI-HF: 2 μL (20 U);

[0062] EcoRI-HF: 2 μL (20 U);

[0063] 10× CutSmart Buffer: 5 μL;

[0064] Nuclease-free water: make up to 50 μL;

[0065] Conditions: Incubate at 37°C for 3 hours and heat at 80°C for 20 minutes to inactivate the enzyme.

[0066] Verification and recovery: The enzyme digestion product was subjected to 1% agarose gel electrophoresis, and the linearized vector was purified by gel excision and dissolved in 30 μL of water (concentration ≥ 50 ng / μL).

[0067] 2. Homologous Recombination Linking

[0068] Recombination reaction

[0069] Reaction system (10 μL):

[0070] Linearized pCDNA3.1 vector: 100 ng;

[0071] Fragment: molar ratio 3:1 (fragment:vector);

[0072] Novozymes ClonExpress MultiS recombinase: 2 μL;

[0073] 5×CE II Buffer: 2 μL;

[0074] Nuclease-free water: make up to 10 μL;

[0075] Conditions: reaction at 37°C for 30 minutes, inactivation at 50°C for 10 minutes.

[0076] 3. Conversion and Screening

[0077] 1. Chemical transformation

[0078] Competent cells: Take 50 μL DH5α competent cells (thawed on ice).

[0079] Conversion steps:

[0080] (1) Add 5 μL of purified recombinant product, mix gently, and incubate on ice for 30 minutes;

[0081] (2) Heat shock at 42°C for 45 seconds, followed by an ice bath for 2 minutes;

[0082] (3) Add 500 μL of resistance-free LB liquid medium and recover at 37°C for 45 minutes;

[0083] (4) Centrifuge (5000 rpm, 2 minutes), discard 400 μL of supernatant, resuspend and apply on Amp + (100 μg / mL) LB agar plates.

[0084] 2. Identification of positive clones

[0085] Sequencing confirmation: Send for single clone sequencing (primers cover the fragment insertion region and homology arms). Only use after sequencing confirms the sequence is correct.

[0086] Verify and amplify the Nanobody synthetic library: Verify the correctness of the ligated plasmid DNA vector through sequencing and PCR amplification. Ensure that the CDR3 region DNA fragment has been correctly inserted into the Nanobody backbone vector and has no mutations or deletions. Amplify the verified plasmid DNA vector to form the final Nanobody synthetic library.

[0087] By randomly combining bases to generate a library of CDR3 region DNA fragments, the diversity of nanobodies is ensured, encompassing a large number of nanobodies with different sequences, providing a rich set of candidate sequences for screening nanobodies against EphA2. This synthetic nanobody library can be used to screen for nanobodies against a variety of disease-related targets, not just EphA2. High-throughput screening techniques can rapidly identify and optimize nanobodies with high affinity and specificity, providing new tools and approaches for disease diagnosis and treatment.

[0088] 2.isPLA-seq screening of nanobodies

[0089] The gene fragments of the artificial synthesis library of nanobodies were cloned into an expression vector with a Flag tag, and EphA2 cDNA was cloned into an expression vector with HA (pCMV-HA, as shown in the figure). Figure 3 As shown), HEK293T cells were co-transfected. After 48 hours of transfection, the cells were fixed and isPLA was performed to obtain cells with positive signals. Positive cells were sorted by flow cytometry, and the plasmids in the positive cells were PCR amplified. The amplified DNA fragments were recovered and subjected to subsequent high-throughput second-generation sequencing, thereby obtaining the CDR3 region DNA fragment sequences of the candidate nanoantibodies that bind to EphA2 and their abundance. The top 8 nanoantibodies with the highest abundance were recombined into a protein expression vector and affinity tested, and finally, nanoantibodies with high affinity and specificity against EphA2 were obtained.

[0090] Detailed steps:

[0091] 2.1 Cloning and co-transfection of nanobody gene fragments

[0092] Cloning of Nanobody gene fragments: The gene fragments of the Nanobody artificial synthesis library were cloned into an expression vector with a Nanobody backbone region and a 3×Flag tag to construct a Nanobody expression plasmid. The specific experimental conditions are as follows:

[0093] 1. CDR3 fragment and vector linearization

[0094] A mixture of nanobody CDR3 fragments with 15-30 bp homology arms (synthesized by Qingke) was prepared. 1 μg of the fragment was dissolved in 20 μL of nuclease-free water and the fragment size was verified by 1% agarose gel electrophoresis (the expected length must match the target insertion region) and set aside.

[0095] pCDNA3.1-Nb-3 X flag vector double enzyme digestion

[0096] Reaction system (50 μL):

[0097] pCDNA3.1 plasmid: 5 μg;

[0098] HindIII-HF: 2 μL (20 U);

[0099] BsaI-HF: 2 μL (20 U);

[0100] 10× CutSmart Buffer: 5 μL;

[0101] Nuclease-free water: make up to 50 μL.

[0102] Conditions: Incubate at 37°C for 3 hours and heat at 80°C for 20 minutes to inactivate the enzyme.

[0103] Verification and recovery: The enzyme digestion product was subjected to 1% agarose gel electrophoresis, and the linearized vector was purified by gel excision and dissolved in 30 μL of water (concentration ≥ 50 ng / μL).

[0104] 2. Homologous Recombination Linking

[0105] Recombination reaction

[0106] Reaction system (10 μL):

[0107] Linearized pCDNA3.1-Nb-3 X flag vector: 100 ng;

[0108] CDR3 fragment mixture: molar ratio 3:1 (fragment:vector);

[0109] Novozymes ClonExpress MultiS recombinase: 2 μL;

[0110] 5×CE II Buffer: 2 μL;

[0111] Nuclease-free water: make up to 10 μL;

[0112] Conditions: reaction at 37°C for 30 minutes, inactivation at 50°C for 10 minutes.

[0113] 3. Conversion and Screening

[0114] 1. Chemical transformation

[0115] Competent cells: Take 50 μL DH5α competent cells (thawed on ice).

[0116] Conversion steps:

[0117] (1) Add 5 μL of purified recombinant product, mix gently, and incubate on ice for 30 minutes;

[0118] (2) Heat shock at 42°C for 45 seconds, followed by an ice bath for 2 minutes;

[0119] (3) Add 500 μL of resistance-free LB liquid medium and recover at 37°C for 45 minutes;

[0120] (4) Centrifuge (5000 rpm, 2 minutes), discard 400 μL of supernatant, resuspend and spread on Amp + (100 μg / mL) LB agar plate.

[0121] Plasmid extraction of positive clones

[0122] After collecting the positive clones on the agar plate, the DNA was extracted using a plasmid extraction kit to obtain the artificial synthesis library of nanobodies.

[0123] Cloning of EphA2 cDNA: EphA2 cDNA was cloned into an expression vector (pCMV-HA) with an HA tag to construct an EphA2 expression plasmid.

[0124] Co-transfection of HEK293T cells: The constructed nanobody expression plasmid and the EphA2 expression plasmid were co-transfected into HEK293T cells. 48 hours after transfection, the cells were fixed and the isPLA assay was performed.

[0125] 2.2isPLA experiment and positive cell sorting

[0126] isPLA Experimental Principle: isPLA (in situ proximity ligation assay) is a highly sensitive molecular detection method used to visualize protein interactions at the single-cell level. This technique uses specific antibodies to recognize and bind to target proteins. A PLA probe containing a segment of oligodeoxynucleotide (single-stranded DNA) then recognizes and binds to the primary antibody. When two target proteins come into proximity, the DNA fragments of the PLA probes pair and complement each other. Ligase then ligates the DNA fragments on the PLA probes, forming a circular structure that generates a detectable signal through rolling circle amplification (RCA).

[0127] Experimental steps:

[0128] Fixation and permeabilization: Fix the treated cell slides with 4% paraformaldehyde and then permeabilize with 0.2% TritonX-100.

[0129] Blocking: Add blocking solution dropwise onto the cell slide, ensuring that the blocking solution evenly covers the entire tissue area, and incubate at 37°C for 1 hour.

[0130] Incubate with primary antibody: Add the diluted primary antibody evenly onto the blocked cell slide, place in a humidified chamber, and incubate at 37°C for 2-3 hours.

[0131] Incubate with PLA probe: Mix the PLUS and MINUS PLA probes and dilute according to the kit instructions. Aspirate the primary antibody solution and wash the slides twice with 1x Wash Buffer A for 5 minutes each. Aspirate the excess wash buffer, then add the PLA probe solution dropwise and incubate at 37°C for 1 hour.

[0132] Ligation and amplification: Add oligodeoxynucleotides complementary to the probes (hybridization solution) and ligase to form a closed circle. Add polymerase, using one of the probes as a template, and perform rolling circle replication to continuously form new closed circles.

[0133] Detection: Add fluorescein-labeled oligonucleotides (detection solution) to react with the circularized DNA to form a detectable fluorescent signal.

[0134] Flow cytometry sorting: Cells with positive red fluorescent signals were sorted using flow cytometry. These positive cells indicate that the nanobody successfully binds to EphA2.

[0135] PCR amplification and DNA fragment recovery: PCR amplification is performed on the plasmids in the sorted positive cells, and the amplified DNA fragments are recovered.

[0136] 2.3 High-throughput second-generation sequencing: The recovered DNA fragments were subjected to high-throughput second-generation sequencing, thereby obtaining the CDR3 region DNA fragment sequence and abundance of the candidate nanoantibody factors that bind to EphA2.

[0137] According to the isPLA-seq screening method of previous studies ( Figure 4 A), the present invention constructed an Nb library, wherein the antibody characteristic determining domain CDR3 region contains 20 amino acids. First, EphA2-HA and Nbs-Flag artificial synthetic library were transiently overexpressed in HEK239T cells simultaneously. In situ red fluorescence signal was obtained by isPLA, and positive cells were sorted out by cell flow cytometry. The positive rate of the control group was 0, and the positive rate of the experimental group was 24.7% ( Figure 4 B). Then, under a fluorescence microscope, the PLA-positive cells were observed to emit specific red fluorescence on their organelle membranes ( Figure 4 C) and PCR amplification by designing forward and reverse primers of CDR3 ( Figure 4 D) The present invention obtained a 108-bp CDR3 mixture, which was recovered and subjected to next-generation sequencing to obtain the DNA sequence of the CDR3 region. The top eight Nanobodies with the most sequence numbers were first selected for subsequent validation and experiments. These data fully demonstrate the effectiveness and efficiency of this Nanobody screening technology.

[0138] Affinity testing:

[0139] The top eight nanobodies with the highest abundance were recombined into a protein expression vector and affinity tested, ultimately obtaining an anti-EphA2 nanobody with high affinity and specificity.

[0140] Advantages: isPLA technology can amplify protein signals a thousand-fold, offering high sensitivity and specificity, enabling the detection of trace samples, weak or transient interactions, and low-abundance expression. The technology is simple to use and quick to perform, with results available in just one day. isPLA technology can visualize protein interactions, allowing observation of the cellular regions where interacting proteins occur. It can also be combined with pathological techniques to observe protein interactions within specific cells. This technology is not only suitable for screening EphA2 but can also be used to detect the interactions, localization, and quantification of other proteins, providing new tools and approaches for disease diagnosis and treatment.

[0141] 3. Expression and Purification of Nanobodies

[0142] The screened nanobody gene sequence was cloned into the pET-28a-SUMO expression vector, pET-28a-SUMO-nanobody, which contained 8 histidine His8, tobacco mosaic virus TEV protease cleavage site, transmembrane peptide TAT domain, nanobody sequence recognizing EphA2 and 3×Flag tag. The constructed vector map is shown below. Figure 5 The protein is shown in Figure 1 (excluding the CDR3 region), with a molecular weight of 39 kDa. The protein was then transformed into competent E. coli BL21 (DE3) cells. Under IPTG induction, the nanobody protein was expressed. Following nickel-bead affinity purification and TEV protease cleavage, a highly purified nanobody protein recognizing EphA2 with a C-terminal TAT transmembrane peptide domain and a Flag tag was obtained. The protein has a molecular weight of 19 kDa and is used for subsequent experimental research and drug development.

[0143] Detailed steps:

[0144] 3.1 Construction of expression vector

[0145] Cloning of nanobody gene sequence: The screened nanobody gene sequence was cloned into the pET-28a-SUMO expression vector to construct an expression vector containing the following elements: 8 histidine (His8) tags, tobacco mosaic virus (TEV) protease cleavage site, transmembrane peptide TAT domain, nanobody sequence recognizing EphA2, 3×Flag tag. The molecular weight of the construct is 39 kDa.

[0146] The specific experimental conditions are as follows:

[0147] 1. Preparation of vector and insert

[0148] The nanobody DNA containing the complete framework region and CDR3 region was synthesized by Qingke Company and included the 15bp homology arms required for cloning.

[0149] 1. Linearization of pET-28a-SUMO vector

[0150] Double enzyme digestion reaction system (50 μL):

[0151] pET-28a-SUMO plasmid: 2 μg;

[0152] BamHI-HF: 2 μL (20 U);

[0153] EcoRI-HF: 2 μL (20 U);

[0154] 10× CutSmart Buffer: 5 μL;

[0155] Nuclease-free water: make up to 50 μL;

[0156] Conditions: incubate at 37°C for 3 hours and inactivate at 80°C for 20 minutes.

[0157] Purification: 1% agarose gel electrophoresis was used to verify linearization (expected band ∼6.8 kb), and the purified vector was recovered from the gel (dissolved in 30 μL TE buffer, concentration ≥50 ng / μL).

[0158] 2. Seamless cloning (homologous recombination)

[0159] Recombination reaction system (10 μL):

[0160] Linearized pET-28a-SUMO vector: 50ng

[0161] Nanobody gene fragment: molar ratio 3:1 (fragment: vector, calculated by length);

[0162] Novozymes ClonExpress MultiS recombinase: 2 μL;

[0163] 5×CE II Buffer: 2 μL;

[0164] Nuclease-free water: make up to 10 μL;

[0165] Reaction conditions:

[0166] Incubate at 37°C for 30 minutes → inactivate at 50°C for 10 minutes → cool on ice.

[0167] 3. Transformation and Positive Clone Screening

[0168] Chemical transformation:

[0169] Competent cells: DH5a.

[0170] step:

[0171] Take 5 μL of recombinant product and add 50 μL of competent cells and incubate on ice for 30 minutes;

[0172] Heat shock at 42°C for 45 seconds → ice bath for 2 minutes;

[0173] Add 500 μL SOC medium and recover at 37°C for 1 hour;

[0174] The cells were centrifuged (5000 rpm, 2 minutes), 400 μL of supernatant was discarded, and the cells were resuspended and spread on LB plates containing kanamycin (50 μg / mL).

[0175] Clone verification:

[0176] Sequencing confirmation: Send for single clone sequencing (primers cover the insertion site and homology arm regions).

[0177] 3.2 Transformation and Expression

[0178] Transformation into E. coli: The constructed expression vector was transformed into E. coli BL21 (DE3) competent cells.

[0179] Induced expression: Nanobody protein was expressed under IPTG (isopropyl-β-D-thiogalactopyranoside) induction conditions of 0.2 mM IPTG at 16°C for 16 hours.

[0180] 3.3 Purification and enzyme digestion

[0181] Nickel bead affinity purification: harvest the bacterial culture after expression, disrupt the cells by sonication, and collect the supernatant.

[0182] Nickel beads (Ni-NTA) affinity chromatography column was used for purification, and the high affinity of His8 tag to nickel beads was utilized to specifically bind to the nanobody protein.

[0183] The chromatography column is washed with an equilibration buffer (eg, 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0) to remove impurities.

[0184] The target protein was eluted with an elution buffer (eg, 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, pH 8.0).

[0185] TEV protease digestion: The purified protein solution is mixed with TEV protease in a specific ratio and digested, typically overnight at 4°C. After digestion, the C-terminus of the nanobody protein will contain a TAT transmembrane peptide domain and a 3×Flag tag with a molecular weight of 19 kDa.

[0186] Purification again: Use affinity chromatography columns (such as Flag-M2 resin) to further purify the enzymatically cleaved nanobody protein to remove uncleaved protein and TEV protease.

[0187] The column is washed with an equilibration buffer (eg, 20 mM Tris-HCl, 150 mM NaCl, pH 8.0) to remove impurities.

[0188] The target protein was eluted with elution buffer (eg, 20 mM Tris-HCl, 150 mM NaCl, 1 mg / mL 3×Flag peptide, pH 8.0).

[0189] Note: However, in the purification of nanobodies and bivalent nanobodies in animal experiments, this application removed TAT and 3XFlag from the nanobodies to obtain naked antibodies to prevent toxicity and side effects on mice in vivo.

[0190] 3.4 Purity testing and storage

[0191] Purity detection: The purity of the purified nanobody protein was detected by SDS-PAGE and HPLC to ensure that its purity reached more than 95%.

[0192] Storage: The purified nanobody protein is packaged and stored at -80°C or freeze-dried for subsequent experimental research and drug development.

[0193] Advantages

[0194] Efficient expression: Using the pET-28a-SUMO expression vector, nanobody proteins can be efficiently expressed, improving protein stability and solubility.

[0195] High-purity purification: Through nickel-bead affinity chromatography and TEV protease cleavage, high-purity nanobody proteins can be obtained, with a purity of more than 95%.

[0196] Functional integrity: The nanobody protein with a TAT transmembrane peptide domain and a 3×Flag tag at the C-terminus not only retains the specific binding ability to EphA2, but also has good cell penetration ability, making it suitable for a variety of biomedical applications.

[0197] Application: This nanoantibody protein can be used in a variety of biomedical research, including cell experiments, animal experiments and preclinical studies, providing new tools and methods for the diagnosis and treatment of EphA2-positive tumors.

[0198] Example 2 Nanobody Affinity Verification

[0199] 1. GST Pull-Down Experiment

[0200] In order to verify whether the eight candidate nanobodies with the highest sequence abundance directly bind to EphA2, a GST pull-down experiment was performed in this example.

[0201] The specific steps are as follows:

[0202] Protein Extraction and Purification: After purifying the GST-EphA2 fusion protein expressed in prokaryotic cells, add an appropriate volume of 50% glutathione-Sepharose 4B and gently shake on a shaker at 4°C for 30-60 minutes. Centrifuge at 4000 rpm for 5 minutes at 4°C and discard the supernatant. Wash the beads with pre-chilled PBS and repeat this step three times. Aspirate the liquid from the beads, but be careful not to remove the beads themselves. This will yield the GST-EphA2-bound Sepharose.

[0203] Incubation and Pull-Down: Combine solutions containing GST-EphA2 protein and candidate nanobody, rotate and incubate overnight at 4°C. Centrifuge at 4000 rpm for 5 minutes at 4°C, discard the supernatant, and wash three times with pre-chilled buffer. Aspirate the aqueous layer above the agarose gel and add 1x protein electrophoresis loading buffer. Boil the protein sample, aliquot, and freeze at -80°C for subsequent testing.

[0204] 2. SPR Experiment

[0205] Furthermore, the present invention detected the affinity of these seven candidate nanobodies through surface plasmon resonance (SPR) experiments.

[0206] The specific steps are as follows:

[0207] Experimental design: at least 8 concentration gradients, low coupling, high flow rate, affinity K D The value must fall within the concentration range. Set at least one concentration of sample to be repeated (interval completion), and set a zero concentration sample.

[0208] Kinetic analysis: By fitting all curves, kinetic ka, kd and affinity K are obtained D . K D =kd / ka.

[0209] Measure the response when steady state is reached, with high ligand coupling levels (high ligand concentration, coupling flow rate, coupling loading time).

[0210] Results: The dissociation constants (KD) of these seven candidate nanobodies were 84.93nM, 151.2nM, 248.6nM, 120.8nM, 57.77nM, 299.6nM and 3718nM, respectively.

[0211] Among them, Nb EphA2#1 and Nb EphA2#6 It has the highest specificity and affinity.

[0212] 3. Molecular Docking Prediction

[0213] In addition, the present invention also performs molecular docking prediction to further verify the specific binding of the nanobody to EphA2.

[0214] The specific steps are as follows:

[0215] 3D model construction: BLAST was used to search the PDB_95 database and select a suitable template. Based on the alignment results, the MODELER program was used to construct a 3D structural model of the nanobody.

[0216] Molecular docking: Use software such as ZDOCK or HADDOCK to perform molecular docking and predict the binding mode of nanoantibodies with EphA2.

[0217] Experimental results: In order to verify whether the eight candidate nanobodies with the highest sequence abundance directly bind to EphA2, the present invention conducted a GST pull-down experiment. The results showed that except for Nb EphA2#2 Except for , the remaining 7 nanobodies all directly bind to EphA2 ( Figure 6 A and B), including Nb EphA2#1 , Nb EphA2#3 , Nb EphA2#4 , Nb EphA2#5 , Nb EphA2#6 , Nb EphA2#7 and Nb EphA2#8 Furthermore, the present invention tested the affinity of these 7 candidate nanobodies through SPR experiments, and their dissociation constants (K D ) were 84.93nM, 151.2nM, 248.6nM, 120.8nM, 57.77nM, 299.6nM and 3718nM ( Figure 6 C). Among them, Nb EphA2#1 and Nb EphA2#6 Has the strongest affinity ( Figure 6 C). The above results indicate that the EphA2 nanobody binds specifically to EphA2 with strong affinity. In addition, the molecular docking prediction model shows that all seven nanobodies to EphA2 bind to it through the CDR3 region ( Figure 6 D), further proved the specificity of EphA2 nanobody. In summary, through isPLA-seq technology, this application obtained 7 candidate nanobodies that specifically bind to EphA2. In subsequent studies, the present invention will further study the nanobodies with the strongest affinity. EphA2#1 and Nb EphA2#6 Functions of 2 nanobodies.

[0218] Nanobody Nb EphA2#1 、Nb EphA2#3 、Nb EphA2#4 、Nb EphA2#5 、Nb EphA2#6 、Nb EphA2#7 and Nb EphA2#8 The amino acid sequence is shown in Table 1.

[0219] Table 1 Amino acid sequences of the CDR regions of Nanobodies

[0220]

[0221] Summary: Through isPLA-seq technology, the present invention obtained 7 candidate nanobodies that specifically bind to EphA2. In subsequent studies, this application will further study the Nb with the strongest affinity. EphA2#1 and Nb EphA2#6 Functions of the two nanobodies.

[0222] Example 3 Activity Verification of Nanobodies

[0223] 1. Cell Experiment

[0224] 1.1 CCK8 Experiment

[0225] In order to detect whether the EphA2 nanobody has killing activity on tumor cells, the present invention detected the proliferation of HCC1806 and MDA-MB-231 cells under the condition of nanobody treatment by CCK8 experiment.

[0226] The specific steps are as follows:

[0227] Cell culture: HCC1806 and MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Experimental treatments were performed when cells reached 70-80% confluence.

[0228] Nanobody Treatment: Cells were seeded at a density of 5,000-10,000 cells / well in a 96-well plate with 100 μL of culture medium per well. After cell attachment, different concentrations of EphA2 nanobody (0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM) were added, with triplicate wells for each concentration. An equal volume of PBS was added to the control group.

[0229] CCK8 Assay: After 24 hours of treatment, add 10 μL of CCK8 reagent to each well and continue incubation for 1-2 hours (the specific time depends on the cell type and experimental conditions). Measure the absorbance (OD value) at 450 nm using a microplate reader.

[0230] Data analysis: The ratio of the OD value of the nanobody-treated group to the OD value of the control group at each concentration was calculated, and a concentration-response curve was plotted. The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression analysis.

[0231] Experimental results:

[0232] The results showed that all seven EphA2 nanobodies exhibited strong cytotoxicity and killed tumor cells in a concentration-dependent manner. The specific IC50 values ​​are shown in Table 2 and Figure 8 As shown in A.

[0233] Table 2 IC50 of EphA2 nanobody against MDA-MB-231 cells and HCC1806 cells

[0234] EphA2 nanobody IC50 in MDA-MB-231 cells IC50 of HCC1806 cells <![CDATA[Nb EphA2#1 ]]> 113.63nM 80.32nM <![CDATA[Nb EphA2#3 ]]> 11.56nM 71.53nM <![CDATA[Nb EphA2#4 ]]> 55.21nM 53.63nM <![CDATA[Nb EphA2#5 ]]> 88.16nM 47.57nM <![CDATA[Nb EphA2#6 ]]> 149.89nM 76.63nM <![CDATA[Nb EphA2#7 ]]> 30.36nM 135.47nM <![CDATA[Nb EphA2#8 ]]> 218.95nM 67.52nM

[0235] Through CCK8 experiments, the present invention verified the killing activity of 7 EphA2 nanobodies against triple-negative breast cancer cells HCC1806 and MDA-MB-231. The results showed that these nanobodies have strong cytotoxicity and kill tumor cells in a concentration-dependent manner. In particular, Nb EphA2#1 and Nb EphA2#6 , they showed high anti-tumor activity in both cell lines with low IC50 values. These nanobodies provide new drug candidates for the treatment of EphA2-positive tumors.

[0236] 1.2 Clone formation assay

[0237] In order to identify the nanoantibodies with the best anti-tumor activity, the present invention selected Nb based on their dissociation constant and IC50. EphA2#1 and Nb EphA2#6 Nanobodies are being studied in depth.

[0238] The specific steps are as follows:

[0239] Cell culture: HCC1806 and MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Experimental treatments were performed when cells reached 70-80% confluence.

[0240] Nanobody treatment: Cells were seeded into 6-well plates at a density of 500 cells / well with 2 mL of culture medium per well.

[0241] After the cells adhered to the wall, different concentrations of Nb EphA2#1 and Nb EphA2#6 Nanobodies (0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL and 10 μg / mL) were added, with 3 replicate wells set for each concentration, and an equal volume of PBS was added to the control group.

[0242] Colony formation assay:

[0243] After treatment, cells were cultured for 10-14 days until visible colonies formed.

[0244] Cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.

[0245] The number of colonies was counted under a microscope, and a colony was defined as a colony of at least 50 cells.

[0246] Data analysis: Calculate the ratio of the number of clones in the nanobody-treated group to the number of clones in the control group at each concentration.

[0247] Experimental results:

[0248] The results of the previous studies of this application indicate that EphA2 can accelerate the progression of triple-negative breast cancer by promoting tumor proliferation and maintaining tumor stemness. Therefore, the present invention hypothesizes that EphA2 nanobodies can have anti-tumor effects by blocking its effects on tumor proliferation, stemness maintenance, etc. To verify this hypothesis, the present invention fused the TAT transmembrane peptide to the N-terminus of the EphA2 nanobody to allow it to enter the cell with maximum efficiency and exert its effect. The nanobody with a purity greater than 95% was obtained by prokaryotic expression ( Figure 7 A). Indeed, EphA2 nanobody can enter the triple-negative breast cancer cell lines HCC1806 and HCC1937 under the action of TAT transmembrane peptide ( Figure 7 BC).

[0249] Nb EphA2#1 and Nb EphA2#6 Both nanobodies inhibited the colony formation of tumor cells in a concentration-dependent manner.

[0250] The specific results are as follows:

[0251] HCC1806 cells: at 2 μg / mL, Nb EphA2#1 and Nb EphA2#6 Both inhibited more than 70% of colony formation ( Figure 8 B).

[0252] MDA-MB-231 cells: at 2 μg / mL, Nb EphA2#1 and Nb EphA2#6 Both inhibited more than 70% of colony formation ( Figure 8 B).

[0253] These results indicate that Nb EphA2#1 and Nb EphA2#6 At a low concentration (2 μg / mL), they can significantly inhibit the colony formation of HCC1806 and MDA-MB-231 cells, indicating that they have strong anti-tumor activity. This further confirms that these two nanobodies have a significant effect in inhibiting tumor cell proliferation and colony formation, providing strong evidence for subsequent mechanism studies and preclinical experiments.

[0254] 1.3 Cell apoptosis detection: Annexin V / PI

[0255] To further verify that Nb EphA2#1 and Nb EphA2#6The anti-tumor activity of nanobodies was investigated using different concentrations of Nb EphA2#1 and Nb EphA2#6 HCC1806 and MDA-MB-231 cells were treated with nanoantibodies, and tumor cell apoptosis was detected by Annexin V and PI staining.

[0256] The specific steps are as follows:

[0257] Cell culture: HCC1806 and MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Experimental treatments were performed when cells reached 70-80% confluence.

[0258] Nanobody Treatment: Cells were seeded at a density of 1000-2000 cells / well in 6-well plates with 2 mL of culture medium per well. After cell attachment, different concentrations of NbEphA2#1 and NbEphA2#6 nanobodies (0 μg / mL, 4 μg / mL, and 8 μg / mL) were added, with triplicate wells per concentration. An equal volume of PBS was added to the control group.

[0259] Cell treatment and staining: After 48 hours of treatment, cells were collected, digested with EDTA-free trypsin, and collected in a centrifuge tube.

[0260] Wash the cells twice with pre-cooled PBS, centrifuging at 4°C and 300g for 5 minutes each time. Add 1× Binding Buffer and adjust the cells to the same concentration (usually 1×10 6 / mL). Take 1-2×10 5 Place the cell suspension on a glass slide, add 5 μL of FITC-labeled Annexin V and 5 μL of PI, and mix gently. Incubate at room temperature in the dark for 15-20 minutes. Cover the cells with a coverslip and immediately observe under a fluorescence microscope.

[0261] Data analysis: The cells were observed under a fluorescence microscope using a dual-color filter. Annexin V-FITC fluorescence signal was green, and PI fluorescence signal was red. + / PI - ) showed green fluorescence, and late apoptotic cells (Annexin V + / PI + ) showed green and red dual fluorescence, dead cells (PI + ) exhibits red fluorescence.

[0262] Experimental results: The present invention further combines Nb EphA2#1 and Nb EphA2#1Nanobodies were added to HCC1806 and MDA-MB-231 cells at concentrations of 4 μg / mL and 8 μg / mL. The results showed that Nb EphA#1 and Nb EphA#6 Nanobodies can significantly induce tumor cell apoptosis ( Figure 9 The specific results are as follows:

[0263] HCC1806 cells: at 4 μg / mL, Nb EphA2#1 and Nb EphA2#6 Both significantly induced cell apoptosis, and the proportions of early apoptotic cells and late apoptotic cells increased significantly.

[0264] MDA-MB-231 cells: at 4 μg / mL, Nb EphA2#1 and Nb EphA2#6 Both significantly induced cell apoptosis, and the proportions of early apoptotic cells and late apoptotic cells increased significantly.

[0265] It can be seen that EphA2 nanoantibodies can inhibit tumor cell proliferation and promote tumor cell apoptosis under low concentration conditions, indicating that Nb EphA#1 and Nb EphA#6 The two nanobodies have good anti-tumor efficacy.

[0266] These results indicate that Nb EphA2#1 and Nb EphA2#6 Nanobodies not only inhibited tumor cell proliferation at low concentrations, but also significantly induced tumor cell apoptosis, indicating that these two nanobodies have good anti-tumor efficacy. EphA2#1 and Nb EphA2#6 It has significant effects in inhibiting tumor cell proliferation and promoting tumor cell apoptosis, providing strong evidence for subsequent mechanism studies and preclinical experiments.

[0267] 1.4 Cell stemness detection experiments: ALDH, WB, mamosphere detection experiments

[0268] One of the most important roles of EphA2 in promoting the progression of triple-negative breast cancer is maintaining the stemness of tumor cells. Therefore, developing nanobodies that can inhibit tumor cell stemness is particularly important for the development of cancer therapeutics. Next, the present invention examined the effects of EphA2 nanobodies on tumor cell stemness.

[0269] (1) ALDH detection

[0270] First, the present invention detected 2μg / ml Nb EphA2#1 and Nb EphA2#6 ALDH expression in HCC1806 and MDA-MB-231 cells + Effect of cell ratio.

[0271] (2) Mamosphere detection

[0272] Secondly, the present invention detects Nb EphA2#1 and Nb EphA2#6 Effects on the formation of stem-like tumor spheres in HCC1937 cells.

[0273] (3) Western Blot

[0274] In terms of molecular mechanism, the present invention detected Nb by Western Blot. EphA2#1 and Nb EphA2#6 Effects on the expression of tumor cell stemness markers.

[0275] The present invention examined the effect of EphA2 nanobody on tumor cell stemness. EphA2#1 and Nb EphA2#6 Significantly reduced ALDH in HCC1806 and MDA-MB-231 + The cell ratio decreased by about 60-70% ( Figure 10 A). Secondly, Nb EphA2#1 and Nb EphA2#6 Significantly inhibited the formation of HCC1937 cell stem-like tumor spheres ( Figure 10 B) In terms of molecular mechanism, Nb EphA2#1 and Nb EphA2#6 Significantly reduced the expression of tumor cell stemness markers, including Nanog, SOX2, CD44, and OCT4, and showed a concentration-dependent trend. The higher the concentration of nanoantibodies, the lower the expression of these markers ( Figure 10 C) In summary, EphA2 nanobody Nb EphA2#1 and Nb EphA2#6 The results significantly inhibited the maintenance of the stemness characteristics of triple-negative breast cancer cells. This further confirmed that the two nanoantibodies have significant effects in inhibiting tumor cell proliferation and promoting tumor cell apoptosis, providing strong evidence for subsequent mechanism studies and preclinical experiments.

[0276] 1.5 Downstream pathway detection: Western blot experiment

[0277] Previous studies of this application have found that EphA2 activates the Jak-STAT3 pathway by promoting Jak phosphorylation, promoting the transcription of tumor-promoting factors such as triple-negative breast cancer oncogenes C-myc, CyclinD1, and KLF5, thereby promoting tumor progression. In order to explore the molecular mechanism of EphA2 nanoantibodies in inhibiting tumors, this invention uses Nb EphA2#1 and Nb EphA2#6HCC1806 and MDA-MB-231 cells were treated and the expression of related proteins was detected by Western Blot. The specific steps are as follows:

[0278] Cell culture: HCC1806 and MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Experimental treatments were performed when cells reached 70-80% confluence.

[0279] Nanobody treatment: cells were seeded into 6-well plates at a density of 5,000-10,000 cells / well, with 2 mL of culture medium per well. After the cells adhered to the wall, different concentrations of Nb EphA2#1 and Nb EphA2#6 Nanobodies (0 μg / mL, 2 μg / mL, 4 μg / mL, 6 μg / mL, 8 μg / mL, and 10 μg / mL) were added to the wells in triplicate for each concentration. An equal volume of PBS was added to the control group.

[0280] Protein Extraction: After 48 hours of treatment, harvest cells and wash twice with pre-chilled PBS, centrifuging at 300g for 5 minutes at 4°C each time. Add an appropriate amount of RIPA lysis buffer, PMSF (protease inhibitor) and phosphatase inhibitor, and lyse on ice for 30 minutes. Centrifuge at 12,000g for 15 minutes at 4°C, and collect the supernatant for the total protein extract.

[0281] Protein quantification: The protein concentration was determined using a BCA protein quantification kit to ensure consistency among the groups.

[0282] Western Blot Assay: Equal amounts of protein samples were collected and subjected to SDS-PAGE electrophoresis to separate the proteins. The proteins were transferred to a PVDF membrane and blocked with 5% skim milk powder for 1 hour. Primary antibodies (EphA2, JAK1, JAK2, STAT3, p-JAK1, p-JAK2, p-STAT3, C-myc, CyclinD1, KLF5, cleaved-caspase3, and BCL2) were added and incubated overnight at 4°C. The membranes were washed three times with TBST for 10 minutes each. Secondary antibodies (HRP-conjugated anti-rabbit or anti-mouse IgG) were added and incubated at room temperature for 1 hour. The membranes were washed three times with TBST for 10 minutes each. The membranes were developed using an ECL luminescence kit and imaged using a chemiluminescence imaging system.

[0283] Experimental results: The previous study of this application found that EphA2 activated the Jak-STAT3 pathway by promoting Jak phosphorylation, promoting the transcription of tumor-promoting factors such as C-myc, CyclinD1, and KLF5 in triple-negative breast cancer, and thus promoting tumor progression. In order to explore the molecular mechanism of EphA2 nanoantibodies in inhibiting tumors, the present invention used Nb EphA2#1 and Nb EphA2#6 Treat HCC1806 and MDA-MB-231 cells, WB experimental results show that: First, Nb EphA2#1 and Nb EphA2#6 The expression of EphA2 was reduced in a concentration-dependent manner ( Figure 11 AB). Secondly, Nb EphA2#1 and Nb EphA2#6 Significantly inhibited the phosphorylation of JAK1, JAK2 and STAT3 in a concentration-dependent manner ( Figure 11 AB). In addition, Nb EphA2#1 and Nb EphA2#6 It significantly inhibited the expression of downstream genes of the JAK-STAT3 pathway in a concentration-dependent manner, including C-myc, CyclinD1, KLF5, etc. Figure 11 AB). Finally, Nb EphA2#1 and Nb EphA2#6 It promotes the expression of apoptosis-related protein cleaved-caspase3 in a concentration-dependent manner and inhibits the expression of anti-apoptotic protein BCL2 ( Figure 11 AB). The above results show three points: EphA2 nanobody Nb EphA2#1 and Nb EphA2#6 1) By downregulating the expression of EphA2; 2) by inhibiting the activation of the JAK-STAT3 pathway to downregulate the expression of oncogenes C-myc, CyclinD1, and KLF5; 3) by increasing the expression of apoptosis-related proteins and decreasing the expression of anti-apoptosis-related proteins, thereby inhibiting tumor progression.

[0284] Example 4 Animal Experiment

[0285] A subcutaneous xenograft tumor model of triple-negative breast cancer was established in nude mice, and the nanobody was administered via intraperitoneal injection. Tumor volume was measured regularly, and the results showed that the nanobody significantly inhibited tumor growth.

[0286] Detailed steps:

[0287] Cell line preparation: HCC1806 and MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C, 5% CO2 incubator. Experimental treatments were performed when cells reached 70-80% confluence.

[0288] Construction of bivalent nanobodies

[0289] In order to enhance the inhibitory efficiency of EphA2 nanobody and prolong its half-life in mice, the present invention designed BiNb EphA2#1 and BiNb EphA2#6 The specific construction method is as follows:

[0290] Construction of bivalent nanobodies:

[0291] The same nanobody of EphA2 was connected head to tail through the GGGS linker (BiNb EphA2#1 With BiNb EphA2#1 Connection, BiNb EphA2#6 With BiNb EphA2#6 This connection ensures the appropriate spacing between the two nanobodies, thereby maintaining their binding activity.

[0292] Establishment of orthotopic tumor model: HCC1806 and MDA-MB-231 cell suspensions (1×10 6 cells / 100 μL) were injected into the mammary fat pad of mice to establish an orthotopic tumor model.

[0293] Six days after the tumor began to grow, bivalent nanobody treatment was started.

[0294] Evaluation of treatment effect: Every other day, the long diameter and short diameter of the tumor were measured with a caliper, and the tumor volume (V = 0.5 × long diameter × short diameter) was calculated. 2 After the treatment, tumor tissues were collected and the tumor weight was measured.

[0295] EphA2-based nanobody Nb EphA2#1 and Nb EphA2#6 Inhibitory effect in triple-negative breast cancer, the present invention designed BiNb EphA2#1 and BiNb EphA2#6 The bivalent antibody was expressed in prokaryotes and the purity of the bivalent antibody was greater than 95%. Figure 12 A). It is intended to enhance the inhibitory efficiency of bivalent nanobodies on EphA2 and prolong the half-life of nanobodies in mice to achieve better therapeutic effects ( Figure 12 B) Consistent with the in vitro test results, the bivalent nanobody BiNb EphA2#1 and BiNb EphA2#6 Significantly reduced the volume and weight of in situ tumors formed by HCC1806 and MDA-MB-231 cells in the mouse mammary gland ( Figure 12 C, D, F and G), in contrast, BiNb EphA2#1 and BiNb EphA2#6There was no significant effect on the body weight of mice ( Figure 12 E and H). The above results show that the bivalent nanobody BiNb EphA2#1 and BiNb EphA2#6 The ability to effectively treat triple-negative breast cancer in vivo by inhibiting EphA2, without significantly affecting the body weight of mice, demonstrates its good safety and therapeutic efficacy. This further confirms that the bivalent nanobody has a significant effect in inhibiting tumor cell proliferation and promoting tumor cell apoptosis, providing strong evidence for subsequent mechanism studies and preclinical experiments.

[0296] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A nanobody against human EphA2, characterized in that The nanobody is nanobody NbEphA2#1 or nanobody NbEphA2#6; the amino acid sequence of NbEphA2#1 is shown in SEQ ID NO.1; the amino acid sequence of NbEphA2#6 is shown in SEQ ID NO.

2.

2. A multivalent nanobody against human EphA2 constructed based on the nanostructure according to claim 1, characterized in that: The multivalent nanobody is constructed by connecting NbEphA2#1 and NbEphA2#1 end to end, or connecting NbEphA2#6 and NbEphA2#6 end to end through a GGGS linker.

3. Use of the nanobody according to claim 1 or the multivalent nanobody according to claim 2 in the preparation of an anti-tumor drug, characterized in that: The tumor is breast cancer.

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