Humanized targeting vegf single-domain antibody sdve05 and application thereof

Abstract

The application belongs to the field of biological medicine, and relates to a single-domain antibody, and more particularly to a humanized single-domain antibody sdVE05 targeting VEGF and application thereof. The amino acid sequence of the single-domain antibody sdVE05 comprises three complementarity determining regions CDR1-CDR3 and four framework regions FR1-FR4. The application further provides a recombinant protein comprising the single-domain antibody sdVE05, a nucleic acid molecule encoding the single-domain antibody sdVE05 or the recombinant protein, a vector containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the vector, and application of the single-domain antibody sdVE05 or the recombinant protein in preparation of a medicament for treating diseases with VEGF as a target.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a humanized VEGF-targeting single-domain antibody sdVE05 and its applications. Background Technology

[0002] Vascular endothelial growth factor (VEGF) is a family of dimeric glycoproteins, including various isoforms such as VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PIGF). VEGF initiates a series of intracellular signal transduction pathways by binding to specific receptors on endothelial cells (such as VEGFR-1, VEGFR-2, and VEGFR-3), primarily involved in increased vascular permeability, extracellular matrix degeneration, intravascular cell migration and proliferation, and angiogenesis. Based on the dominant role of VEGF-A in regulating angiogenesis and vascular permeability, the term VEGF usually refers to VEGF-A. Among the VEGF-A isoforms, VEGF165 is dominant in both quantity and biological activity. Under physiological conditions, it plays a crucial role in tissue growth, maintenance of vascular health, and pregnancy support. However, in certain disease states, abnormal expression or dysfunction of VEGF can lead to excessive or insufficient angiogenesis, which is closely related to the occurrence and development of various diseases. For example, during tumor growth, tumor cells can stimulate surrounding normal cells to secrete large amounts of VEGF, thereby promoting tumor angiogenesis and providing the tumor with sufficient nutrients and oxygen. This abnormal angiogenesis not only accelerates tumor growth but also increases the risk of tumor metastasis. Abnormal VEGF expression is also closely related to neovascular eye diseases, such as diabetic retinopathy and age-related macular degeneration. In these diseases, abnormally elevated VEGF levels lead to excessive retinal angiogenesis, which in turn triggers a series of pathological changes, such as vascular leakage, hemorrhage, and edema, severely impairing the patient's vision. In cardiovascular diseases, excessive VEGF expression can also lead to the occurrence and development of diseases such as atherosclerosis and vasculitis. In addition, in autoimmune diseases such as rheumatoid arthritis, abnormal VEGF expression can promote the infiltration of inflammatory cells and angiogenesis, exacerbating the inflammatory response and destruction of joints. Therefore, VEGF has become a key cytokine for clinical detection of diseases such as tumors, neovascular eye diseases, cardiovascular diseases, and rheumatoid arthritis, and a major target for therapeutic drugs.

[0003] Currently, approved anti-VEGF macromolecular drugs for clinical use mainly fall into three categories: antibodies or antibody fragments, such as bevacizumab (Avastin), ranibizumab (Lucentis), brolucizumab (Beovu), and faricimab-svoa (Vabysmo), a bispecific antibody targeting VEGF and Ang2; fusion proteins, such as aflibercept (Eylea) and conbercept (Lambu); and nucleic acid aptamers, such as pegaptanib (Macugen). In 1993, Hamers et al. discovered a naturally occurring active antibody lacking the light chain in camel serum, termed a heavy chain antibody, whose variable region is called a single-domain antibody (sdAb or VHH antibody). Subsequently, similar heavy chain antibodies were also found in some cartilaginous fish. Single-domain antibodies are currently the smallest functional antigen-binding fragments available, with a molecular weight of approximately 15 kDa, about 1 / 10 that of conventional antibodies, and are also known as nanobodies. Nanobodies possess advantages such as small molecular size and strong penetrability, high specificity and affinity, high solubility and stability, low immunogenicity, the ability to recognize antigenic epitopes with unique conformations, ease of expression, and ease of modification. They are poised to overcome the bottlenecks in the development of existing monoclonal antibody drugs and become a new favorite for the miniaturization and functionalization of monoclonal antibody drugs. In recent years, several single-domain antibody drugs have been successfully approved for marketing. For example, the first single-domain antibody drug, Caplacizumab (Cablivi), has been approved in the European Union for the treatment of thrombotic thrombocytopenic purpura; in China, KN-035, a PD-L1 single-domain antibody Fc fusion protein developed by KNJ Biopharma, has also been approved for marketing, becoming the world's first subcutaneously injectable single-domain antibody drug; and Ozoralizumab, a bispecific single-domain antibody, has been approved in Japan for the treatment of rheumatoid arthritis. These findings fully demonstrate the enormous potential of single-domain antibodies in drug development.

[0004] Based on the above background, this invention develops a humanized single-domain antibody targeting VEGF, providing a new option for the development of VEGF blocking drugs and the diagnosis and treatment of related diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a humanized VEGF-targeting single-domain antibody sdVE05 and its applications.

[0006] The technical solution adopted in this invention is as follows:

[0007] A first aspect of the present invention provides a humanized VEGF-targeting single-domain antibody sdVE05, wherein the amino acid sequence of the single-domain antibody sdVE05 includes three complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are as follows:

[0008] CDR1: YRINYENMA

[0009] CDR2: TILRPN

[0010] CDR3: TRRHRSNLGWRSSAPVQF.

[0011] The aforementioned single-domain antibody sdVE05 can specifically bind to VEGF and has high affinity.

[0012] Furthermore, the aforementioned single-domain antibody sdVE05 also includes four backbone regions FR1, FR2, FR3, and FR4, the amino acid sequences of which are as follows:

[0013] FR1: EVQLVESGGGLVQPGGSLRLSCAASG,

[0014] FR2: WVRQAPGKGLEWVS

[0015] FR3:GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA,

[0016] FR4: WGQGTLVTVSS.

[0017] Furthermore, the structure of the aforementioned single-domain antibody sdVE05 is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0018] Furthermore, the aforementioned single-domain antibody sdVE05 can be fused with other peptides, proteins, or functional substances to achieve diverse application goals.

[0019] A second aspect of the present invention provides a recombinant protein comprising the aforementioned single-domain antibody sdVE05.

[0020] A third aspect of the present invention provides a nucleic acid molecule that encodes the aforementioned single-domain antibody sdVE05 or recombinant protein.

[0021] A fourth aspect of the present invention provides a carrier containing the above-described nucleic acid molecules.

[0022] A fifth aspect of the present invention provides a host cell containing the aforementioned nucleic acid molecules or carriers.

[0023] A sixth aspect of the present invention provides a method for preparing the above-described single-domain antibody sdVE05 or recombinant protein, the method comprising culturing the above-described host cells and isolating and purifying the single-domain antibody sdVE05 or recombinant protein from the culture.

[0024] The seventh aspect of the present invention provides the use of the above-mentioned single-domain antibody sdVE05 and recombinant protein in the preparation of a medicament targeting VEGF to treat diseases.

[0025] The eighth aspect of the present invention provides the use of the above-described single-domain antibody sdVE05 and recombinant protein in the preparation of VEGF inhibitors.

[0026] The ninth aspect of the present invention provides the application of the above-described single-domain antibody sdVE05 and recombinant protein in the preparation of angiogenesis inhibitors.

[0027] The significant advantages of this invention are:

[0028] This invention provides a single-domain antibody sdVE05 targeting VEGF and a recombinant protein containing said single-domain antibody sdVE05, which has a unique CDR sequence capable of specifically binding to VEGF. Their discovery provides new drug candidates for treating diseases related to abnormal VEGF, such as neovascular eye disease, tumors, and rheumatoid arthritis, and offers new methods for the diagnosis and detection of these diseases. Attached Figure Description

[0029] Figure 1 Polyclonal phage ELISA was used to detect the enrichment of phages after four rounds of screening. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0030] Figure 2 Amino acid sequence alignment of the single-domain antibody targeting VEGF (sdVE). CDR1~CDR3 are complementarity-determining regions, and FR1~FR4 are backbone regions.

[0031] Figure 3 ELISA analysis of sdVE monoclonal phage. ** P<0.01, *** P<0.001, **** P<0.0001.

[0032] Figure 4 : Identification of sdVE expression in 293T cells (Western blotting analysis). Lane 1, intracellular; Lane 2, extracellular.

[0033] Figure 5Prokaryotic expression, purification, and affinity analysis of recombinant sdVE05 protein. A. SDS-PAGE analysis of the purification and desalting effect of recombinant sdVE05 protein (1. Protein molecular weight standard, 2. Before induction, 3. After induction, 4. Lysis supernatant, 5. Lysis precipitation, 6. Magnetic bead purification, 7. Desalting and lyophilization, 8. Renaturation); B. HiPrep™ 26 / 10 desalting plot; ELISA analysis of sdVE05 (C) and VHHL (D) binding to hVEGF. 165 Its affinity.

[0034] Figure 6 Effects of sdVE05 on HUVEC cell viability and in vitro migration ability. A, Effect of sdVE05 detected by CCK8 assay; B, Cell scratch assay; C, Cell migration assay by Transwell assay; D, Quantitative analysis of wound healing rate; E, Quantitative analysis of cell migration number. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs control.

[0035] Figure 7 :sdVE05 against hVEGF 165 Effects of stimulation on the horizontal and vertical migration ability of HUVEC vascular endothelial cells in vitro. A and C: Cell scratch assay and quantitative analysis of wound area healing rate; B and D: Cell migration assay and quantitative analysis of cell migration number. #### P<0.0001 vs hVEGF 165 Group; * P<0.05, ** P<0.005, *** P<0.0005, **** P<0.0001 vs Control group.

[0036] Figure 8 Effects of sdVE05 on HUVEC cell cast formation. A, in the absence of hVEGF 165 Formation of tubular structures in HUVEC cells under stimulation; B, ImageJ analysis quantifies the absence of hVEGF 165 The number of tubes formed under stimulation; C, in hVEGF 165 Formation of tubular structures in HUVEC cells under stimulation; D, ImageJ analysis to quantify hVEGF 165 Number of tubes formed under stimulation. Cell counts were obtained from three random fields of view and expressed as mean ± SD. #### P<0.0001 vs hVEGF 165 Group; * P<0.05, ** P<0.005, *** P<0.0005, **** P<0.0001 vs Control group. Detailed Implementation

[0037] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0038] Example 1: Biopaneling of VEGF-targeting single-domain antibodies

[0039] 1. First round of screening

[0040] (1) Coating antigen: The target antigen, human vascular endothelial growth factor 165 (hVEGF), is coated onto the target antigen. 165 Dilute to 10 µg / ml with coating buffer, coat ELISA plates at 100 μl / well, and incubate overnight at 4°C.

[0041] (2) Blocking: Wash 5 times with 0.1% PBST, then wash once with PBS, and block with 3% PBSM at 37℃ for 2 hours to block non-specific binding sites.

[0042] (3) Antibody incubation: After blocking, wash 6 times with 0.1% PBST, wash once with PBS, and add 100 μl / well of humanized single-domain antibody phage library (10 13 (pfu / mL), incubate at 37℃ for 2 hours.

[0043] (4) Washing and elution: Wash 5 times with 0.1% PBST, wash once with PBS, add 200 μl of elution buffer per well, and elute on a shaker at room temperature for 10 min. Transfer the elution buffer to a new tube and immediately add 100 μl of neutralization buffer and mix well. Take half of the solution for the amplification and titer determination of the new phage library.

[0044] 2. Second, third and fourth rounds of screening

[0045] Based on the phage library obtained in the previous round of screening, the steps of the first round of screening were repeated, except that the antigen coating concentration was reduced in each round (5.0 µg / ml, 2.5 µg / ml and 1.0 µg / ml) and the dilution factor was increased in each round of plating to enrich high-affinity phage clones.

[0046] Results Explanation:

[0047] As the number of screening rounds increased, the P / N value also increased accordingly, indicating the effectiveness of the enrichment screening and the increased specificity of the phages (see Table 1). This embodiment successfully screened high-affinity phage clones targeting VEGF from a humanized single-domain antibody phage library. (Note: In each round of screening, the amount of phage screened is recorded as Input, and the amount of phage obtained after elution is recorded as Output.)

[0048] Table 1. Enrichment effect of biopharmaceutical targeting hVEGF165 single-domain antibody phage

[0049]

[0050] Example 2: Polyclonal ELISA detection of phage library specific enrichment after 4 rounds of screening

[0051] (1) Coating: The target antigen hVEGF is coated. 165 Dilute to 5 µg / ml with coating buffer, coat ELISA plates at 100 μl / well, and incubate overnight at 4°C.

[0052] (2) Blocking: Wash 5 times with 0.1% PBST and block with 2% BSA at room temperature for 2 hours.

[0053] (3) Incubation with primary antibody: Wash twice with 0.1% PBST, take 50µl of the phage solution amplified after each round of screening and mix with 50µl of 2% BSA, react at room temperature for 15 minutes, add to wells and incubate at room temperature for 2 hours.

[0054] (4) Incubation of secondary antibody: Wash 5 times with 0.1% PBST, add 100µl of HRP-M13 antibody diluted 5000 times with 2% BSA to each well, incubate at room temperature for 1 hour, and wash 4 times again with 0.1% PBST.

[0055] (5) Colorimetric reaction: Add 100µl / well of TMB chromogenic substrate and incubate at room temperature in the dark for 15 min.

[0056] (6) Termination of reaction: Add 50µl / well of 1M H2SO4 to terminate the colorimetric reaction, and measure the absorbance of each well at OD450nm using an ELISA reader.

[0057] Results Explanation:

[0058] Biopharmaceutical targeting of hVEGF 165 In the single-domain antibody screening process, polyclonal ELISA was used to detect the binding ability of the products to the antigen in each round of screening. Results showed that the antigen targeting hVEGF was selected. 165 The absorbance values ​​of single-domain antibodies showed a trend of increasing with each round, with the strongest binding ability observed in the third round (see...). Figure 1 In the fourth round, the absorbance value decreased, possibly because the amount of positive phages had reached saturation. Therefore, screening was stopped after the fourth round. The polyclonal phages that passed the four rounds of screening were then tested for their effectiveness against hVEGF. 165 The specificity of the antigen was significantly improved, and single clones were selected from the products of the fourth round for subsequent analysis.

[0059] Example 3: Obtaining an effective single-domain antibody targeting VEGF (sdVE) based on PCR-based DNA sequencing analysis.

[0060] 1. Preparation of monoclonal bacteriophages:

[0061] The phage library selected in the fourth round of screening was used to infect JM101 *E. coli*, and plated at an appropriate density on 2×YT-AG plates (approximately 100-300 clones / plate), and incubated overnight at 37°C. Single colonies were picked using sterile toothpicks and transferred to 96-well cell culture plates containing 200 µl of 2×YT-AG medium. The plates were then incubated at 37°C with shaking at 250 rpm until the medium became turbid, indicating successful phage amplification.

[0062] 2. Colony PCR amplification of bacteriophage-specific DNA fragments:

[0063] Using 2×PCR Master Mix reagent, and specific primers Forward Primer L1 and Reverse Primer S6, PCR amplification was performed on monoclonal phage-specific DNA fragments, and the PCR reaction system was prepared as shown in Table 2.

[0064] Table 2 PCR reaction solution preparation

[0065]

[0066] Forward Primer L1: 5'-TGGAATTGTGAGCGGATAACAATT-3'

[0067] Reverse Primer S6: 5'-GTAAATGAATTTTCTGTATGAGG-3'

[0068] PCR program settings: 94℃ for 1 min; 94℃ for 30 s, 60℃ for 30 s, 72℃ for 2 min, 30 cycles; 72℃ for 5 min; store at 4℃.

[0069] 3. Agarose gel electrophoresis and DNA sequencing analysis:

[0070] The PCR products were subjected to 1.5% agarose gel electrophoresis, and the results were observed and photographed. PCR products with band molecular weights matching the expected values ​​(approximately 700 bp) were selected for DNA sequencing.

[0071] Results Explanation:

[0072] After DNA sequencing and sequence analysis, 36 single-domain antibody (sdVE) sequences targeting VEGF with complete open reading frames (ORFs) were obtained and named sdVE01 to sdVE36, respectively. The amino acid sequences of single-domain antibodies sdVE01 to sdVE36 are shown in Table 3 (the underlined parts are the amino acid sequences of the complementarity-determining region, and the rest are the amino acid sequences of the backbone region).

[0073] Table 3

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] The amino acid sequences of sdVE01 to sdVE36 were compared using CLC sequence viewer 6.0 software to obtain information on the similarities and differences between these sequences, such as... Figure 2 As shown.

[0080] Example 4: sdVE monoclonal phage ELISA analysis

[0081] 1. Preparation of sdVE monoclonal phage: See the relevant section of Example 3 for the specific method.

[0082] 2. Monoclonal phage ELISA analysis:

[0083] (1) Coating and sealing: See the relevant part of Example 2 for specific methods.

[0084] (2) Antigen-antibody binding: Wash twice with 0.1% PBST, add 75µl / well of 2% BSA blocking buffer and 25µl / well of monoclonal phage supernatant, and incubate at room temperature for 2h.

[0085] (3) Incubation with primary antibody: Wash 5 times with 0.1% PBST, add 100µl / well of Anti-fdbacteriophage antibody produced in rabbit diluted 5000 times with 2% BSA, incubate at room temperature for 1h, and wash 4 times with 0.1% PBST.

[0086] (4) Incubation of secondary antibody: Wash 5 times with 0.1% PBST, add 100µl / well of VHH Anti-Rabbit-HRP antibody diluted 5000 times with 2% BSA, incubate at room temperature for 1h, and wash 4 times with 0.1% PBST.

[0087] (5) Color development: Add 100µl / well of TMB color development substrate and incubate at room temperature in the dark for 15 min.

[0088] (6) Termination: Add 50µl / well of 1M H2SO4 to terminate the reaction, measure the OD450nm value, plot the graph and perform statistical analysis.

[0089] Results Explanation:

[0090] hVEGF 165 ELISA analysis of sdVE monoclonal phage was performed using PBS as the negative control and PBS as the antigen. Figure 3 As shown, compared with the blank PBS control, except for sdVE23, the other 35 sdVE monoclonal phages showed resistance to hVEGF. 165 They all have a certain degree of binding specificity.

[0091] Example 5: Expression and Identification of VEGF-Targeting Single-Domain Antibody (sdVE) in Mammalian Cells

[0092] 1. Construction and preparation of sdVE eukaryotic expression plasmid

[0093] Based on the codon bias of eukaryotic mammalian cells, the sdVE05 gene sequence was optimized, and an NheI restriction endonuclease sequence, a Kozak sequence, and a human albumin signal peptide sequence were added to its 5' segment. At its 3' end, a three-amino acid AAA sequence, a 6×His tag sequence, and an XhoI restriction endonuclease sequence were added, resulting in the sdVE05 fusion gene with the structure: 5'-NheI restriction endonuclease sequence-Kozak sequence-human albumin signal peptide sequence-optimized sdVE05 gene sequence-three-amino acid AAA sequence- A 6×His tag sequence-XhoI restriction endonuclease sequence was obtained. The sdVE05 fusion gene was synthesized and cloned into the NheI / XhoI restriction endonuclease site of the pCDNA3.1(+) vector to construct a recombinant expression vector. This vector was transformed into competent E. coli (TOP10) to obtain genetically engineered bacteria capable of expressing sdVE05. An endotoxin-free plasmid large-scale extraction kit was used to prepare the sdVE05 eukaryotic expression plasmid. The concentration and purity were determined by Nanodrop assay and identified by 1% agarose gel electrophoresis. The heavy chain variable region genes of Avastin (GenBank: LQ506318.1) and Lucentis (GenBank: HC869890.1) were used as positive controls.

[0094] NheI restriction endonuclease sequence: 5'-G↑CTAGC-3',

[0095] Kozak sequence: 5'-GCCGCCACC-3',

[0096] Human albumin signal peptide sequence:

[0097] 5'-ATGAAGTGGGTGACTTTTATCAGTCTACTATTTCTGTTTCTCCAGCGCCTACTCC-3',

[0098] The AAA sequence consists of three amino acids: 5'-GCCGCTGCC-3'.

[0099] 6×His tag sequence: 5'-CACCATCACCATCACCAT-3',

[0100] XhoI restriction endonuclease sequence: 5'-C↑TCGAG-3',

[0101] The nucleotide sequences of the sdVE05 fusion gene, Avastin fusion gene, and Lucentis fusion gene are shown in Table 4.

[0102] Table 4

[0103]

[0104]

[0105] 2. Expression and identification of sdVE recombinant protein in mammalian cells

[0106] Using PEI4000 as the transfection reagent, the eukaryotic expression plasmid of the single-domain antibody sdVE05 was transfected into HEK293T cells. Forty-eight hours after transfection, cells were centrifuged at 1000g for 5 min, and the cell pellet and supernatant were collected separately. 1 ml of the supernatant was concentrated to 40 μl using an ultrafiltration tube, followed by the addition of 10 μl of 5× Loading Buffer, and denatured at 98℃ for 8 min as the extracellular sample. 1×10⁻⁶ cells were collected from the cell pellet. 6(Number of cells) were washed twice with PBS, and then lysed with 40 μl of RIPA cell lysis buffer on ice for 30 min. The cells were then centrifuged at 12000g for 10 min to remove the precipitate. The supernatant was collected, and 10 μl of 5×Loading Buffer was added. The supernatant was then denatured at 98℃ for 8 min to obtain the intracellular sample. Protein expression in the extracellular and intracellular samples was analyzed using SDS-PAGE electrophoresis and Western blot. In the Western blot experiment, recombinant HRP Anti-6×His tag antibody was used as the primary antibody. Protein expression was detected by ECL chemiluminescence immunoassay, and exposure, development, and image storage were performed according to the imaging system's operating instructions.

[0107] Results Explanation:

[0108] like Figure 4 As shown, no expression product of the sdVE05 recombinant protein was detected intracellularly or extracellularly 48 h after plasmid transfection.

[0109] Example 10: Prokaryotic expression preparation and activity analysis of LM-sdVE05

[0110] Preparation of SdVE05 genetically engineered bacteria: Based on the codon bias of *E. coli*, the sdVE05 gene sequence was optimized, and NdeI (CA↑TATG) and XhoI (C↑TCGAG) restriction endonuclease sites were introduced at its 5' and 3' ends, respectively, to obtain the LM-sdVE05 gene. The LM-sdVE05 gene was synthesized using artificial gene synthesis technology and cloned into the NdeI / XhoI restriction endonuclease site space of the pET-28a expression vector to construct a recombinant expression vector, which was then transformed into *E. coli* BL21(DE3) competent cells. Positive clones were screened from the transformed cells using colony PCR and DNA sequencing analysis. The LM-sdVE05 gene sequence is shown in Table 5.

[0111] Table 5

[0112]

[0113] Recombinant protein was efficiently expressed by SdVE05 genetically engineered bacteria using 1 mM IPTG, with most of it existing in the form of inclusion bodies. Inclusion bodies were washed with a combination of DOC washing and gradient washing with urea (2 M and 3 M urea). The inclusion bodies were dissolved in a binding buffer containing 8 M urea (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M Urea, and 5 mM imidazole adjusted to pH 8.0), and further purified by nickel ion chelate affinity chromatography. The protein was diluted and renatured overnight at 4°C using a 1:10 volume ratio renaturing buffer. After high-speed centrifugation and renaturation, the supernatant was collected and the precipitate discarded. The supernatant was then concentrated by ultrafiltration and desalted using an HPrep™ 26 / 10 column. The LM-sdVE05 recombinant protein solution was freeze-dried to obtain lyophilized powder and stored at -80°C for later use. The expression and purification process of the recombinant protein was detected by conventional SDS-PAGE gel electrophoresis.

[0114] EC of LM-sdVE05 50 Analysis (ELISA): hVEGF at a concentration of 2 μg / ml was used. 165 100 µl of antigen was used to coat the immunoassay plate, incubated overnight at 4°C, and washed twice with PBST. 300 µl of 2% BSA was added to each well, and the plate was blocked at room temperature for 2 hours, followed by two washes. The LM-sdVE05 recombinant protein was then diluted 1:10 with 2% BSA, and a blank control was added. 100 µl of different concentrations of LM-sdVE05 recombinant protein were incubated in 12 wells, with 3 replicates per concentration gradient, for 1 hour at room temperature, followed by 5 washes with 0.05% PBST. 100 µl of anti-rabbit antibody carrying the His-Tag was added to each well, and the plate was incubated at room temperature for 1 hour, followed by 5 washes. 100 µl of enzyme-labeled secondary antibody diluted 1:5000 with PBS was added to each well, and the plate was incubated at 37°C for 1 hour. 100 µl of TMB substrate was added to each well, and the reaction was allowed to proceed for 5 minutes. 50 µl of 1M chromogenic substrate was then added to each well. The reaction was terminated with H2SO4 solution, and the absorbance at OD450nm was measured; statistical analysis of the data was performed to calculate EC. 50 .

[0115] Effect of LM-sdVE05 recombinant protein on HUVEC cell viability: Logarithmic growth phase HUVECs were collected and prepared into cell suspensions, with 2*102 4Cells were seeded at a density of 6 replicates per well in 96-well plates. After overnight culture, the cells were transferred to medium containing 1% FBS and incubated for 24 hours. The supernatant was discarded, and serum-free medium containing different concentrations (0, 0.246, 1.23, and 6.15 μmol / L LM-sdVE05 recombinant protein) was added to each well. After 24 hours of culture, 10 µL of CCK8 was added to each well, and the cells were cultured for another 2 hours. The absorbance of each well was measured at 450 nm using an ELISA reader. Cell viability % was calculated as: (Experimental group OD value / Control group OD value) × 100%.

[0116] LM-sdVE05 inhibits hVEGF 165 Analysis of the migration ability of stimulated HUVEC cells: For the specific methods and steps of the HUVEC cell scratch assay and Transwell chamber assay, please refer to Example 9.

[0117] Analysis of LM-sdVE05 inhibition of HUVEC cell cast formation: BD Matrigel was removed from a -20℃ freezer and thawed overnight at 4℃; the 96-well plates and pipette tips used the next day were pre-cooled overnight at 4℃; the pre-cooled 96-well plates were placed on ice, kept horizontal, and 50µL of liquid Matrigel was evenly spread in each well, incubated at 37℃ for 30 min to allow the Matrigel to polymerize, taking care to maintain a horizontal position and avoid air bubbles, otherwise it would affect cell tube formation and photographic results; HUVECs in the logarithmic growth phase were prepared into single-cell suspensions containing complete culture medium and counted, at a ratio of 2*10-1. 4 Cells were seeded at a density of 1 / well in 96-well plates and cultured in an incubator. After the cells adhered (about 8 hours), the supernatant was aspirated and the medium was replaced with 1% FBS medium and cultured for 12 hours. When starvation was terminated, 100 µL of 1% FBS medium containing different concentrations of LM-sdVE05 recombinant protein was added to each well and the cells were cultured for another 12 hours. At 6-8 hours, the tubular structures formed by the junctions between endothelial cells were continuously observed under a microscope, counted, and photographed. At least 4 random fields of view were selected for photographing each well.

[0118] Results Explanation:

[0119] like Figure 5 As shown, the SdVE05 genetically engineered bacteria can highly express the target recombinant protein and obtain effective purification; ELISA analysis showed that the LM-sdVE05 recombinant protein and hVEGF... 165 EC 50 The value was 3.91 μM, which was higher than the EC50 of the positive control recombinant protein VHHL (ranibizumab variable region). 50 =1.363μM.

[0120] like Figure 6As shown, CCK8 assay results indicated that the LM-sdVE05 recombinant protein did not significantly alter the viability of HUVEC cells, meaning it was non-cytotoxic. Figure 6 A); Scratch assay results showed that the healing rates of HUVEC cells in the high, medium, and low dose groups of LM-sdVE05 (6.15μM, 1.23μM, and 0.246μM) were 40.71%, 50.52%, and 58.57%, respectively, which were significantly lower than the 74.42% healing rate in the blank group (Ctrl group) (P<0.05 or P<0.01). Figure 6 B, 6D); Transwell results showed that the number of migrated cells in the high, medium, and low dose groups of LM-sdVE05 recombinant protein were 47.67±8.74, 79.00±4.36, and 101.01±1.14, respectively, which were significantly reduced compared with the blank group (Ctrl group) of 103.7±4.73. There was no statistically significant difference in the healing area (46.76%±2.74) and the number of migrated cells (48.00±9.54) of the high-dose group of LM-sdVE05 recombinant protein compared with the positive control VHHL at the same dose. Analysis of variance showed statistically significant differences in healing rate and number of migrated cells among the high, medium, and low dose groups of LM-sdVE05 recombinant protein (P<0.05), indicating a dose-dependent relationship. Figure 6 (C, 6D). These results indicate that sdVE05 can inhibit the migration of HUVEC endothelial cells by suppressing VEGF secreted by HUVEC cells.

[0121] like Figure 7 As shown in A and 7C, hVEGF 165 The healing rate of HUVEC cells stimulated by the virus was 69.60%, significantly higher than that of the control group (Ctrl group) (53.93%), indicating that hVEGF significantly enhanced the healing ability. 165 It can stimulate the migration of HUVEC cells; after administration of LM-sdVE05 recombinant protein, the healing rates of the high, medium, and low dose groups were 45.96%, 52.96%, and 58.28%, respectively, compared with hVEGF. 165 The healing ability of the groups was significantly lower than that of the control group (P<0.05 or P<0.01), and the healing rate of the high-dose group was even lower than that of the control group (P<0.0001). Transwell results ( Figure 7 B, 7D) are displayed in hVEGF 165 Under stimulation, the number of migrating cells in the high, medium, and low dose groups of LM-sdVE05 recombinant protein were 48.00±7.55, 66.33±5.77, and 90.33±3.51, respectively, compared with hVEGF. 165The number of migrating cells in the stimulation group was significantly reduced (157.01±2.29), and the healing rate in the high- and medium-dose groups was also lower than that in the control group (P<0.0001). Furthermore, there was no statistically significant difference in healing rate (47.39%) and cell migration number (47.33±10.12) between the high-dose group and the equivalent dose of the positive control drug VHHL. Analysis of variance showed statistically significant differences in healing rate and number of migrating cells among the high, medium, and low-dose LM-sdVE05 recombinant protein groups (P<0.05), indicating a dose-dependent relationship. These results suggest that sdVE05 can also inhibit the migration of HUVEC endothelial cells by inhibiting VEGF secreted by HUVECs in a dose-dependent manner.

[0122] like Figure 8 As shown, SdVE05 inhibits HUVEC cell cast formation. In the absence of hVEGF... 165 Under stimulation, the number of tubes formed in the high, medium, and low dose groups of LM-sdVE05 recombinant protein were 6.750±1.26, 10.33±2.08, and 16.00±3.16, respectively, which were significantly lower than those in the blank group (29.33±2.52) (P<0.0001). Figure 8 A, 8B). In hVEGF 165 The number of angiogenesis in HUVECs stimulated by VEGF was 21.00±2.16, which was significantly higher than that in the control group (17.00±1.00) (P<0.05), indicating that VEGF stimulates angiogenesis in HUVEC cells. Figure 8 C, 8D). In hVEGF 165 Under stimulation, the number of tubes formed in the high, medium, and low dose groups of LM-sdVE05 recombinant protein were 7.000±1.00, 11.00±2.00, and 19.33±2.08 per field of view, respectively, compared with hVEGF. 165 The number of tubes formed was significantly lower in the high-dose group than in the control group (P<0.0001), and the number of tubes formed in the high-dose group was even lower than that in the control group (P<0.0001). Figure 8 C, 8D). Additionally, the high-dose LM-sdVE05 recombinant protein group and the equivalent-dose VHHL positive control group were in the absence of hVEGF. 165 The number of tubes formed under stimulation (9.25±2.22) was related to hVEGF. 165 There was no statistically significant difference in the number of tubes formed under stimulation (7.667±0.58). Analysis of variance showed statistically significant differences in the number of tubes formed at high, medium, and low doses of LM-sdVE05 recombinant protein (P<0.01 or P<0.0001), indicating a dose-dependent relationship. Therefore, it can be concluded that sdVE05 can also inhibit hVEGF secreted by HUVECs in a dose-dependent manner. 165 Stimulation-induced angiogenesis and endocrine hVEGF 165Stimulating angiogenesis, thereby inhibiting angiogenesis in HUVEC endothelial cells.

[0123] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A humanized VEGF-targeting single-domain antibody sdVE05, characterized in that: The amino acid sequence of the single-domain antibody sdVE05 includes three complementarity-determining regions (CDR1, CDR2, and CDR3), and the amino acid sequences of CDR1, CDR2, and CDR3 are as follows: CDR1: YRINYENMA CDR2: TILRPN CDR3: TRRHRSNLGWRSSAPVQF.

2. The single-domain antibody sdVE05 according to claim 1, characterized in that: The single-domain antibody also includes four backbone regions FR1, FR2, FR3, and FR4, and the amino acid sequences of the backbone regions FR1, FR2, FR3, and FR4 are as follows: FR1: EVQLVESGGGLVQPGGSLRLSCAASG, FR2: WVRQAPGKGLEWVS FR3:GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA, FR4: WGQGTLVTVSS.

3. A recombinant protein, characterized in that: The recombinant protein comprises the single-domain antibody sdVE05 as described in any one of claims 1 to 2.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the single-domain antibody sdVE05 or recombinant protein as described in any one of claims 1 to 3.

5. A carrier, characterized in that: The carrier contains the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that: The host cell contains the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A method for preparing the single-domain antibody sdVE05 or recombinant protein according to any one of claims 1 to 3, characterized in that: The method includes the steps of culturing the host cells of claim 6 and isolating and purifying the single-domain antibody sdVE05 or recombinant protein from the culture.

8. The use of the single-domain antibody sdVE05 according to any one of claims 1-2 and the recombinant protein according to claim 3 in the preparation of a drug targeting VEGF to treat a disease.

9. The use of the single-domain antibody sdVE05 according to any one of claims 1-2 and the recombinant protein according to claim 3 in the preparation of VEGF inhibitors.

10. The use of the single-domain antibody sdVE05 according to any one of claims 1-2 and the recombinant protein according to claim 3 in the preparation of angiogenesis inhibitors.

Images