Humanized single-domain antibody targeting vegf and use thereof

Abstract

This invention belongs to the field of biomedicine, and relates to single-domain antibodies, more specifically to a humanized single-domain antibody targeting VEGF and its applications. The amino acid sequence of the single-domain antibody includes three complementarity-determining regions (CDR1-CDR3) and four backbone regions (FR1-FR4). This invention also provides a recombinant protein containing the single-domain antibody, a nucleic acid molecule encoding the single-domain antibody or recombinant protein, a vector containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or vector, and the application of the single-domain antibody or recombinant protein in the preparation of drugs targeting VEGF to treat diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a humanized single-domain antibody targeting VEGF 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 plays a dominant role 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, triggering a series of pathological changes, such as vascular leakage, hemorrhage, and edema, severely impairing the patient's vision. In cardiovascular diseases, excessive VEGF expression may also lead to the occurrence and development of diseases such as atherosclerosis and vasculitis. Furthermore, in autoimmune diseases such as rheumatoid arthritis, abnormal VEGF expression can promote the infiltration of inflammatory cells and angiogenesis, exacerbating joint inflammation and destruction. Therefore, VEGF has become a key cytokine for clinical detection in 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 faricin (a bispecific antibody targeting VEGF and Ang2, Faricimab-svoa, Vabysmo); 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, becoming 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 single-domain antibody targeting VEGF and its application.

[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, wherein the amino acid sequence of the single-domain antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are any one of the following 1) to 36):

[0008] 1)CDR1: FSINDEAMS, CDR2: GIRSPS, CDR3: AARHPDSIHQEVAY;

[0009] 2) CDR1: VKVSHQYMA, CDR2: GITTQD, CDR3: TSPVRWLWGAQHLAF;

[0010] 3) CDR1: VTVSNQYMG, CDR2: SILNRN, CDR3: GVSRMGCEATIPY;

[0011] 4) CDR1: YSLSAQDMS, CDR2: GISDTD, CDR3: GTGLSAEGGAVDYSRQGQV;

[0012] 5) CDR1: YRINYENMA, CDR2: TILRPN, CDR3: TRRHRSNLGWRSSAPVQF;

[0013] 6) CDR1: VRFNHEFMS, CDR2: GINSRS, CDR3: SARRAPFRTKYIKF;

[0014] 7) CDR1: FTVNTKDMA, CDR2: TIEMAN, CDR3: RCVGIVCLNENIPF;

[0015] 8) CDR1: VRVSDEVMS, CDR2: SITRGN, TGYWGRLPSRWENKQVGF;

[0016] 9) CDR1: DSISPENMG, CDR2: TIYSPN, CDR3: TYHNREASSALKS;

[0017] 10) CDR1: FTFIYEAMA, CDR2: GINKGS, CDR3: GSLGGETSTVGF;

[0018] 11) CDR1: DNISPEYMG, CDR2: GIHRSD, CDR3: RSGPPMESKLWS;

[0019] 12)CDR1:YNVSNENMG,CDR2:GISTDN,CDR3:GIIELNTTLWF;

[0020] 13)CDR1:VMINNEYMA,CDR2:TIKIQS,CDR3:RPDWAYNQSIGY;

[0021] 14)CDR1:YSVINQDMS,CDR2:GIATGN,CDR3:GVSEDRSRLKEYGPHDMKS;

[0022] 15)CDR1:FTVSSEDMA,CDR2:SIQAAN,CDR3:ANGVERLGMHGPSPFRY;

[0023] 16)CDR1: DNISTEDMT, CDR2: AIENPD, CDR3: GLSDNQVTRDYNQLTF;

[0024] 17)CDR1:VTLSYENMS,CDR2:SITTPD,CDR3:AEPVELVSDVRTKQAHMSY;

[0025] 18)CDR1:VKVNSYNMG,CDR2:SILDAD,CDR3:SGTHRSTEELPF;

[0026] 19)CDR1:DSISDEVMT,CDR2:SIKGQN,CDR3:ASELCFEGKILSSEVKY;

[0027] 20)CDR1:DMFNYNNMS,CDR2:TIHTND,CDR3:SVMDSVTWADAAPEALHF;

[0028] 21)CDR1:DRFTPEVMT,CDR2:SISNGG,CDR3:GNDLGSRELSATQQLKY;

[0029] 22)CDR1:YNISDETMG,CDR2:GIKGRD,CDR3:GNLIFAPEAALGY;

[0030] 23)CDR1:DRVGAENMA,CDR2:GISVDN,CDR3:GYESWRCLAMNWEPPDVCF;

[0031] 24)CDR1:DRVSNNTMS,CDR2:GIENRG,CDR3:SRSVRDEKDVIRPNTKVEF;

[0032] 25)CDR1:DSISSEYMA,CDR2:TIKDTD,CDR3:GCCDIDFSDAQTIGS;

[0033] 26)CDR1:DTVSDDSMG,CDR2:GIRANG,CDR3:APGREKHNAVHY;

[0034] 27)CDR1:YRINDEVMS,CDR2:TINNAN,CDR3:GINSTISEEVGS;

[0035] 28)CDR1:YTFSTKNMG,CDR2:TIDNDN,CDR3:RATATEQLKKEVKY;

[0036] 29)CDR1:DMVTDETMA,CDR2:GIYNRG,CDR3:GPGNVLIQSTYELSS;

[0037] 30)CDR1:DKVIDENMG,CDR2:GIRESD,CDR3:RELRLPSYLCY;

[0038] 31)CDR1:VKIIAEDMG,CDR2:TISDTS,CDR3:SPKRQASFPQELNS;

[0039] 32)CDR1:DKITAEAMG,CDR2:SIGTPS,CDR3:GLDMDFGGKAANYDKEAEVPF;

[0040] 33)CDR1:FTFSAEDMS,CDR2:AIIVNS,CDR3:TAYKRDGIPEDAPLGF;

[0041] 34)CDR1:VTVSDEDMG,CDR2:SIEGTG,CDR3:RPIVNSISTSENMRF;

[0042] 35)CDR1:VKFSSENMS,CDR2:TITVAN,CDR3:ADEEWCTTPMRS;

[0043] 36)CDR1:FSINNENMG,CDR2:GITAKN,CDR3:SLESAQTRKKQDMEF。

[0044] Preferably, the above-mentioned single-domain antibodies can specifically bind to VEGF and have high affinity, especially single-domain antibodies having the complementary determinant regions shown in items 1), 5), 10), 12), 13), 25), 34) and 36) above.

[0045] Furthermore, the aforementioned single-domain antibody also includes four backbone regions FR1, FR2, FR3, and FR4, the amino acid sequences of which are as follows:

[0046] FR1: EVQLVESGGGLVQPGGSLRLSCAASG,

[0047] FR2: WVRQAPGKGLEWVS

[0048] FR3:GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA,

[0049] FR4: WGQGTLVTVSS.

[0050] Furthermore, the structure of the above-mentioned single-domain antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0051] Furthermore, the aforementioned single-domain antibodies can be fused with other peptides, proteins, or functional substances to achieve diverse application goals. For example, fusion with protein tags (such as 6xHis, albumin signal peptide, GFP, RFP, etc.), enzymes (such as horseradish peroxidase, alkaline phosphatase, etc.), or radioisotope markers can significantly improve the convenience and sensitivity of VEGF target detection or facilitate protein purification. Another example is the use of tandem fusion technology to link multiple identical single-domain antibody fragments into bivalent or multivalent antibodies, which can not only effectively enhance the binding affinity of the antibody to VEGF but also potentially prolong its half-life in vivo, thereby enhancing therapeutic efficacy and reducing dosing frequency. Yet another example is the linking of single-domain antibodies with bioactive small molecule drugs via specific linkers (such as cleavable linkers, non-cleavable linkers, etc.) to form antibody-drug conjugates (ADCs) for more efficient drug efficacy.

[0052] A second aspect of the present invention provides a recombinant protein comprising the single-domain antibody described above.

[0053] A third aspect of the present invention provides a nucleic acid molecule that encodes the aforementioned single-domain antibody or recombinant protein.

[0054] A fourth aspect of the present invention provides a carrier containing the above-described nucleic acid molecules.

[0055] A fifth aspect of the present invention provides a host cell containing the aforementioned nucleic acid molecules or carriers.

[0056] A sixth aspect of the present invention provides a method for preparing the above-described single-domain antibody or recombinant protein, the method comprising the steps of culturing the above-described host cells and isolating and purifying the single-domain antibody or recombinant protein from the culture.

[0057] The seventh aspect of the present invention provides the use of the above-described single-domain antibody and recombinant protein in the preparation of a medicament targeting VEGF to treat diseases, including neovascular ophthalmopathy, tumors, cardiovascular diseases, and rheumatoid arthritis.

[0058] An eighth aspect of the present invention provides a kit for detecting VEGF, the kit comprising the above-described single-domain antibody or recombinant protein.

[0059] The significant advantages of this invention are:

[0060] This invention provides single-domain antibodies targeting VEGF and recombinant proteins containing said single-domain antibodies. These antibodies or proteins have unique CDR sequences that enable them to specifically bind 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

[0061] 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.

[0062] 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.

[0063] Figure 3 ELISA analysis of sdVE monoclonal phage. **P<0.01, ***P<0.001, ****P<0.0001.

[0064] Figure 4 : Identification of sdVE expression in 293T cells (Western Blotting analysis). Lane 1, intracellular; Lane 2, extracellular.

[0065] Figure 5Expression and purification of sdVE in 293T cells (SDS-PAGE with Coomassie Brilliant Blue staining). Lane M: Protein molecular weight standard; Lane 1: Intracellular; Lane 2: Extracellular; Lane 3: Purified protein.

[0066] Figure 6 ELISA analysis of the affinity of recombinant sdVE protein (expressed in 293T cells) (A) and its normalized comparison with AVST as a control (B). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control.

[0067] Figure 7 LM-sdVE (293T cell expression system) inhibits hVEGF 165 Stimulation of HUVEC cell proliferation (CCK-8 assay).

[0068] Figure 8 Prokaryotic expression, purification, detection, and identification of LM-sdVE recombinant protein (SDS-PAGE and Coomassie Brilliant Blue staining). A, SDS-PAGE and Coomassie Brilliant Blue staining; B, Imagej software analysis of the purity of LM-sdVE recombinant protein in A. Lane M, protein molecular weight standard; Lane 1, uninduced; Lane 2, after IPTG induction for 5 hours; Lane 3, supernatant after bacterial lysis and centrifugation; Lane 4, precipitate after bacterial lysis and centrifugation; Lane 5, supernatant after washing inclusion bodies with 3M urea; Lane 6, precipitate after washing inclusion bodies with 3M urea; Lane 7, inclusion body supernatant dissolved in TGE buffer; Lane 8, inclusion body precipitate dissolved in TGE buffer; Lane 9, purified LM-sdVE recombinant protein.

[0069] Figure 9 Representative diagram of HiPrep 26 / 10 Desalting for LM-sdVE.

[0070] Figure 10 ELISA analysis of the affinity of LM-sdVE recombinant protein (expressed in E. coli). ****P<0.0001 vs PBS.

[0071] Figure 11 sdVE01 inhibits hVEGF 165Stimulation of HUVEC vascular endothelial cells at the in vitro horizontal (A, C) and vertical (B, D) migration levels. Observation under a 10× inverted microscope. ####P<0.0001, ##P<0.01 vs control. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, NS: no statistical significance vs model.

[0072] Figure 12 Prokaryotic 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.

[0073] Figure 13 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.

[0074] Figure 14 :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.

[0075] Figure 15 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 165Formation 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.

[0076] Figure 16 Evaluation of the in vivo therapeutic effect of sdVE01 on a corneal alkali burn model in SD rats (slit-lamp observation). A, Schematic diagram of the treatment cycle; B, Slit-lamp examination on days 1, 4, 7, and 14 after corneal alkali burn (scale bar 1 mm); C, Changes in corneal neovascularization area in each group on days 4, 7, and 14 after corneal burn (AB group vs. treatment group **P<0.01, ***P<0.001); D, Changes in corneal opacity grade in each group on days 4, 7, and 14 after corneal burn (AB group vs. treatment group **P<0.01).

[0077] Figure 17 Evaluation of the in vivo therapeutic effect of sdVE01 on corneal alkali burn model in SD rats (HE staining and fluorescence staining of ocular tissue). A, H&E staining of corneal sections on days 4, 7, and 14 after different drug treatments (scale bar 100 μm); B, Immunofluorescence staining of corneal sections on days 4, 7, and 14 after different drug treatments (blue: DPAI, red: anti-VEGF antibody, scale bar: 100 μm); C, Changes in mean corneal thickness in each group on days 4, 7, and 14 after injury (AB group vs. treatment group *P<0.5, **P<0.01); D, Changes in mean expression of corneal endothelial growth factor (VEGF) in each group on days 4, 7, and 14 after injury (AB group vs. treatment group *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001).

[0078] Figure 18 Safety of sdVE01 was observed and analyzed using a slit lamp.

[0079] Figure 19 H&E staining analysis of tissues to assess the safety of sdVE01. A, H&E staining of the eyeball; B, H&E staining of major organs. Detailed Implementation

[0080] 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.

[0081] Example 1: Biopaneling of VEGF-targeting single-domain antibodies

[0082] 1. First round of screening

[0083] (1) Coating antigen: The target antigen human vascular endothelial growth factor 165 (hVEGF) is coated onto the target antigen. 165 Dilute with coating buffer to 10 μg / ml, coat microplates with 100 μl / well, and incubate overnight at 4°C.

[0084] (2) Blocking: Wash 5 times with 0.1% PBST, then wash once with PBS, and block with 3% PBSM at 37°C for 2 hours to block non-specific binding sites.

[0085] (3) Antibody incubation: After blocking, wash 6 times with 0.1% PBST, wash once with PBS, and add 100 μl / well titer of humanized single-domain antibody phage library (10 13 (pfu / mL), incubate at 37℃ for 2 hours.

[0086] (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 a new round of phage library.

[0087] 2. Second, third and fourth rounds of screening

[0088] 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.

[0089] Results explanation:

[0090] As the number of screening rounds increased, the P / N ratio also increased accordingly, indicating the effectiveness of the enrichment screening and the increased specificity of the phages (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.)

[0091] Table 1. Enrichment effect of biopharmaceutical targeting hVEGF165 single-domain antibody phage

[0092]

[0093] Example 2: Polyclonal ELISA detection of phage library specific enrichment after 4 rounds of screening

[0094] (1) Coating: The target antigen hVEGF is coated. 165 Dilute to 5 μg / ml with coating buffer, coat microplates at 100 μl / well, and incubate overnight at 4°C.

[0095] (2) Blocking: Wash 5 times with 0.1% PBST and block with 2% BSA at room temperature for 2 hours.

[0096] (3) Incubation with primary antibody: Wash twice with 0.1% PBST, take 50 μl of the phage solution amplified after each round of screening and mix with 50 μl of 2% BSA, react at room temperature for 15 minutes, add to wells, and incubate at room temperature for 2 hours.

[0097] (4) Incubation of secondary antibody: Wash 5 times with 0.1% PBST, add 100 μl 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.

[0098] (5) Colorimetric reaction: Add 100 μl / well of TMB chromogenic substrate and incubate at room temperature in the dark for 15 min.

[0099] (6) Termination of reaction: Add 50 μl of 1M H2SO4 per well to terminate the colorimetric reaction, and measure the absorbance of each well at OD450 nm using an ELISA reader.

[0100] Results explanation:

[0101] 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. 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 effective 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.

[0102] Example 3: Obtaining an effective VEGF-targeting single-domain antibody (sdVE) based on PCR-based DNA sequencing analysis.

[0103] 1. Preparation of monoclonal bacteriophages:

[0104] 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.

[0105] 2. Colony PCR amplification of bacteriophage-specific DNA fragments:

[0106] 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.

[0107] Table 2 PCR reaction solution preparation

[0108]

[0109] Forward Primer L1: 5'-TGGAATTGTGAGCGGATAACAATT-3'

[0110] Reverse Primer S6: 5'-GTAAATGAATTTTCTGTATGAGG-3'

[0111] 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℃.

[0112] 3. Agarose gel electrophoresis and DNA sequencing analysis:

[0113] 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.

[0114] Results explanation:

[0115] 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).

[0116] Table 3

[0117]

[0118]

[0119]

[0120] 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.

[0121] Example 4: sdVE monoclonal phage ELISA analysis

[0122] 1. Preparation of sdVE monoclonal phage: See the relevant section of Example 3 for the specific method.

[0123] 2. Monoclonal phage ELISA analysis:

[0124] (1) Coating and sealing: See the relevant section of Example 2 for specific methods.

[0125] (2) Antigen-antibody binding: Wash twice with 0.1% PBST, add 75 μl / well of 2% BSA blocking buffer and 25 μl / well of monoclonal phage supernatant, and incubate at room temperature for 2 h.

[0126] (3) Incubation with primary antibody: Wash 5 times with 0.1% PBST, add 100 μl / well of Anti-fdbacteriophage antibody produced in rabbit diluted 5000 times with 2% BSA, incubate at room temperature for 1 h, and wash 4 times with 0.1% PBST.

[0127] (4) Incubation of secondary antibody: Wash 5 times with 0.1% PBST, add 100 μl / well of VHH Anti-Rabbit-HRP antibody diluted 5000 times with 2% BSA, incubate at room temperature for 1 h, and wash 4 times with 0.1% PBST.

[0128] (5) Color development: Add 100 μl / well of TMB color development substrate and incubate at room temperature in the dark for 15 min.

[0129] (6) Termination: Add 50 μl / well of 1M H2SO4 to terminate the reaction, measure the OD450nm value, plot the graph and perform statistical analysis.

[0130] Results explanation:

[0131] 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.

[0132] Example 5: Expression and Identification of VEGF-Targeting Single-Domain Antibody (sdVE) in Mammalian Cells

[0133] 1. Construction and preparation of sdVE eukaryotic expression plasmid

[0134] Based on the codon bias of eukaryotic mammalian cells, the gene sequences of sdVE01 to sdVE36 were optimized. An NheI restriction endonuclease sequence, a Kozak sequence, and a human albumin signal peptide sequence were added to the 5' segment. A three-amino acid AAA sequence, a 6×His tag sequence, and an XhoI restriction endonuclease sequence were added to the 3' end to obtain the sdVE fusion gene. Its structure is: 5'-NheI restriction endonuclease sequence-Kozak sequence-human albumin signal peptide sequence-optimized sdVE gene sequence-three amino acid AAA sequences. The AA sequence-6×His tag sequence-XhoI restriction endonuclease sequence were used to synthesize the sdVE fusion gene. The gene was cloned into the NheI / XhoI restriction endonuclease site of the pCDNA3.1(+) vector to construct a recombinant expression vector. This vector was then transformed into competent *E. coli* (TOP10) to obtain a genetically engineered bacterium capable of expressing sdVE. An endotoxin-free plasmid large-scale extraction kit was used to prepare the sdVE eukaryotic expression plasmid. Concentration and purity were determined using Nanodrop assays, and identification was performed using 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.

[0135] NheI restriction endonuclease sequence: 5'-G↑CTAGC-3',

[0136] Kozak sequence: 5'-GCCGCCACC-3',

[0137] Human albumin signal peptide sequence:

[0138] 5'-ATGAAGTGGGTGACTTTTATCAGTCTACTATTTCTGTTTCTCCAGCGCCTACTCC-3',

[0139] The AAA sequence consists of three amino acids: 5'-GCCGCTGCC-3'.

[0140] 6×His tag sequence: 5'-CACCATCACCATCACCAT-3',

[0141] XhoI restriction endonuclease sequence: 5'-C↑TCGAG-3',

[0142] The nucleotide sequences of the sdVE01 fusion gene to the sdVE36 fusion gene, the Avastin fusion gene, and the Lucentis fusion gene are shown in Table 4.

[0143] Table 4

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] 2. Expression and identification of sdVE recombinant protein in mammalian cells

[0152] Using PEI4000 as the transfection reagent, the sdVE eukaryotic expression plasmids of the 36 single-domain antibodies sdVE01–sdVE36 were 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 Cells were washed twice with PBS, then 40 μl of RIPA cell lysis buffer was added, and the cells were lysed on ice for 30 min. After centrifugation at 12000g for 10 min, the precipitate was removed, and the supernatant was collected. 10 μl of 5×Loading Buffer was added, and the supernatant was 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×Histag antibody was used as the primary antibody, and protein expression was detected by ECL chemiluminescence. Exposure, development, and image storage were performed according to the imaging system's operating instructions.

[0153] Results explanation:

[0154] like Figure 4 As shown, 48 hours after plasmid transfection, most of the recombinant proteins were detected in the culture medium as secreted proteins. Among them, no expression product was detected in sdVE05 recombinant protein, either intracellularly or extracellularly. No extracellular secreted products were detected in sdVE10, sdVE29, sdLuc and sdAVST recombinant proteins, which remained as intracellular proteins.

[0155] Example 6: Purification and ELISA Analysis of sdVE Recombinant Protein (Mammalian Cell Expression System) 1. Purification of sdVE Recombinant Protein Since the human albumin signal peptide sequence and 6×His tag sequence were introduced during the design of the sdVE eukaryotic expression plasmid in Example 5, the subsequent preliminary purification of the sdVE recombinant protein involved the following steps: For secretory recombinant proteins, initial enrichment was performed using 75% ammonium sulfate precipitation, followed by purification using nickel ion chelating magnetic beads; for intracellular recombinant proteins, the protein was first released via cell lysis, followed by purification using nickel ion chelating magnetic beads. The preliminarily purified sdVE recombinant protein was desalted using a HiPrep 26 / 10 desalting column to remove salts and other impurities from the solution, and then freeze-dried to obtain lyophilized powder, which was stored at -80°C for later use.

[0156] 2. ELISA analysis of sdVE recombinant protein

[0157] (1) Coating and sealing: See the relevant section of Example 2 for specific methods.

[0158] (2) Antigen-antibody binding: Discard the liquid, add 200 μl / well of 0.1% PBST and let stand for 30 seconds. Discard the liquid and repeat the washing process twice. Add 50 ng / 100 μl of sdVE recombinant protein to each well and react at 37°C for 2 hours.

[0159] (3) Incubate primary antibody: Discard the liquid, add 200 μl / well of 0.1% PBST and let stand for 30 s. Discard the liquid, repeat the washing 4 times, add 100 μl / well of mouse anti-His anti-body (1:1000 dilution) and react at 37℃ for 1 h.

[0160] (4) Incubation with secondary antibody: Discard the liquid, add 200 μl / well of 0.05% PBST and let stand for 30 seconds. Discard the liquid and repeat the washing process 4 times. Add 100 μl of anti-mouse IgG1-HRP (1:2000 dilution) to each well and react at 37°C for 1 hour.

[0161] (5) Color development: Discard the liquid, add 200 μl / well of 0.1% PBST and let stand for 30 s. Discard the liquid and repeat the washing 4 times. Add 100 μl / well of TMB color development solution and incubate at room temperature in the dark. When the blank wells start to turn blue, add 50 μl / well of stop solution to terminate the reaction and measure the absorbance at 450 nm.

[0162] Results explanation:

[0163] Thirty-six sdVE recombinant proteins and two positive recombinant proteins were initially purified. Intracellular and extracellular proteins, as well as purified protein samples, were subjected to SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining to observe the protein expression and purification processes. Figure 5 As shown, the content of impurity proteins was significantly reduced after purification. ELISA affinity analysis was performed on the preliminarily purified sdVE recombinant protein to select sdVE molecules with high affinity. Due to the large number of recombinant proteins, they were divided into different groups for batch expression, purification, and analysis. Protein products from the same batch were subjected to the same ELISA analysis. The recombinant protein with the highest absorbance value in that group was selected and added to the next group for further batch expression, purification, and ELISA analysis as a control, until all recombinant proteins were analyzed. Figure 6 A). Further, the control sdVAST was set at 100%, and normalization was performed accordingly. The 35 recombinant sdVE proteins (sdVE05 not expressed) were sorted according to affinity, as follows: Figure 6 As shown in B, the recombinant proteins with greater affinity than sdAVST include sdVE01, sdVE02, sdVE03, sdVE09, sdVE10, sdVE11, sdVE12, sdVE13, sdVE14, sdVE21, sdVE25, sdVE34, and sdVE36. Among them, sdVE01, sdVE10, sdVE25, and sdVE36 exhibited excellent affinity and were selected as lead molecules (LM-sdVE).

[0164] Example 7: LM-sdVE (293T cell expression system) inhibits hVEGF 165 Stimulation of HUVEC cell proliferation (detected by CCK-8 assay)

[0165] The sdVE recombinant protein was prepared and purified according to the methods in Examples 5 and 6.

[0166] 1. Take HUVEC cells in the logarithmic growth phase, prepare a single-cell suspension and count them. Seed them in 96-well plates at a density of 2000 cells / well. Add 200 μl of ECM medium (containing 10% FBS, 1% antibiotics, and 1% growth factors) to each well and continue culturing.

[0167] 2. Prepare each component according to the table below. After mixing the drugs, place them at 37℃ for 3 hours to fully combine.

[0168]

[0169] 3. After the cells in the 96-well plate adhered for 24 hours, replace the medium with 180 μl of fresh ECM medium (containing 1% FBS and 1% antibiotics), add 20 μl of the mixed drug to each well, and continue culturing at 37°C and 5% CO2.

[0170] 4. 72 h after drug administration, add 20 μl of CCK-8 solution to each well of the 96-well plate, incubate at 37 °C for another 4 h, and measure the absorbance at 450 nm.

[0171] Results explanation:

[0172] CCK-8 contains WST-8, which, under the action of the electron carrier dimethyl sulfate, is reduced by dehydrogenases in the cell mitochondria to a highly water-soluble yellow formazan product. The amount of formazan produced is directly proportional to the number of live cells. IC50 is detected by measuring absorbance values. 50 From smallest to largest, they are: sdLuc recombinant protein (15.01 ng / ml), sdAVST recombinant protein (9.896 ng / ml), sdVE01 recombinant protein (36.12 ng / ml), sdVE10 recombinant protein (55.59 ng / ml), sdVE36 recombinant protein (99.14 ng / ml), and sdVE25 recombinant protein (114.6 ng / ml). Figure 7 As shown, all four sdVE recombinant proteins have a certain inhibitory effect on the proliferation of HUVEC cells, among which the sdVE01 recombinant protein has the best inhibitory effect.

[0173] Example 8: Preparation of LM-sdVE recombinant protein (prokaryotic expression system) and its ELISA analysis

[0174] 1. Construction of the LM-sdVE genetically engineered bacterium: Based on the codon bias of *E. coli*, the gene sequences of sdVE01, sdVE10, sdVE25, and sdVE36 were optimized, and NdeI (5'-CA↑TATG-3') and XhoI (5'-C↑TCGAG-3') restriction endonuclease sites were introduced at their 5' and 3' ends, respectively, to obtain the LM-sdVE gene. The LM-sdVE 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.

[0175] The LM-sdVE gene sequence is shown in Table 5.

[0176] Table 5

[0177]

[0178]

[0179] 2. Induction of LM-sdVE recombinant protein expression: Resuscitate LM-sdVE genetically engineered bacteria and expand culture in LB-K (20 μg / mL kanamycin) medium (37℃ / 225 rpm) until the bacterial culture reaches OD. 600nm =0.6~0.8; after inducing culture with 1mM IPTG for 5 hours, collect the bacterial culture and centrifuge (5000g / 10min) to collect the precipitated bacterial cells. The wet bacteria can be stored in a -80℃ refrigerator for later use.

[0180] 3. Cell sonication disruption: Resuspend the collected wet bacterial pellet in TGE Buffer (20 ml TGE Buffer is needed for 1 g of wet bacterial solution), let it stand on ice for 10 min, and then sonicate (200 W power, 5 s on / 5 s off, 4 min / time, for a total of 2 sonication disruptions). After disruption, centrifuge the solution (10000 g / 10 min), discard the supernatant and collect the inclusion body pellet.

[0181] 4. Inclusion body washing, dissolution, and renaturation: The inclusion body precipitate (corresponding to 1g of the original wet bacteria) was resuspended in 4ml of 3M urea, placed on ice for 10min, and then centrifuged (10000g / 10min) to collect the precipitate. The washing was repeated once to obtain high-purity inclusion bodies. After dissolving the inclusion bodies in NaOH (10 or 20mM), 0.3 times the volume of TGE neutralization solution was immediately added, and the supernatant was collected by centrifugation (10000g / 10min). The supernatant was placed in a dialysis bag (molecular weight cutoff of 3kDa), and then placed in 1L of 100 times the volume of dialysis buffer (IBR) and dialyzed overnight at 4℃ for renaturation.

[0182] 5. Desalting of LM-sdVE recombinant protein: Centrifuge the above refolding solution (12000g / 10min), collect the supernatant, and add saturated ammonium sulfate solution to precipitate the target protein; dissolve the precipitate with an appropriate amount of PBS and filter it through a 0.22μm filter membrane; desalt using a HiPrep 26 / 10 Desalting column, collect the sample and freeze-dry it under vacuum to obtain lyophilized powder, and store it in a -80℃ freezer for later use.

[0183] 6. Identification of LM-sdVE recombinant protein: Take 10 μl of equal volume from all samples (including pre- / post-induction, cell lysis supernatant / precipitate, supernatant / precipitate from 3M urea washings of inclusion bodies twice, TGE neutralization supernatant / precipitate, and PBS-dissolved target protein sample) for SDS-PAGE gel electrophoresis. Then, place the PAGE gel in 100 ml of Coomassie Brilliant Blue staining solution, microwave for 20 seconds, and then stain on a shaker at 80–90 rpm for 15 minutes. Replace the staining solution with 100 ml of destaining solution, and shake on a shaker at room temperature at 80–90 rpm. Replace the destaining solution with fresh destaining solution every hour until the bands are clear. Analyze the SDS-PAGE results using Imagej software and calculate the protein purity.

[0184] 7. LM-sdVE recombinant protein ELISA analysis: Refer to Example 5 for specific methods and steps, except that the LM-sdVE recombinant protein in this example is used only in the antigen-antibody binding step.

[0185] Results explanation:

[0186] All four LM-sdVE genetically engineered bacteria strains constructed were able to be effectively induced to express the LM-sdVE recombinant protein. Figure 8 A) Through a series of purification steps including ultrasonic disruption, inclusion body washing, dialysis refolding, and desalting, the purity of LM-sdVE recombinant protein reached over 90%. Figure 8 B to Figure 9 ELISA analysis of LM-sdVE affinity activity yielded the following results: Figure 10 As shown, with PBS as the control group, LM-sdVE can effectively bind hVEGF. 165 Compared to other targets, LM-sdVE01 has a higher affinity activity.

[0187] Example 9: sdVE01 inhibits the migration ability of hVEGF165-stimulated HUVEC cells

[0188] The LM-sdVE01 recombinant protein was prepared according to the method in Example 8.

[0189] 1. Scratch test

[0190] (1) Use a marker pen to draw a marking line on the back of the 24-well plate at 1cm intervals;

[0191] (2) Take HUVEC cells in the logarithmic growth phase and use 1×10 5 Cells were seeded at a density of 3 ml of ECM medium containing 10% FBS in each well of a 24-well plate and cultured for another 3 ml.

[0192] (3) After about 24 hours, observe under a microscope that each well has formed a monolayer with about 90% fusion. Discard the culture medium and replace it with culture medium containing 1% FBS for 12 hours to eliminate the interference of serum-induced proliferation on migration.

[0193] (4) When the starvation time ends, use a 200μl sterile pipette tip that has been sterilized by high pressure to scratch the HUVEC single cell layer with a vertical marking line. The intersection of the scratch and the horizontal line on the Marker pen on the well plate is the fixed point for observation.

[0194] (5) Gently rinse the detached cell debris three times with PBS buffer, and add 2 ml of 1% FBS medium (containing 20 ng / ml hVEGF) to each rinse. 165 The protein was cultured with different concentrations of LM-sdVE01 recombinant protein, with 1500 nM Ranibizumab as the positive control group.

[0195] (6) After 24 hours, four fixed points were taken from each well, and the cell migration in each well was observed and images were acquired under a microscope. The remaining area of ​​the scratched area was determined using ImageJ image analysis software. Cell migration ability is expressed as migration rate: Migration rate (%) = (Initial scratch area - 24-hour residual area) / Initial scratch area × 100%.

[0196] 2. Transwell experiment

[0197] (1) When HUVEC cells reach 80-90% confluence, digest the cells and count them. Prepare a single-cell suspension using ECM basal medium, count the cells, and adjust the cell density to 2.5 × 10⁻⁶. 5 pcs / ml;

[0198] (2) In the lower chamber of the Transwell chamber, 600 μl of culture medium with different drug treatments were added according to the experimental group and intervention. In the upper chamber of the Transwell chamber, 200 μl of HUVEC cell suspension was added per well.

[0199] (3) After adding the sample, place the Transwell chamber in an incubator and continue culturing for 24 hours;

[0200] (4) After 24 hours, aspirate the liquid in the Transwell chamber, gently wipe away the unmigrated cells in the chamber with a cotton swab, wash twice with PBS solution to remove residual cells, fix in 4% paraformaldehyde for 30 min, and then stain the chamber in 0.1% crystal violet staining solution for 20 min.

[0201] (5) After staining, wash three times with PBS to remove excess crystal violet and background. Observe, photograph and count the number of cells that have migrated under a microscope.

[0202] (6) The obtained data were statistically analyzed using SPSS software, and the measurement data were expressed as mean ± SD.

[0203] Results explanation:

[0204] like Figure 11 As shown in A and 11C, hVEGF can be seen from the model group. 165 At a concentration of 20 ng / ml, it significantly promoted the growth and migration of HUVEC cells; the healing rates of the high, medium, and low dose LM-sdVE01 groups were 44.06% ± 5.800, 49.27% ​​± 4.254, and 54.14% ± 1.285, respectively, compared with hVEGF. 165 The healing rate of the control group under stimulation was 76.25% ± 3.501%, which was significantly lower than that of the control group (P < 0.05 or P < 0.01); the healing rate of the positive drug was 60.33% ± 8.235%. Transwell assay results showed ( Figure 11 The number of cells that migrated in the high, medium, and low dose groups of LM-sdVE01 (B and 11D) were 86.33±6.110, 410.33±8.021, and 510.0±6.083, respectively; compared with hVEGF 165 Compared with the control group (522.0±38.22), the number of migrating cells in the stimulated group was significantly reduced, and the number of migrating cells showed a dose-dependent relationship with the administered dose. However, the number of migrating cells in the low-dose LM-sdVE01 group was not statistically significant compared with the model group; the number of migrating cells in the positive control group was 156.67±29.77. In conclusion, sdVE01 effectively inhibits the horizontal and vertical migration ability of HUVEC cells by antagonizing VEGF.

[0205] Example 10: Prokaryotic expression preparation and activity analysis of LM-sdVE05

[0206] Preparation of SdVE05 genetically engineered bacteria: Based on the codon bias of *E. coli*, the gene sequence of sdVE05 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 6.

[0207] Table 6

[0208]

[0209]

[0210] The 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. The 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 refolded overnight at 4°C using a 1:10 volume ratio refolding buffer. After high-speed centrifugation, 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.

[0211] 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 coated 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 PCR solution 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 .

[0212] 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 LLM-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%.

[0213] 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.

[0214] Analysis of LM-sdVE05 inhibition of HUVEC cell tubule 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 50 μL 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 tubule 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 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.

[0215] Results explanation:

[0216] like Figure 12 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.

[0217] like Figure 13As 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 13 A); Scratch assay results showed that the healing rates of HUVEC cells in the high, medium, and low dose groups of LM-sdVE05 (6.15 μM, 1.23 μM, and 0.246 μM) 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 13 B, 13D); Transwell results showed that the number of migrating 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 migrating cells (48.00±9.54) of the high dose group of LM-sdVE05 recombinant protein compared with the positive control VHHL of the same dose; analysis of variance showed that the healing rate and the number of migrating cells in the high, medium, and low dose groups of LM-sdVE05 recombinant protein were statistically different among the groups (P<0.05), showing a dose-dependent relationship. Figure 13 (C, 13D). These results indicate that sdVE05 can inhibit the migration of HUVEC endothelial cells by suppressing VEGF secreted by HUVEC cells.

[0218] like Figure 14 As shown in A, 14C, 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 two 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 14 B, 14D) is shown 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.

[0219] like Figure 15 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 15 A, 14B). 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 15 C, 15D). 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 15 C, 15D). 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.

[0220] Example 11: Inhibitory effect of subconjunctival injection of sdVE01 on corneal neovascularization in alkali-burned rats. LM-sdVE01 recombinant protein was prepared according to the method in Example 8.

[0221] Construction of a rat model of corneal neovascularization caused by alkali burn: 8-9 week old male SD rats were placed under light anesthesia. The rats were anesthetized intraperitoneally with 0.3% sodium pentobarbital, and the ocular surface was locally anesthetized with 0.5% promecaine hydrochloride eye drops before surgery. Circular filter paper with a diameter of 3 mm was soaked in a 1M sodium hydroxide solution and precisely adhered to the central corneal surface of the right eye of each mouse for 45 seconds to induce alkali burn. The eyes were then gently rinsed twice with 10 mL of 1xPBS using a 10 mL syringe to remove residual 1M NaOH. After inducing injury, the mice were randomly divided into a control group or an experimental group.

[0222] Corneal opacity scoring: Slit-lamp images were acquired with two light sources placed to avoid reflections; one light source was placed at the 12 o'clock position, and the second at the 6 o'clock position to enhance visualization of corneal opacity. Two examiners scored the severity of corneal opacity based on the slit-lamp images. Two observers, randomly assigned to two groups, scored the slit-lamp images twice, one week apart. The severity and affected area of ​​corneal opacity were graded according to the Fantes grading scale, with grade 4 being the most severe. See the table below for details.

[0223]

[0224]

[0225] Experimental grouping: Unmodeled SD rats served as the normal control group. After animal modeling, SD rats were randomly divided into four groups of 15 rats each (see table below) using a random number table. Drug administration was performed via subconjunctival injection, with ranibizumab (lucentis) serving as the positive control; subconjunctival injection was administered on days 1 and 7.

[0226]

[0227] H&E staining: After euthanizing mice and removing the eyeballs, several small holes were made at the angular sclera using a 1mL syringe needle. The eyeballs were then fixed in 4% paraformaldehyde solution at 4°C for 24 hours. The fixed eyeballs were then placed in 20% sucrose solution and 30% sucrose solution at 4°C to allow them to settle. The dehydrated eyeballs were removed and, under a dissecting microscope, cut along the angular sclera to remove the anterior segment tissue. The lens and vitreous body were carefully removed, while the posterior segment of the eye cup was preserved. The cryostat was pre-cooled to -20°C. Liquid on the specimen surface was absorbed with filter paper. OCT embedding medium was added to a special specimen holder, and the specimen was placed on it. The specimen was adjusted so that the concave surface of the eye cup was perpendicular to the plane of the holder. The specimen was then placed on the cryostat stage. After complete freezing, more embedding medium was added to ensure complete embedding. Sections were then prepared with a thickness of 10μm. m; The sections were warmed to room temperature for 15 min, stained with hematoxylin for 3 min, washed with water for 30 s, stained with 0.5-1% ethanol-eosin for 5 s, washed with water for 30 s; 70% ethanol for 1 s, 80% ethanol for 1 s, 90% ethanol for 1 s, 95% ethanol for 1 s, anhydrous ethanol for 1 s; xylene for 2 s, xylene for 2 s; mounted with neutral resin; 3 eyes per group, sagittal plane parallel to the optic nerve, continuous sections, 6 regions selected at approximately 30 μm intervals for each retinal section (3 sections symmetrically arranged on each side with the optic nerve as the center), section thickness 5 μm; Image J software was used to measure the thickness of the outer nuclear layer of the retina, the mean was calculated, and statistical analysis was performed.

[0228] Corneal fluorescein staining: Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital; fluorescein test paper was moistened with physiological saline and fluorescein fluorescent dye was evenly applied to the corneal surface of mice; images of mouse corneal fluorescein staining were acquired using a handheld slit-lamp microscope (10× eyepiece, 2× objective lens, 5mm slit lamp width, 5mm spot diameter, cobalt blue light).

[0229] Immunofluorescence staining of the eyeball: Following the steps described above, fix, embed, and section the eyeball. Then, bake the tissue microarray in a 60°C oven for 60 minutes. Remove the dried tissue microarray and immerse it in a glass staining jar filled with xylene for 30 minutes. Replace the xylene and immerse again for 30 minutes. Remove the tissue microarray and immerse it in another staining jar filled with anhydrous ethanol for 10 minutes. Replace the anhydrous ethanol and immerse again for 10 minutes. Then, immerse it in 95% ethanol and 85% ethanol for 10 minutes each. After immersion, wash three times with distilled water for 5 minutes each time, followed by three washes with 1×PBS for 5 minutes each time. Place the tissue microarray in a staining jar filled with EDTA antigen retrieval solution and place it in a preheated 95°C water bath for heat retrieval for 20 minutes. After completion, the tissue microarray was washed three times with 1×PBS for 5 min each time; the tissue microarray was blocked with serum for 30 min, and then placed in a dark box with water. Primary antibody (VEGF: primary antibody diluent = 1:50) was added to the tissue surface and the microarray was placed in a 4°C refrigerator overnight. The next day, the tissue microarray was removed from the refrigerator and washed three times with 1×PBS for 5 min each time at room temperature. After washing, FITC-labeled goat anti-rabbit secondary antibody was added to the tissue surface and the microarray was placed in a dark box at room temperature for 30 min. Then, the microarray was washed three times with 1×PBS for 5 min each time. The tissue cell nuclei were counterstained with DAPI. After that, an anti-fluorescence quencher was added to the tissue microarray and the microarray was mounted. Images were acquired under a fluorescence microscope.

[0230] Results explanation:

[0231] The inhibitory effect of sdVE01 on angiogenesis in vivo was evaluated by constructing a rat corneal neovascularization model using alkali cauterization. Figure 16 As shown in Figure B, the rat corneas exhibited distinct white circular burn marks after alkali cauterization. On the 4th day after cauterization, rats in the alkali cauterization model group showed obvious anterior chamber hemorrhage, and limbal plexus neovascularization was observed. Over time, the anterior chamber hemorrhage in the model group tended to subside, while corneal neovascularization began at the limbus and gradually extended towards the central cornea. On the 7th day after alkali burn, the central cornea of ​​rats in both the alkali cauterization and saline injection groups showed a positive fluorescein staining reaction, indicating corneal epithelial defects. Meanwhile, the corneas of rats receiving other drug treatments showed a negative fluorescein staining reaction, indicating that these treatments, to some extent, preserved the integrity of the corneal epithelium. Figure 16 B). The results of corneal neovascularization area analysis on days 4, 7, and 14 showed that the corneal neovascularization area in the LM-sdVE01 recombinant protein injection group and the ranibizumab group was approximately 27.9%–56.5% of that in the saline injection group, exhibiting a statistically significant difference (**p<0.01 or ***p<0.001), indicating that the drug treatment groups all showed a significant inhibitory effect on corneal neovascularization. Figure 16 C). Furthermore, continued administration until day 14 significantly reduced corneal opacity caused by burns and promoted corneal recovery. Figure 16 D).

[0232] In a rat model of corneal neovascularization induced by alkali cauterization, alkali cauterization not only promoted the formation of pathological neovascularization in the cornea but also led to corneal stromal hyperplasia and corneal thickening. For example... Figure 17 As shown in A and 17C, the corneal layer in the cauterization group and the saline injection group was significantly thickened, with a loose and disordered stromal structure, reflecting the destructive effect of alkali cauterization on corneal tissue. In contrast, the corneal layer treated with LM-sdVE01 recombinant protein and ranibizumab was thinner overall, approximately 49.05%-76.25% of that in the cauterization group and the saline injection group, with a compact and orderly stromal structure, demonstrating the protective effect of these treatments on corneal tissue structure. To investigate the changes in VEGF in corneal tissue during corneal neovascularization, we also performed VEGF immunofluorescence staining of corneal tissue. The results showed that the VEGF level in the rat corneal layer significantly increased after alkali cauterization, further illustrating that VEGF, as a key factor in angiogenesis, directly leads to the subsequent massive formation of pathological corneal neovascularization. In the corneal tissue of eyes treated with LM-sdVE01 recombinant protein and ranibizumab, the VEGF level significantly decreased, approximately 5.73%-45.27% of that in the model group. This result indicates that these treatments can effectively control the content of VEGF in tissues, thereby inhibiting the formation of pathological angiogenesis. Figure 17 B, 17D).

[0233] Example 13: Safety evaluation of single-domain antibody sdVE01

[0234] The LM-sdVE01 recombinant protein was prepared according to the method in Example 8.

[0235] Twenty healthy male SD rats aged 7-8 weeks (300±20g, SPF grade) were selected and, after one week of acclimatization, randomly divided into four groups of five rats each (see table below). Administration was performed via subconjunctival injection or ocular instillation. Subconjunctival injection was administered on days 1, 4, 7, and 14; ocular instillation was administered twice daily throughout the treatment period. The safety of sdVE01 was evaluated using slit-lamp photography, corneal fluorescein staining, ocular immunofluorescence staining, and H&E staining of immune organs and other toxic target organs. Specific experimental procedures and corneal opacity scoring are detailed in the relevant section of Example 10.

[0236]

[0237] Results explanation:

[0238] The safety evaluation experiment used the same dosage and administration cycle as normal treatment. During the treatment cycle, slit-lamp photography was continuously used to record the anterior segment tissue of the rats. After the treatment cycle, the rats' eyeballs and major organs were collected for H&E staining. Figure 18 As shown, slit-lamp analysis revealed no significant differences in the bright field and cobalt blue light fields of the corneas in each group, which preliminarily indicates that these drugs did not cause significant abnormal changes under normal ocular conditions.

[0239] After the drug treatment cycle ended, healthy SD rats were sacrificed, and their eyeballs, immune organs, and potential target organs for toxicity (heart, liver, spleen, lung, and kidney) were collected for H&E staining to further assess the safety of sdVE01. Figure 19 As shown in Figure A, H&E-stained sections of the eyeballs revealed that the corneal and retinal tissue structures of rats in all groups were intact, with no obvious defects or inflammatory exudates observed in the cornea, outer nuclear layer, inner nuclear layer, and retinal pigment epithelium. This result is consistent with previous slit-lamp observations, further confirming the good tolerability of sdVE01 in healthy eyes. Simultaneously, observations of H&E staining of major organs showed ( Figure 19 B) After treatment with sdVE01, no significant histological changes were observed in these organs, indicating that these drugs have no significant toxic effects on vital organs under normal physiological conditions and have good biocompatibility and safety.

[0240] 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, characterized in that: The amino acid sequence of the single-domain antibody includes three complementarity-determining regions, CDR1, CDR2, and CDR3, and the amino acid sequences of CDR1, CDR2, and CDR3 are as follows: CDR1: FSINDEAMS CDR2: GIRSPS CDR3: AARHPDSIHQEVAY.

2. The single-domain antibody 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 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 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 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 or recombinant protein from the culture.

8. The use of the single-domain antibody according to any one of claims 1 to 2 and the recombinant protein according to claim 3 in the preparation of a medicament targeting VEGF to treat a disease, characterized in that: The disease in question is corneal neovascularization.

9. A kit for detecting VEGF, characterized in that: The kit comprises the single-domain antibody as described in any one of claims 1 to 2 or the recombinant protein as described in claim 3.

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