Humanized targeted VEGF (vascular endothelial growth factor) single-domain antibody and application thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a single-domain antibody, in particular to a humanized targeted VEGF (vascular endothelial growth factor) single-domain antibody and application thereof. The amino acid sequence of the single-domain antibody comprises three complementary determining regions (CDR1-CDR3) and four skeleton regions (FR1-FR4). The invention also provides a recombinant protein containing the single-domain antibody, a nucleic acid molecule for coding the single-domain antibody or the recombinant protein, a carrier containing the nucleic acid molecule, a host cell containing the nucleic acid molecule or the carrier, and an application of the single-domain antibody or the recombinant protein in preparation of a medicine for treating diseases by taking the VEGF as a target spot.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a humanized VEGF-targeted single-domain antibody and an application thereof. Background Art

[0002] Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein family, including multiple subtypes, such as VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta growth factor (PIGF). VEGF initiates a series of intracellular signal transduction by binding to specific receptors on endothelial cells (such as VEGFR-1, VEGFR-2 and VEGFR-3), mainly involved in the increase of vascular permeability, degeneration of extracellular matrix, migration and proliferation of intravascular cells and formation of blood vessels. Based on the dominant role of VEGF-A in regulating angiogenesis and vascular permeability, VEGF usually refers to VEGF-A. Among the VEGF-A subtypes, VEGF165 occupies a dominant position in terms of both quantity and biological activity. Under physiological conditions, it plays a vital role in tissue growth, maintenance of vascular health and pregnancy protection. However, in certain disease states, abnormal expression or dysfunction of VEGF may 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 a large amount of VEGF, thereby promoting the formation of new tumor blood vessels and providing sufficient nutrition and oxygen for the tumor. This abnormal angiogenesis not only accelerates tumor growth, but also increases the risk of tumor metastasis. Abnormal expression of VEGF is also closely related to neovascular eye diseases, such as diabetic retinopathy and age-related macular degeneration. In these diseases, abnormal increase in VEGF can lead to excessive retinal angiogenesis, which in turn triggers a series of pathological changes, such as vascular leakage, hemorrhage, edema, etc., which seriously damage the patient's vision. In terms of cardiovascular disease, excessive VEGF expression may also lead to the occurrence and development of diseases such as atherosclerosis and vasculitis. In addition, in autoimmune diseases such as rheumatoid arthritis, abnormal expression of VEGF can promote the infiltration and angiogenesis of inflammatory cells, aggravating the inflammatory response and destruction of joints. Therefore, VEGF has become a key cytokine and a heavyweight target for clinical detection of diseases such as tumors, neovascular eye diseases, cardiovascular diseases and rheumatoid arthritis and their therapeutic drugs.

[0003] At present, the anti-VEGF macromolecular drugs approved for clinical use mainly include three categories: antibodies or antibody fragments, such as Bevacizumab (Avastin), Ranibizumab (Lucentis), Brolucizumab (Beovu) and Faricimab (bispecific antibody targeting VEGF and Ang2, Faricimab-svoa, Vabysmo); fusion proteins, such as Ablifercept (Eylea) and Conbercept (Langmu) and nucleic acid aptamers such as Pegaptanib (Macugen). In 1993, Hamers et al. found that there was an active antibody with a natural lack of light chain in camel serum, called a heavy chain antibody, and its variable region was 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 the smallest functional antigen-binding fragments currently available, with a molecular weight of about 15kD, which is 1 / 10 of that of conventional antibodies. They are also called nanobodies. Nanobodies have the advantages of small molecules and strong penetration, high specificity and high affinity, high solubility and stability, low immunogenicity, the ability to recognize antigenic epitopes with unique conformations, easy expression, and easy modification. They will break through the bottleneck of the development of existing monoclonal antibody drugs and become the new favorite for the miniaturization and functionalization of monoclonal antibody drugs. In recent years, many single-domain antibody drugs have been successfully approved for marketing. For example, the first single-domain antibody drug, Caplacizumab (Cablivi), has been approved by the European Union for the treatment of thrombotic thrombocytopenic purpura; Alphamab's PD-L1 single-domain antibody Fc fusion protein KN-035 has also been approved for marketing in China, becoming the world's first single-domain antibody drug for subcutaneous injection; Ozoralizumab, as a bispecific single-domain antibody, has been approved in Japan for the treatment of rheumatoid arthritis. These fully demonstrate the huge potential of single-domain antibodies in drug development.

[0004] Based on the above background, the present invention develops a humanized single-domain antibody targeting VEGF, which provides 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 the present invention is to provide a humanized VEGF-targeted single-domain antibody and its application.

[0006] The technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a humanized single-domain antibody targeting VEGF, wherein the amino acid sequence of the single-domain antibody includes three complementary 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): 1)CDR1: FSINDEAMS, CDR2: GIRSPS, CDR3: AARHPDSIHQEVAY; 2) CDR1: VKVSHQYMA, CDR2: GITTQD, CDR3: TSPVRWLWGAQHLAF; 3) CDR1: VTVSNQYMG, CDR2: SILNRN, CDR3: GVSRMGCEATIPY; 4) CDR1: YSLSAQDMS, CDR2: GISDTD, CDR3: GTGLSAEGGAVDYSRQGQV; 5) CDR1: YRINYENMA, CDR2: TILRPN, CDR3: TRRHRSNLGWRSSAPVQF; 6) CDR1: VRFNHEFMS, CDR2: GINSRS, CDR3: SARRAPFRTKYIKF; 7) CDR1: FTVNTKDMA, CDR2: TIEMAN, CDR3: RCVGIVCLNENIPF; 8) CDR1: VRVSDEVMS, CDR2: SITRGN, TGYWGRLPSRWENKQVGF; 9) CDR1: DSISPENMG, CDR2: TIYSPN, CDR3: TYHNREASSALKS; 10) CDR1: FTFIYEAMA, CDR2: GINKGS, CDR3: GSLGGETSTVGF; 11) CDR1: DNISPEYMG, CDR2: GIHRSD, CDR3: RSGPPMESKLWS; 12) CDR1: YNVSNENMG, CDR2: GISTDN, CDR3: GIIELNTTLWF; 13) CDR1: VMINNEYMA, CDR2: TIKIQS, CDR3: RPDWAYNQSIGY; 14) CDR1: YSVINQDMS, CDR2: GIATGN, CDR3: GVSEDRSRLKEYGPHDMKS; 15)CDR1:FTVSSEDMA,CDR2:SIQAAN,CDR3:ANGVERLGMHGPSPFRY; 16)CDR1:DNISTEDMT,CDR2:AIENPD,CDR3:GLSDNQVTRDYNQLTF; 17)CDR1:VTLSYENMS,CDR2:SITTPD,CDR3:AEPVELVSDVRTKQAHMSY; 18)CDR1:VKVNSYNMG,CDR2:SILDAD,CDR3:SGTHRSTEELPF; 19)CDR1:DSISDEVMT,CDR2:SIKGQN,CDR3:ASELCFEGKILSSEVKY; 20)CDR1:DMFNYNNMS,CDR2:TIHTND,CDR3:SVMDSVTWADAAPEALHF; 21)CDR1:DRFTPEVMT,CDR2:SISNGG,CDR3:GNDLGSRELSATQQLKY; 22)CDR1:YNISDETMG,CDR2:GIKGRD,CDR3:GNLIFAPEAALGY; 23)CDR1:DRVGAENMA,CDR2:GISVDN,CDR3:GYESWRCLAMNWEPPDVCF; 24)CDR1:DRVSNNTMS,CDR2:GIENRG,CDR3:SRSVRDEKDVIRPNTKVEF; 25)CDR1:DSISSEYMA,CDR2:TIKDTD,CDR3:GCCDIDFSDAQTIGS; 26)CDR1:DTVSDDSMG,CDR2:GIRANG,CDR3:APGREKHNAVHY; 27)CDR1:YRINDEVMS,CDR2:TINNAN,CDR3:GINSTISEEVGS; 28)CDR1:YTFSTKNMG,CDR2:TIDNDN,CDR3:RATATEQLKKEVKY; 29)CDR1:DMVTDETMA,CDR2:GIYNRG,CDR3:GPGNVLIQSTYELSS; 30) CDR1: DKVIDENMG, CDR2: GIRESD, CDR3: RELRLPSYLCY; 31) CDR1: VKIIAEDMG, CDR2: TISDTS, CDR3: SPKRQASFPQELNS; 32) CDR1: DKITAEAMG, CDR2: SIGTPS, CDR3: GLDMDFGGKAANYDKEAEVPF; 33) CDR1: FTFSAEDMS, CDR2: AIIVNS, CDR3: TAYKRDGIPEDAPLGF; 34) CDR1: VTVSDEDMG, CDR2: SIEGTG, CDR3: RPIVNSISTSENMRF; 35) CDR1: VKFSSENMS, CDR2: TITVAN, CDR3: ADEEWCTPTPMRS; 36) CDR1: FSINNENMG, CDR2: GITAKN, CDR3: SLESAQTRKKQDMEF.

[0007] Preferably, the above-mentioned single domain antibody can specifically bind to VEGF with high affinity, especially the single domain antibody having the complementary determining regions shown in items 1), 5), 10), 12), 13), 25), 34) and 36) above.

[0008] Furthermore, the above-mentioned single domain antibody also includes four framework regions FR1, FR2, FR3, and FR4, and the amino acid sequences of the framework regions FR1, FR2, FR3, and FR4 are as follows: FR1: EVQLVESGGGLVQPGGSLRLSCAASG, FR2: WVRQAPGKGLEWVS, FR3:GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA, FR4:WGQGTLVTVSS.

[0009] Furthermore, the structure of the above single domain antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0010] Furthermore, the above-mentioned single-domain antibodies can be fused with other polypeptides, proteins or functional substances to achieve diversified application goals. For example, fusion with markers such as protein tags (such as 6xHis, albumin signal peptide, GFP, RFP, etc.), enzymes (such as horseradish peroxidase, alkaline phosphatase, etc.) or radioactive isotopes can significantly improve the convenience and sensitivity of VEGF target detection or facilitate protein purification; for example, through tandem fusion technology, multiple identical single-domain antibody fragments are connected into a bivalent or multivalent antibody form, which can not only effectively improve the binding affinity of antibodies to VEGF, but also prolong their half-life in the body, thereby enhancing the therapeutic effect and reducing the frequency of administration; for example, single-domain antibodies can be connected to small molecule drugs with biological activity through specific linkers (such as cleavable linkers, non-cleavable linkers, etc.) to form antibody-coupled drugs to more efficiently exert their efficacy.

[0011] The second aspect of the present invention provides a recombinant protein, which comprises the above-mentioned single domain antibody.

[0012] The third aspect of the present invention provides a nucleic acid molecule encoding the above-mentioned single domain antibody or recombinant protein.

[0013] The fourth aspect of the present invention provides a vector, which contains the above-mentioned nucleic acid molecule.

[0014] The fifth aspect of the present invention provides a host cell, wherein the host cell contains the above-mentioned nucleic acid molecule or vector.

[0015] The sixth aspect of the present invention provides a method for preparing the above-mentioned single domain antibody or recombinant protein, which comprises the steps of culturing the above-mentioned host cell and isolating and purifying the single domain antibody or recombinant protein from the culture.

[0016] The seventh aspect of the present invention provides the use of the above-mentioned single domain antibodies and recombinant proteins in the preparation of drugs targeting VEGF for treating diseases, wherein the diseases include neovascular eye diseases, tumors, cardiovascular diseases, and rheumatoid arthritis.

[0017] The eighth aspect of the present invention provides a kit for detecting VEGF, the kit comprising the above-mentioned single domain antibody or recombinant protein.

[0018] The significant advantages of the present invention are: The present invention provides a single domain antibody targeting VEGF and a recombinant protein containing the single domain antibody, wherein the antibodies or proteins have a unique CDR sequence and can specifically bind to VEGF. Their discovery provides new candidate drugs for treating related diseases caused by abnormal VEGF such as neovascular eye disease, tumors and rheumatoid arthritis, and provides a new means for the diagnosis and detection of such diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Polyclonal phage ELISA was used to detect the enrichment of phages after 4 rounds of screening. *P<0.05, **P<0.01, ***P<0.001.

[0020] Figure 2 : Alignment of amino acid sequences of single domain antibodies targeting VEGF (sdVE). CDR1 to CDR3 are complementary determining regions, and FR1 to FR4 are framework regions.

[0021] Figure 3 : ELISA analysis of sdVE monoclonal phage. **P<0.01, ***P<0.001, ****P<0.0001.

[0022] Figure 4 : Identification of sdVE expression in 293T cells (Western Blotting analysis). Lane 1, intracellular; Lane 2, extracellular.

[0023] Figure 5 : Expression of sdVE in 293T cells and purification identification (SDS-PAGE Coomassie Brilliant Blue staining). Lane M, protein molecular weight standard; Lane 1, intracellular; Lane 2, extracellular; Lane 3, purified protein.

[0024] Figure 6 : ELISA analysis of the affinity of sdVE recombinant protein (expressed in 293T cells) (A) and normalized comparative analysis using AVST as control (B). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs control.

[0025] Figure 7 LM-sdVE (293T cell expression system) inhibits hVEGF 165 Stimulate the proliferation of HUVEC cells (CCK-8 assay).

[0026] Figure 8:Prokaryotic expression of LM-sdVE recombinant protein and its purification detection and identification (SDS-PAGE Coomassie Brilliant Blue staining). A, SDS-PAGE Coomassie Brilliant Blue staining; B, Image j software analysis of the purity of LM-sdVE recombinant protein in A. Lane M, protein molecular weight standard; Lane 1, uninduced; Lane 2, 5 hours after IPTG induction; Lane 3, supernatant after bacterial lysis and centrifugation; Lane 4, precipitate after bacterial lysis and centrifugation; Lane 5, supernatant after inclusion body 3M urea washing; Lane 6, precipitate after inclusion body 3M urea washing; Lane 7, supernatant of inclusion body dissolved by TGE buffer and alkali; Lane 8, precipitate of inclusion body dissolved by TGE buffer and alkali; Lane 9, purified LM-sdVE recombinant protein.

[0027] Fig. 9 : Representative images of HiPrep 26 / 10 Desalting of LM-sdVE.

[0028] Fig.10 : ELISA analysis of the affinity of LM-sdVE recombinant protein (expressed in E. coli). ****P<0.0001vs PBS. Fig.11 :sdVE01 inhibits hVEGF 165 Stimulate the horizontal (A, C) and vertical migration (B, D) of HUVEC vascular endothelial cells in vitro. Observed under 10× inverted microscope, ####P<0.0001, ##P<0.01vs control. ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, NS no statistical significance vs model.

[0029] Fig.12 :Prokaryotic expression, purification detection and affinity analysis of sdVE05 recombinant protein. A, SDS-PAGE analysis of the purification and desalting effect of recombinant protein sdVE05 (1. Protein molecular weight standard, 2. Before induction, 3. After induction, 4. Lysis supernatant, 5. Lysis precipitate, 6. Magnetic bead purification, 7. Desalting and freeze-drying, 8. Renaturation); B, HiPrepTM 26 / 10 desalting diagram; ELISA analysis of sdVE05 (C) and VHHL (D) binding to hVEGF 165 affinity.

[0030] Fig.13:Effects of sdVE05 on HUVEC cell viability and in vitro migration ability. A, CCK8 assay for the effect of sdVE05; B, cell scratch assay; C, Transwell assay for cell migration; D, quantitative analysis of the healing rate of the scratch area; E, quantitative analysis of the number of migrated cells. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001vs control.

[0031] Fig.14 :sdVE05 against hVEGF 165 Effects of stimulating the horizontal and vertical migration of HUVEC vascular endothelial cells in vitro. A and C, cell scratch test and quantitative analysis of the healing rate of the scratch area; B and D, cell migration test and quantitative analysis of the number of migrated cells. ####P<0.0001vs hVEGF 165 group; *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001vs Control group.

[0032] Fig.15 :The effect of sdVE05 on HUVEC cell cast formation. A, in the absence of hVEGF 165 The formation of HUVEC cell tubular structures under stimulation; B, ImageJ analysis and quantification of the absence of hVEGF 165 The number of tubes formed under stimulation; C, in the presence of hVEGF 165 The formation of HUVEC cell tubular structures under stimulation; D, ImageJ analysis and quantification of hVEGF 165 Number of tubes formed under stimulation. Cell counts were obtained from 3 random fields and expressed as mean ± SD. ####P<0.0001vs hVEGF 165 group; *P<0.05, **P<0.005, ***P<0.0005, ****P<0.0001vs Control group.

[0033] Fig.16 :In vivo evaluation of the therapeutic effect of sdVE01 on the corneal alkali burn model of 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 of 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 of corneal opacity grade in each group on days 4, 7, and 14 after corneal burn (AB group vs treatment group **P<0.01).

[0034] Fig.17:In vivo evaluation of the therapeutic effect of sdVE01 on the corneal alkali burn model of SD rats (HE staining and fluorescence staining of eye tissues). A, H&E staining of corneal sections on days 4, 7, and 14 after treatment with different drugs (scale bar 100 μm); B, Immunofluorescence staining of corneal sections on days 4, 7, and 14 after treatment with different drugs (blue: DPAI, red: anti-VEGF antibody, scale bar: 100 μm); C: Changes of the average corneal thickness of each group on days 4, 7, and 14 after injury (AB group vs treatment group *P<0.5, **P<0.01); D: Changes of the average expression of corneal endothelial growth factor (VEGF) on days 4, 7, and 14 after injury in each group (AB group vs treatment group *P<0.5, **P<0.01, ***P<0.001, ****P<0.0001).

[0035] Fig.18 : Slit lamp observation and analysis of the safety of sdVE01.

[0036] Fig.19 :Tissue H&E staining analysis of the safety of sdVE01. A, H&E staining of eyeballs; B, H&E staining of major organs. DETAILED DESCRIPTION

[0037] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0038] Example 1: Biopanning of single domain antibodies targeting VEGF 1. First round of screening (1) Coating antigen: The target antigen human vascular endothelial growth factor 165 (hVEGF 165 ) was diluted to 10 μg / ml with coating buffer and coated on ELISA plate at 100 μl / well at 4°C overnight.

[0039] (2) Blocking: Wash 5 times with 0.1% PBST, then wash once with PBS, and block with 3% PBSM at 37°C for 2 h to block nonspecific binding sites.

[0040] (3) Antibody incubation: After blocking, wash the cells 6 times with 0.1% PBST and once with PBS, and add 100 μl / well of a humanized single domain antibody phage library (10 13 pfu / mL) and incubated at 37°C for 2 h.

[0041] (4) Washing and elution: Wash 5 times with 0.1% PBST and once with PBS, add 200 μl / well of elution buffer, elute on a shaker at room temperature for 10 min, transfer the eluate to a new tube, and immediately add 100 μl of neutralization buffer to mix evenly. Take half of it for a new round of phage library amplification and titer determination.

[0042] 2. Second, third and fourth rounds of screening Based on the phage library obtained in the previous round of screening, the above first round of panning steps were repeated, with the difference 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 multiple when coating the plates was increased in each round to enrich for high-affinity phage clones.

[0043] Result description: As the number of screening rounds increases, the P / N value also increases accordingly, indicating the effectiveness of enrichment screening and the increase in phage specificity (Table 1). This example successfully screened a high-affinity phage clone targeting VEGF from a humanized single-domain antibody phage library. (Note: In each round of screening, the amount of screened phage is recorded as Input, and the amount of eluted phage is recorded as Output.) Table 1 Enrichment effect of biopanning targeting hVEGF165 single domain antibody phage Example 2: Polyclonal ELISA to detect specific enrichment of phage library after 4 rounds of screening (1) Coating: Target antigen hVEGF 165 Dilute to 5 μg / ml with coating buffer, coat ELISA plate at 100 μl / well at 4°C overnight.

[0044] (2) Blocking: Wash 5 times with 0.1% PBST and block with 2% BSA at room temperature for 2 h.

[0045] (3) Incubation with primary antibody: Wash twice with 0.1% PBST, take 50 μl of the amplified phage solution after each round of screening and mix it with 50 μl of 2% BSA, react at room temperature for 15 minutes, add it to the wells, and incubate at room temperature for 2 hours.

[0046] (4) Incubation with 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 again with 0.1% PBST for 4 times.

[0047] (5) Color development reaction: Add 100 μl / well TMB color development substrate and incubate at room temperature in the dark for 15 min.

[0048] (6) Termination of reaction: Add 50 μl / well of 1 M H2SO4 to terminate the color development reaction, and measure the absorbance of each well at OD450 nm using an ELISA reader.

[0049] Result description: Targeting hVEGF in biopanning 165 In the process of single-domain antibody, polyclonal ELISA was used to detect the antigen binding ability of the products in each round of screening. The results showed that the screening of hVEGF-targeted 165 The absorbance values ​​of single domain antibodies showed a trend of increasing in each round, and the round with the strongest binding ability was the third round ( Figure 1 ), the absorbance value decreased in the fourth round, which may be due to the saturation of the positive phages. Therefore, the screening was stopped after the fourth round. The polyclonal phages after four rounds of screening were 165 The specificity of the antigen was significantly improved, and single clones were picked from the fourth round of products for subsequent analysis.

[0050] Example 3: PCR-based DNA sequencing analysis to obtain the effectiveness of single-domain antibody targeting VEGF (sdVE) 1. Preparation of monoclonal phage: The phage library after the fourth round of screening was infected with JM101 E. coli, plated on 2×YT-AG plates at an appropriate density (about 100-300 clones / plate), and cultured overnight in a 37°C incubator. A single colony was picked with a sterile toothpick and transferred to a 96-well cell culture plate containing 200 μl of 2×YT-AG medium, and cultured at 37°C and 250 rpm with shaking until the medium became turbid, indicating that the phage was amplified.

[0051] 2. Colony PCR amplification of phage-specific DNA fragments: PCR amplification was performed on the monoclonal phage-specific DNA fragment using 2×PCR Master Mix reagent and specific primers Forward Primer L1 and Reverse Primer S6. The PCR reaction system was prepared as shown in Table 2.

[0052] Table 2 Preparation of PCR reaction solution Forward Primer L1: 5'-TGGAATTGTGAGCGGATAACAATT-3' Reverse Primer S6: 5'-GTAAATGAATTTTCTGTATGAGG-3' PCR program settings: 94°C for 1 min; 94°C for 30 s, 60°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 5 min; store at 4°C.

[0053] 3. Agarose gel electrophoresis and DNA sequencing analysis: The PCR products were subjected to 1.5% agarose gel electrophoresis, and the results were observed and photographed. The PCR products with the expected molecular weight (about 700 bp) were selected for DNA sequencing.

[0054] Result description: After DNA sequencing and sequence analysis, 36 VEGF-targeting single domain antibody (sdVE) sequences with complete open reading frames (ORFs) were obtained, which were named sdVE01 to sdVE36. The amino acid sequences of single domain antibodies sdVE01 to sdVE36 are shown in Table 3 (the underlined part is the amino acid sequence of the complementary determining region, and the rest is the amino acid sequence of the framework region).

[0055] Table 3 The amino acid sequences of sdVE01 to sdVE36 were aligned using CLC sequence Viewer 6.0 software, and the similarity and difference information between these sequences was obtained, such as Figure 2 shown.

[0056] Example 4: sdVE monoclonal phage ELISA analysis 1. Preparation of sdVE monoclonal phage: See the corresponding part of Example 3 for the specific method.

[0057] 2. Monoclonal phage ELISA analysis: (1) Coating and blocking: For specific methods, see the corresponding part of Example 2.

[0058] (2) Antigen-antibody binding: Wash twice with 0.1% PBST, add 75 μl / well 2% BSA blocking buffer and 25 μl / well monoclonal phage supernatant, and incubate at room temperature for 2 h.

[0059] (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.

[0060] (4) Incubation with 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.

[0061] (5) Color development: Add 100 μl / well TMB color development substrate and incubate at room temperature in the dark for 15 min.

[0062] (6) Termination: Add 50 μl / well of 1 M H2SO4 to terminate the reaction, measure the OD450nm value, draw a graph and perform statistical analysis.

[0063] Result description: hVEGF 165 ELISA analysis of sdVE monoclonal phage was performed with PBS as negative control. Figure 3 As shown, compared with the blank PBS control group, except for sdVE23, the other 35 sdVE monoclonal phages had a significant effect on hVEGF 165 All have certain binding specificity.

[0064] Example 5: Mammalian cell expression of single domain antibody targeting VEGF (sdVE) and its identification 1. Construction and preparation of sdVE eukaryotic expression plasmid According to the codon preference of eukaryotic mammalian cells, the gene sequences of sdVE01 to sdVE36 were optimized respectively, and NheI restriction endonuclease sequence, Kozak sequence and human albumin signal peptide sequence were added to the 5' segment, and 3 amino acid AAA sequence, 6×His tag sequence and XhoI restriction endonuclease sequence were added to the 3' end to obtain the sdVE fusion gene, whose structure is 5'-NheI restriction endonuclease sequence-Kozak sequence-human albumin signal peptide sequence-optimized sdVE gene sequence-3 amino acid AAA sequence AA sequence-6×His tag sequence-XhoI restriction endonuclease sequence; synthesize the sdVE fusion gene, clone it into the NheI / XhoI restriction endonuclease site of the pCDNA3.1(+) vector, construct a recombinant expression vector, transform competent E. coli (TOP10), and obtain genetically engineered bacteria capable of expressing sdVE; use an endotoxin-free plasmid extraction kit to prepare the sdVE eukaryotic expression plasmid; determine the concentration and purity by Nanodrop and identify it by 1% agarose gel. The heavy chain variable region genes of Avastin (GenBank: LQ506318.1) and Lucentis (GenBank: HC869890.1) were used as positive controls.

[0065] NheI restriction endonuclease sequence: 5'-G↑CTAGC-3', Kozak sequence: 5'-GCCGCCACC-3', Human albumin signal peptide sequence: 5'-ATGAAGTGGGTGACTTTTATCAGTCTACTATTTCTGTTTCTCCAGCGCCTACTCC-3', 3 amino acid AAA sequence: 5'-GCCGCTGCC-3', 6×His tag sequence: 5'-CACCATCACCATCACCAT-3', XhoI restriction endonuclease sequence: 5'-C↑TCGAG-3', 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 .

[0066] Table 4 2. Expression and identification of sdVE recombinant protein in mammalian cells Using PEI4000 as a transfection reagent, the sdVE eukaryotic expression plasmids of the above 36 single-domain antibodies sdVE01 to sdVE36 were transfected into HEK293T cells. 48 hours after transfection, centrifuge at 1000g for 5 minutes, and collect the cell pellet and supernatant respectively. Take 1ml of the supernatant, concentrate it to 40μl using an ultrafiltration tube, then add 10μl of 5× Loading Buffer, denature at 98℃ for 8min, and use it as an extracellular sample. Take the cell pellet (1×10 6cells), washed twice with PBS, added 40μl RIPA cell lysis buffer, lysed on ice for 30min, centrifuged at 12000g for 10min, removed the precipitate, collected the supernatant, added 10μl 5× Loading Buffer, denatured at 98℃ for 8min as intracellular samples. Protein expression analysis of extracellular and intracellular samples was performed by SDS-PAGE electrophoresis and Western blot technology. In the Western blot experiment, recombinant HRP Anti-6×Histag antibody was used as the primary antibody, protein expression was detected by ECL chemiluminescence, and exposure, development and image preservation were performed according to the operating instructions of the imager.

[0067] Result description: like Figure 4 As shown, 48 hours after plasmid transfection, most of the recombinant proteins were detected in the culture medium in the form of secreted proteins, among which no expression products of sdVE05 recombinant protein were detected both inside and outside the cell, and no secreted extracellular products were detected for sdVE10, sdVE29, sdLuc and sdAVST recombinant proteins, which still existed in the form of intracellular proteins.

[0068] Example 6: Purification of sdVE recombinant protein (mammalian cell expression system) and ELISA analysis thereof 1. Purification of sdVE recombinant protein Since the human albumin signal peptide sequence and 6×His tag sequence were introduced in the design of the sdVE eukaryotic expression plasmid in Example 5, in the subsequent preliminary purification of sdVE recombinant protein, for secretory recombinant protein, 75% ammonium sulfate precipitation method was first used for preliminary enrichment, and then the enriched sample was purified using nickel ion chelated magnetic beads; for intracellular recombinant protein, the protein was first released by cell lysis technology, and then purified using nickel ion chelated magnetic beads. The sdVE recombinant protein after preliminary purification was desalted using HiPrep 26 / 10 Desalting column to remove salt and other impurities in the solution, and then freeze-dried powder was obtained by vacuum freeze-drying technology and stored in a -80°C refrigerator for later use.

[0069] 2. ELISA analysis of sdVE recombinant protein (1) Coating and blocking: For specific methods, see the corresponding part of Example 2.

[0070] (2) Antigen-antibody binding: Pour out the liquid, add 200 μl / well 0.1% PBST and let stand for 30 s, then pour out the liquid, repeat the washing process twice, add 50 ng / 100 μl sdVE recombinant protein to each well and react at 37°C for 2 h.

[0071] (3) Incubation with primary antibody: Pour out the liquid, add 200 μl / well 0.1% PBST and let stand for 30 s, then pour out the liquid, repeat the wash for 4 times, add 100 μl / well mouse anti-His anti-body (1:1000 dilution) and react at 37°C for 1 h.

[0072] (4) Incubation with secondary antibody: Pour out the liquid, add 200 μl / well 0.05% PBST and let stand for 30 s, then pour out the liquid, repeat the wash 4 times, add 100 μl anti-mouse IgG1-HRP (1:2000 dilution) to each well and react at 37°C for 1 h.

[0073] (5) Color development: Pour out the liquid, add 200 μl / well 0.1% PBST and let stand for 30 s, pour out the liquid, repeat washing 4 times, add 100 μl / well TMB color development solution, incubate at room temperature away from light, when the blank well begins to appear blue, add 50 μl / well stop solution to terminate the reaction, and detect the absorbance at 450 nm.

[0074] Result description: Thirty-six sdVE recombinant proteins and two positive recombinant proteins were initially purified, and samples of intracellular proteins, extracellular proteins and purified proteins were subjected to SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining to observe the protein expression and purification process. Figure 5 As shown, the content of impurity proteins after purification was significantly reduced. The ELISA affinity analysis was performed using the initially purified sdVE recombinant protein to select sdVE molecules with higher affinity. Due to the large number of recombinant proteins, the recombinant proteins were divided into different groups for expression, purification and analysis in batches. The protein products of the same batch of expression and purification were subjected to the same ELISA analysis experiment. The recombinant protein with the highest absorbance value in this group was selected and added to the next group for expression, purification and ELISA analysis of the same batch as a control until all recombinant proteins were analyzed ( Figure 6 A). The control sdVAST was further set as 100%, and normalization was performed accordingly. The 35 sdVE recombinant proteins (sdVE05 was not expressed) were ranked according to affinity. Figure 6 As shown in B, the recombinant proteins sdVE01, sdVE02, sdVE03, sdVE09, sdVE10, sdVE11, sdVE12, sdVE13, sdVE14, sdVE21, sdVE25, sdVE34 and sdVE36 have affinities greater than those of the sdAVST recombinant protein, among which the recombinant proteins sdVE01, sdVE10, sdVE25 and sdVE36 have excellent affinities and are selected as lead molecules (LM-sdVE).

[0075] Example 7: LM-sdVE (293T cell expression system) inhibits hVEGF165 Stimulate the proliferation of HUVEC cells (detected by CCK-8 method) The sdVE recombinant protein was prepared and purified according to the methods of Examples 5 and 6.

[0076] 1. Take HUVEC cells in the logarithmic growth phase, make a single cell suspension and count, inoculate in a 96-well plate at a density of 2000 cells / well, add 200 μl ECM culture medium (containing 10% FBS, 1% double antibody, 1% growth factor) to each well and continue culturing; 2. Prepare the components according to the table below, mix the drugs and place them at 37℃ for 3 hours to fully combine; 3. After the cells in the 96-well plate adhered for 24 hours, replace with 180 μl of fresh ECM medium (containing 1% FBS and 1% double antibody), add 20 μl of mixed drugs to each well and continue to culture at 37°C and 5% CO2; 4. 72 hours after administration, add 20 μl / well of CCK-8 solution to the 96-well plate, place it in a 37°C incubator for another 4 hours, and measure the absorbance at 450 nm.

[0077] Result description: CCK-8 contains WST-8, which is reduced to a highly water-soluble yellow formazan product by dehydrogenase in the cell mitochondria under the action of dimethyl sulfate, an electron carrier. The amount of formazan produced is proportional to the number of living cells. IC 50 From small to large, they are: sdLuc recombinant protein (15.01ng / ml), sdAVST recombinant protein (9.896ng / ml), sdVE01 recombinant protein (36.12ng / ml), sdVE10 recombinant protein (55.59ng / ml), sdVE36 recombinant protein (99.14ng / ml), sdVE25 recombinant protein (114.6ng / ml). Figure 7 As shown, the four sdVE recombinant proteins all have a certain inhibitory effect on the proliferation of HUVEC cells, among which sdVE01 recombinant protein has the best inhibitory effect.

[0078] Example 8: Preparation of LM-sdVE recombinant protein (prokaryotic expression system) and ELISA analysis 1. Construction of LM-sdVE genetic engineering bacteria: According to the codon preference of Escherichia coli (E. coli), the gene sequences of sdVE01, sdVE10, sdVE25 and sdVE36 were optimized respectively, 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 by artificial gene synthesis technology and cloned into the NdeI / XhoI restriction endonuclease sites of the pET-28a expression vector to construct a recombinant expression vector, which was transformed into E. coli BL21 (DE3) competent cells; positive clones were screened from the transformed cells by colony PCR and DNA sequencing analysis.

[0079] The LM-sdVE gene sequence is shown in Table 5.

[0080] Table 5 2. Induced expression of LM-sdVE recombinant protein: Resuscitate LM-sdVE genetically engineered bacteria and expand the culture in LB-K (20 μg / mL kanamycin) medium (37°C / 225rpm) until the bacterial solution OD 600nm =0.6~0.8; after inducing culture for 5 hours with the final concentration of 1mM IPTG, the bacterial solution was collected and centrifuged (5000g / 10min), the precipitated bacteria were collected, and the wet bacteria could be stored in a -80℃ refrigerator for later use.

[0081] 3. Ultrasonic disruption of cells: resuspend the collected wet bacterial precipitate with TGE Buffer (1 g of wet bacterial solution requires 20 ml of TGE Buffer), place on ice for 10 min, and then ultrasonically disrupt the cells (200 W power, on for 5 s / off for 5 s, 4 min / time, for a total of 2 ultrasonic disruptions). After disruption, centrifuge the solution (10,000 g / 10 min), remove the supernatant, and collect the inclusion body precipitate.

[0082] 4. Inclusion body washing, dissolution and renaturation: The inclusion body precipitate (corresponding to 1g of original wet bacteria) was resuspended with 4ml of 3M urea, placed on ice for 10min, and centrifuged (10000g / 10min) to collect the precipitate; the washing was repeated once to obtain high-purity inclusion bodies; after dissolving the inclusion bodies with NaOH (10 or 20mM), 0.3 times the volume of TGE neutralization solution was immediately added dropwise, and the supernatant was collected by centrifugation (10000g / 10min); the inclusion body was placed in a dialysis bag (molecular weight cutoff 3kDa), placed in 100 times the volume of 1L dialysis solution (IBR), and dialyzed overnight in a 4°C refrigerator for renaturation.

[0083] 5. Desalting of LM-sdVE recombinant protein: centrifuge the above refolding solution (12000g / 10min), collect the supernatant, 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 with a HiPrep 26 / 10Desalting column, collect the sample, and obtain lyophilized powder by vacuum freeze drying, and store it in a -80℃ refrigerator for later use.

[0084] 6. Identification of LM-sdVE recombinant protein: Take 10 μl of equal volume of all samples (including before / after induction, supernatant / precipitate of cell disruption, supernatant / precipitate of inclusion bodies washed twice with 3M urea, supernatant / precipitate neutralized by TGE, and target protein sample dissolved in PBS) for SDS-PAGE gel electrophoresis; then, place the PAGE gel in 100 ml Coomassie brilliant blue staining solution, heat in a microwave oven for 20 seconds, and then stain for 15 minutes on a shaker at 80-90 rpm. Replace the staining solution with 100 ml decolorizing solution, shake at room temperature at 80-90 rpm, and replace with fresh decolorizing solution every hour until the bands are clear; use Imagej software to analyze the SDS-PAGE results and calculate the protein purity.

[0085] 7. ELISA analysis of LM-sdVE recombinant protein: The specific methods and steps refer to Example 5, and the LM-sdVE recombinant protein of this example is used only in the antigen-antibody binding step.

[0086] Result description: The four LM-sdVE genetic engineering bacteria constructed can effectively induce the expression of LM-sdVE recombinant protein ( Figure 8 A), through a series of purification steps such as ultrasonic disruption, inclusion body washing, dialysis renaturation and desalting, the purity of LM-sdVE recombinant protein reached more than 90% ( Figure 8 B to Fig. 9 ). ELISA was used to analyze the affinity activity of LM-sdVE. The results are shown in Fig.10 As shown, LM-sdVE can effectively bind to hVEGF with PBS as the control group. 165 In comparison, LM-sdVE01 has a higher affinity activity for the target.

[0087] Example 9: sdVE01 inhibits the migration ability of HUVEC cells stimulated by hVEGF165 The LM-sdVE01 recombinant protein was prepared according to the method of Example 8.

[0088] 1. Scratch test (1) Use a marker pen to draw a line every 1 cm on the back of the 24-well plate; (2) HUVEC cells in the logarithmic growth phase were collected and 1×10 5Cells were seeded in a 24-well plate at a density of 1.5 cells / well, and 3 ml of ECM medium containing 10% FBS was added to each well and continued to be cultured; (3) After about 24 hours, observe under a microscope that each well has formed a monolayer of cells with a confluence of about 90%. Aspirate the culture medium and replace it with a culture medium containing 1% FBS for 12 hours to eliminate the interference of serum-induced proliferation on migration. (4) When the starvation time is over, the HUVEC monolayer is scratched vertically along the marked line using a 200 μl sterile pipette tip that has been sterilized by high pressure. The intersection of the scratch and the horizontal line on the marker pen on the well plate is the fixed point for observation; (5) Gently rinse the detached cell fragments with PBS buffer three times, and add 2 ml of 1% FBS culture medium (containing 20 ng / ml hVEGF) to each 165 and different concentrations of LM-sdVE01 recombinant protein) were cultured continuously, and 1500 nM Ranibizumab was used as the positive control group; (6) After 24 hours, four fixed points were selected from each well, and the cell migration in each well was observed under a microscope and images were collected. The remaining area of ​​the scratch area was determined using Image J image analysis software. The cell migration ability was expressed as migration rate: migration rate (%) = (initial scratch area - 24h residual area) / initial scratch area × 100%.

[0089] 2. Transwell experiment (1) When HUVEC cells grow to 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 5 Pieces / ml; (2) According to the experimental groups and interventions, 600 μl of culture medium with different drug treatments were added to the lower chamber of the Transwell chamber, and 200 μl / well of HUVEC cell suspension was added to the upper chamber of the Transwell chamber; (3) After adding the sample, place the Transwell chamber in the incubator and continue culturing for 24 h; (4) After 24 hours, the liquid in the Transwell chamber was aspirated, and the non-migrated cells in the chamber were gently wiped with a cotton swab. The residual cells were washed twice with PBS solution, and the chamber was fixed in 4% paraformaldehyde for 30 minutes, and then placed in 0.1% crystal violet staining solution for staining for 20 minutes; (5) After staining, wash with PBS three times to remove excess crystal violet and background, observe under a microscope, take pictures, and count the number of migrated cells; (6) The data were statistically analyzed using SPSS software, and the measurement data were expressed as mean ± SD.

[0090] Result description: like Fig.11 As shown in A and 11C, hVEGF 165 At a concentration of 20 ng / ml, it can significantly promote 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, which is comparable to hVEGF 165 The healing ability of the control group under stimulation was significantly lower than that of 76.25% ± 3.501 (P < 0.05 or P < 0.01); the healing rate of the positive drug was 60.33% ± 8.235%. The results of the Transwell experiment showed that ( Fig.11 B and 11D), the number of migrated cells in the high, medium and low dose groups of LM-sdVE01 were 86.33±6.110, 410.33±8.021, 510.0±6.083, respectively; compared with hVEGF 165 Compared with the control group (522.0±38.22), the number of migrating cells decreased significantly under stimulation, and the number of migrating cells showed a certain dose relationship with the dosage, but 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 drug group was 156.67±29.77. In short, sdVE01 effectively inhibited the horizontal and vertical migration ability of HUVEC cells by antagonizing VEGF.

[0091] Example 10: Prokaryotic expression preparation of LM-sdVE05 and activity analysis Preparation of SdVE05 genetically engineered bacteria: According to the codon preference of Escherichia coli (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' end and 3' end, respectively, to obtain the LM-sdVE05 gene. The LM-sdVE05 gene was synthesized by artificial gene synthesis technology, cloned between the NdeI / XhoI restriction endonuclease sites of the pET-28a expression vector, and a recombinant expression vector was constructed, and transformed into E. coli BL21 (DE3) competent cells; colony PCR method and DNA sequencing analysis were used to screen positive clones from the transformed cells. The LM-sdVE05 gene sequence is shown in Table 6.

[0092] Table 6 1mM IPTG was used to induce SdVE05 genetically engineered bacteria to efficiently express recombinant proteins, most of which existed in the form of inclusion bodies; the inclusion bodies were washed by combining DOC washing and urea gradient washing (2M and 3M urea); the inclusion bodies were dissolved in binding buffer (100mM NaH2PO4, 10mM Tris-HCl, 8M Urea, 5mM imidazole to adjust the pH to 8.0) containing 8M urea, and further purified by nickel ion chelate affinity chromatography; the renaturation solution was diluted with a volume ratio of 1:10 and renaturation was carried out at 4℃ overnight; the renatured protein solution was centrifuged at high speed, the supernatant was taken and the precipitate was discarded, and the supernatant component after renaturation was concentrated by ultrafiltration and desalted by HPrepTM26 / 10 column; the LM-sdVE05 recombinant protein solution was vacuum freeze-dried to obtain lyophilized powder, which was stored in a -80℃ refrigerator for future use; conventional SDS-PAGE gel electrophoresis was used to detect the expression and purification process of the recombinant protein.

[0093] EC of LM-sdVE05 50 Analysis (ELISA): hVEGF was used at a concentration of 2 μg / ml. 165 100μl of antigen was coated on the immune plate, incubated at 4℃ overnight, and washed twice with PBST; 300μl of 2% BSA was added to each well, blocked at room temperature for 2h, and washed twice; LM-sdVE05 recombinant protein was diluted with 2% BSA at a ratio of 1:10 in sequence, and a blank group was added; 100μl of LM-sdVE05 recombinant protein of different concentrations was taken to incubate the 12 coated wells, each concentration gradient contained 3 replicate wells, incubated at room temperature for 1h, and washed 5 times with 0.05% PBST; 100μl of anti-rabbit antibody carrying His-Tag was added to each well, incubated at room temperature for 1h, and washed 5 times; enzyme-labeled secondary antibody diluted with PBS at a ratio of 1:5000 was added to each well, 100μl was added, and incubated at 37℃ for 1h; 100μl of TMB colorimetric substrate was added to each well for 5min, and 50μl of 1M The reaction was terminated with H2SO4 solution, and the absorbance at OD450nm was detected; the data were statistically analyzed and EC was calculated. 50 .

[0094] Effect of LM-sdVE05 recombinant protein on HUVEC cell viability: HUVEC in logarithmic growth phase were prepared into cell suspension and then cultured at 2*10 4Cells were inoculated into 96-well plates at a density of 1% FBS per well, with 6 replicates per well. After overnight culture, the cells adhered to the plate and were replaced with 1% FBS medium. After 24 hours of incubation, the supernatant was discarded and serum-free medium containing different concentrations of drugs (0, 0.246, 1.23, 6.15 μmol / LLM-sdVE05 recombinant protein) was added to each well. After 24 hours of culture, CCK8 10 μL / well was added and cultured for 2 hours. The absorbance of each well at a wavelength of 450 nm was measured on an enzyme-linked immunosorbent assay. Cell viability % = OD value of the experimental group / OD value of the control group × 100%.

[0095] 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, refer to Example 9.

[0096] Analysis of LM-sdVE05 inhibition of HUVEC cell tube formation: BD Matrigel matrix gel was taken out from the -20℃ refrigerator and placed in a 4℃ refrigerator overnight to dissolve; the 96-well plate and pipette tip used the next day were placed in a 4℃ refrigerator overnight to pre-cool; the pre-cooled 96-well plate was placed on ice and kept horizontal, and liquid Matrigel matrix gel was evenly spread in the 96-well plate at 50μL / well, and placed in a 37℃ incubator for 30min to allow Matrigel to polymerize into gel. Pay attention to keeping the level and avoiding the generation of bubbles, otherwise it will affect the cell tube formation and photography effect; HUVECs in the logarithmic growth phase were made into a single cell suspension containing complete culture medium and counted, with 2*10 4 The cells were seeded at a density of 1.5 cells / well in a 96-well plate and cultured in an incubator. After the cells adhered to the wall (about 8 hours), the supernatant was discarded and cultured with 1% FBS medium for 12 hours. When starvation ended, 100 μL of 1% FBS medium containing different concentrations of LM-sdVE05 recombinant protein was added to each well and culture continued. At 6 to 8 hours, the tubular structures formed by the connections between endothelial cells were continuously observed under a microscope, counted, and photographed. At least 4 random fields of view were selected for each well to be photographed.

[0097] Result description: like Fig.12 As shown in the figure, the SdVE05 genetically engineered bacteria can highly express the target recombinant protein and be effectively purified; 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 EC value of the positive control recombinant protein VHHL (ranibizumab variable region) 50 =1.363μM.

[0098] like Fig.13As shown in Figure 2, the CCK8 assay results showed that the LM-sdVE05 recombinant protein had no significant effect on HUVEC cell viability, i.e., no cytotoxicity ( Fig.13 A); The results of the scratch test showed that the healing rates of HUVEC cells in the high, medium and low dose groups (6.15 μM, 1.23 μM and 0.246 μM) of LM-sdVE05 were 40.71%, 50.52% and 58.57%, respectively, which were significantly lower than the 74.42% of the blank group (Ctrl group) (P<0.05 or P<0.01) ( Fig.13 B, 13D); 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 was significantly reduced compared with the blank group (Ctrl group) 103.7±4.73; there was no statistical difference in the healing area 46.76%±2.74 and the number of migrated cells 48.00±9.54 between the high dose group of LM-sdVE05 recombinant protein and the positive control VHHL of the same dose; the healing rate and the number of migrated cells in the high, medium and low dose groups of LM-sdVE05 recombinant protein were statistically different among the groups by variance analysis (P<0.05), and were dose-dependent ( Fig.13 C, 13D). These results indicate that sdVE05 can inhibit the migration of HUVEC endothelial cells by inhibiting the secretion of VEGF in HUVEC cells.

[0099] like Fig.14 A, 14C, hVEGF 165 The healing rate of HUVEC cells under stimulation was 69.60%, which was significantly higher than that of the blank group (Ctrl group) (53.93%), indicating that hVEGF 165 It can stimulate the migration of HUVEC cells. After the 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, which was similar to hVEGF. 165 The healing ability of the two groups was significantly lower than that of the blank group (P<0.05 or P<0.01), and the healing rate of the high and medium dose groups was even lower than that of the blank group (P<0.0001). Fig.14 B, 14D) showed in hVEGF 165 Under stimulation, the number of migrating cells in the high, medium and low dose groups of LM-sdVE05 recombinant protein was 48.00±7.55, 66.33±5.77 and 90.33±3.51, respectively, which was higher than that of hVEGF 165The number of cell migration in the stimulation group (157.01±2.29) was significantly reduced, and the healing rate of the high and medium dose groups was also lower than that of the blank group (P<0.0001); in addition, there was no statistical difference in the healing rate (47.39%) and the number of cell migration (47.33±10.12) between the high dose group and the positive control drug VHHL at the same dose. The healing rate and the number of migrated cells in the high, medium and low dose groups of LM-sdVE05 recombinant protein were statistically different among the groups by variance analysis (P<0.05), and were dose-dependent. These results indicate that sdVE05 can also inhibit the exocytosis of VEGF by HUVEC in a dose-dependent manner, thereby inhibiting the migration of HUVEC endothelial cells.

[0100] like Fig.15 As shown, SdVE05 inhibited HUVEC cell cast formation. 165 Under the condition of stimulation, the number of tubes in the high, medium and low dose groups of LM-sdVE05 recombinant protein was 6.750±1.26, 10.33±2.08 and 16.00±3.16, respectively, which was lower than that in the blank group (29.33±2.52) (P<0.0001) ( Fig.15 A, 14B). In hVEGF 165 The number of HUVEC tubes formed under stimulation was 21.00±2.16, which was significantly higher than that of the blank group (17.00±1.00) (P<0.05), indicating that VEGF stimulated the angiogenesis of HUVEC cells ( Fig.15 C, 15D). In hVEGF 165 The number of tubes formed in the high, medium and low dose groups of LM-sdVE05 recombinant protein under stimulation was 7.000±1.00, 11.00±2.00 and 19.33±2.08 per field of view, respectively, which was similar to that of hVEGF 165 The number of tubes in the high and medium dose groups was significantly lower than that in the blank group (P<0.0001). Fig.15 C, 15D). In addition, the LM-sdVE05 recombinant protein high-dose group and the VHHL positive control group with the same dose in the absence of hVEGF 165 The number of tubes formed under stimulation (9.25±2.22) and hVEGF 165 There was no statistical difference in the number of tubes formed under stimulation (7.667±0.58). The number of tubes formed by high, medium and low doses of LM-sdVE05 recombinant protein was statistically different between the groups by variance analysis (P<0.01 or P<0.0001), which was dose-dependent. Therefore, it was judged that sdVE05 could also inhibit the secretion of hVEGF by HUVEC in a dose-dependent manner. 165 Stimulation of angiogenesis and endocrine hVEGF 165Stimulate angiogenesis, thereby inhibiting the angiogenesis of HUVEC endothelial cells.

[0101] Example 11: Inhibitory effect of subconjunctival injection of sdVE01 on corneal neovascularization in rats with alkali burn. The LM-sdVE01 recombinant protein was prepared according to the method of Example 8.

[0102] Construction of alkali-burned corneal neovascularization rat model: 8-9 week-old male SD rats were placed under light anesthesia, 0.3% sodium pentobarbital was intraperitoneally administered to anesthetize the rats, and 0.5% proparacaine hydrochloride eye drops were given for local anesthesia of the ocular surface before surgery. A circular filter paper with a diameter of 3 mm was soaked in a 1M sodium hydroxide solution, and the filter paper was precisely glued to the central corneal surface of the right eye of each mouse for 45s to induce alkali burns. Using a 10ml syringe, the eyes were gently rinsed twice with 10mL 1xPBS to wash off the residual 1M NaOH. After inducing injury, the mice were randomly divided into a control group or an experimental group.

[0103] Corneal opacity scoring: Slit-lamp photographs were obtained with two light sources placed to avoid reflections, one at the 12 o'clock position and the second at the 6 o'clock position to enhance the visualization of corneal opacity. Two examiners scored the severity of corneal opacity based on the slit-lamp photographs. Two observers scored the slit-lamp photographs twice, one week apart, in a randomized manner. The severity and area of ​​corneal opacity were graded according to the Fantes grading scale, with grade 4 being the most severe, as described in the table below.

[0104] Experimental grouping: SD rats without modeling were used as the normal control group. After animal modeling, SD rats were randomly divided into 4 groups using the random number table method, with 15 rats in each group (see the table below). Drugs were administered by subconjunctival sac injection, and Lucentis was used as the positive control; the time of subconjunctival sac injection was: day 1 and day 7. H&E staining: After killing the mouse and removing the eyeball, use a 1mL syringe needle to pierce several small holes at the limbus of the cornea and sclera, and place it in a 4% paraformaldehyde solution and fix it at 4°C for 24 hours; the fixed eyeball is placed in a 20% sucrose solution and a 30% sucrose solution at 4°C to sink to the bottom. Take out the dehydrated eyeball, cut it along the limbus of the cornea and sclera under a dissecting microscope, remove the anterior segment tissue, carefully remove the lens and vitreous body, and retain the posterior eye cup; turn on the freezing slicer to precool to a temperature of -20°C; use a filter paper strip to absorb the liquid on the surface of the specimen, add OCT embedding agent to a special specimen stand, place the specimen on it, adjust the specimen's placement so that the concave surface of the eye cup is perpendicular to the plane of the support frame, and place it on the freezing table of the freezing slicer. After it is completely frozen, continue to add embedding agent to the specimen so that the specimen is completely embedded, and then it can be sliced. The thickness of the slice is 10μ m; the sections were placed at 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; neutral resin sealing; 3 eyeballs in each group, sagittal plane parallel to the optic nerve continuous section, each retinal section was selected with an interval of about 30 μm (centered on the optic nerve, 3 sections on each side symmetrically), and the section thickness was 5 μm; Image J software was used to measure the thickness of the retinal outer nuclear layer, and the mean was calculated for statistical analysis.

[0106] Corneal fluorescein sodium staining: mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital; sodium fluorescein test paper was moistened with normal saline, and sodium fluorescein fluorescent dye was evenly applied to the corneal surface of the mouse; a handheld slit lamp microscope (eyepiece 10×, objective lens 2×, slit lamp width 5 mm, spot diameter 5 mm, cobalt blue light) was used to collect mouse corneal fluorescent staining images.

[0107] Immunofluorescence staining of the eyeball: fix, embed, and slice the eyeball according to the above steps, and then bake the tissue chip in a constant temperature box at 60°C for 60 minutes. Take out the dried tissue chip, soak it in a glass dye vat filled with xylene for 30 minutes, then replace the xylene in the dye vat and soak it again for 30 minutes; take out the tissue chip, put it in another dye vat filled with anhydrous ethanol, soak it for 10 minutes, replace the anhydrous ethanol, and continue to soak it for 10 minutes, then transfer to 95% ethanol and 85% ethanol and soak it for 10 minutes respectively; after the soaking is completed, wash it with distilled water 3 times, 5 minutes each time, and continue to wash it with 1×PBS 3 times, 5 minutes each time; put the tissue chip into a dye vat filled with EDTA antigen repair solution, and put it in a constant temperature water bath preheated to 95°C in advance, heat repair for 20 minutes, and repair it. After completion, wash with 1×PBS for 3 times, 5 min each time; block the tissue chip with serum for 30 min, then put the blocked tissue chip into a dark box with water, add primary antibody (VEGF: primary antibody diluent = 1:50) on the tissue surface, and place it in a 4°C refrigerator overnight; take out the tissue chip from the refrigerator the next day, wash with 1×PBS for 3 times at room temperature, 5 min each time, and add FITC-labeled goat anti-rabbit secondary antibody on the tissue surface after completion, place it in a dark box at room temperature for 30 min, and then wash with 1×PBS for 3 times, 5 min each time; counterstain the tissue cell nucleus with DAPI, and after completion, add anti-fluorescence quencher on the tissue chip and seal the slide, and collect images under a fluorescence microscope.

[0108] Result description: A rat corneal neovascularization model was constructed to evaluate the inhibitory effect of sdVE01 on neovascularization in vivo. Fig.16 As shown in B, the rat cornea after alkali burn showed obvious white circular burn marks. On the 4th day after burn, the rats in the alkali burn model group showed obvious symptoms of anterior chamber hemorrhage, and the formation of new blood vessels in the limbal plexus was observed. Over time, the anterior chamber hemorrhage in the model group tended to subside, while the corneal neovascularization started from the initial limbus and gradually extended to the center of the cornea. On the 7th day after alkali burn, the central cornea of ​​the rats in the alkali burn group and the saline injection group showed positive sodium fluorescein staining, indicating that there was a defect in the corneal epithelium. At the same time, the corneas of rats receiving other drug treatments showed negative sodium fluorescein staining, indicating that these treatments protected the integrity of the corneal epithelium to a certain extent ( Fig.16 B). The results of counting the corneal neovascularization area on the 4th, 7th and 14th days showed that the corneal neovascularization area in the LM-sdVE01 recombinant protein injection group and the ranibizumab injection group was approximately 27.9% to 56.5% of that in the saline injection group, with significant statistical differences (**p<0.01 or ***p<0.001), indicating that the drug treatment groups showed a significant inhibitory effect on the growth of corneal neovascularization ( Fig.16C). In addition, continued administration until the 14th day significantly reduced corneal opacity caused by burning and promoted corneal recovery ( Fig.16 D).

[0109] In the alkali-burned rat corneal neovascularization model, alkali-burning not only promotes the formation of corneal pathological neovascularization, but also leads to corneal stroma hyperplasia and corneal thickening. Fig.17 As shown in A and 17C, the corneal layer of the burning group and the saline injection group was significantly thickened, and the basal structure was loose and disordered, reflecting the destructive effect of alkali burning on corneal tissue. In contrast, the corneal layer treated with LM-sdVE01 recombinant protein and ranibizumab injection was thinner overall, about 49.05%-76.25% of the burning group and the saline injection group, and the stromal layer structure was tight and orderly, showing the protective effect of these treatments on the corneal tissue structure. In order to explore the changes in VEGF in corneal tissue during corneal neovascularization, we also performed corneal tissue VEGF immunofluorescence staining. The results showed that the VEGF level in the corneal layer of rats after alkali burning increased significantly, further indicating that VEGF, as a key factor in angiogenesis, directly led to the massive generation of subsequent pathological corneal neovascularization. In the corneal tissue of the eyes treated with LM-sdVE01 recombinant protein and ranibizumab injection, the VEGF factor level decreased significantly, about 5.73%-45.27% of the model group. This result indicates that these treatments can effectively control the content of VEGF in tissues, thereby inhibiting the formation of pathological new blood vessels ( Fig.17 B, 17D).

[0110] Example 13: Safety evaluation of single domain antibody sdVE01 The LM-sdVE01 recombinant protein was prepared according to the method of Example 8.

[0111] Twenty healthy male SD rats aged 7-8 weeks, weighing 300±20g, SPF grade, were selected. After 1 week of adaptive culture, the SD rats were randomly divided into 4 groups using a random number table method, with 5 rats in each group (see the table below). The administration method is subconjunctival sac injection or ocular surface instillation. The administration time of subconjunctival sac injection is: 1, 4, 7, and 14 days; the administration time of ocular surface instillation is: instillation twice a day, and drug treatment is given every day during the treatment. The safety of sdVE01 was evaluated by slit lamp photography, corneal fluorescein sodium staining, eyeball immunofluorescence staining, and H&E staining of immune organs and other toxic target organs. For specific experimental methods and steps and corneal turbidity scoring, refer to the corresponding part of Example 10.

[0112] Result description: The safety evaluation experiment used the same dosage and administration cycle as normal treatment. During the treatment cycle, slit lamps were used to continuously take photos and record the anterior segment tissue of the rats. After the treatment cycle, the eyeballs and major organs of the rats were taken for H&E staining. Fig.18 As shown, there were no significant differences in the bright field and cobalt blue light field of the cornea of ​​each group under slit lamp analysis, which preliminarily indicated that these drugs did not cause significant abnormal changes under normal eye conditions.

[0113] After the drug treatment period, healthy SD rats were sacrificed, and the eyeballs, immune organs, and possible toxic target organs (heart, liver, spleen, lung, and kidney) were removed for H&E staining to further evaluate the safety of sdVE01. Fig.19 As shown in A, H&E staining of the eyeballs showed that the cornea and retina of each group of rats had good tissue structure, and no obvious defects or inflammatory exudates were found in the cornea, outer nuclear layer, inner nuclear layer and retinal pigment epithelium. This result is consistent with the previous slit lamp observation results, further confirming the good tolerance of sdVE01 in healthy eyes. At the same time, the observation results of H&E staining of major organs showed ( Fig.19 B) After sdVE01 treatment, these organs did not show obvious histological changes, indicating that these drugs have no significant toxic effects on important organs under normal physiological conditions and have good biocompatibility and safety.

[0114] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A humanized single-domain antibody targeting VEGF, characterized in that: The amino acid sequence of the single-domain antibody includes three complementary 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): 1) CDR1: FSINDEAMS, CDR2: GIRSPS, CDR3: AARHPDSIHQEVAY; 2) CDR1: VKVSHQYMA, CDR2: GITTQD, CDR3: TSPVRWLWGAQHLAF; 3) CDR1: VTVSNQYMG, CDR2: SILNRN, CDR3: GVSRMGCEATIPY; 4) CDR1: YSLSAQDMS, CDR2: GISDTD, CDR3: GTGLSAEGGAVDYSRQGQV; 5) CDR1: YRINYENMA, CDR2: TILRPN, CDR3: TRRHRSNLGWRSSAPVQF; 6) CDR1: VRFNHEFMS, CDR2: GINSRS, CDR3: SARRAPFRTKYIKF; 7) CDR1: FTVNTKDMA, CDR2: TIEMAN, CDR3: RCVGIVCLNENIPF; 8) CDR1: VRVSDEVMS, CDR2: SITRGN, TGYWGRLPSRWENKQVGF; 9) CDR1: DSISPENMG, CDR2: TIYSPN, CDR3: TYHNREASSALKS; 10) CDR1: FTFIYEAMA, CDR2: GINKGS, CDR3: GSLGGETSTVGF; 11) CDR1: DNISPEYMG, CDR2: GIHRSD, CDR3: RSGPPMESKLWS; 12) CDR1: YNVSNENMG, CDR2: GISTDN, CDR3: GIIELNTTLWF; 13) CDR1: VMINNEYMA, CDR2: TIKIQS, CDR3: RPDWAYNQSIGY; 14) CDR1: YSVINQDMS, CDR2: GIATGN, CDR3: GVSEDRSRLKEYGPHDMKS; 15) CDR1: FTVSSEDMA, CDR2: SIQAAN, CDR3: ANGVERLGMHGSPSPFRY; 16) CDR1: DNISTEDMT, CDR2: AIENPD, CDR3: GLSDNQVTRDYNQLTF; 17) CDR1: VTLSYENMS, CDR2: SITTPD, CDR3: AEPVELVSDVRTKQAHMSY; 18) CDR1: VKVNSYNMG, CDR2: SILDAD, CDR3: SGTHRSTEELPF; 19) CDR1: DSISDEVMT, CDR2: SIKGQN, CDR3: ASELCFEGKILSSEVKY; 20) CDR1: DMFNYNNMS, CDR2: TIHTND, CDR3: SVMDSVTWADAAPEALHF; 21) CDR1: DRFTPEVMT, CDR2: SISNGG, CDR3: GNDLGSRELSATQQLKY; 22) CDR1: YNISDETMG, CDR2: GIKGRD, CDR3: GNLIFAPEAALGY; 23) CDR1: DRVGAENMA, CDR2: GISVDN, CDR3: GYESWRCLAMNWEPPDDVCF; 24) CDR1: DRVSNNTMS, CDR2: GIENRG, CDR3: SRSVRDEKDVIRPNTKVEF; 25) CDR1: DSISSEYMA, CDR2: TIKDTD, CDR3: GCCDIDFSDAQTIGS; 26) CDR1: DTVSDDSMG, CDR2: GIRANG, CDR3: APGREKHNAVHY; 27) CDR1: YRINDEVMS, CDR2: TINNAN, CDR3: GINSTISEEVGS; 28) CDR1: YTFSTKNMG, CDR2: TIDDNDN, CDR3: RATATEQLKKEVKY; 29) CDR1: DMVTDETMA, CDR2: GIYNRG, CDR3: GPGNVLIQSTYELSS; 30) CDR1: DKVIDENMG, CDR2: GIRESD, CDR3: RELRLPSYLCY; 31) CDR1: VKIIAEDMG, CDR2: TISDTS, CDR3: SPKRQASFPQELNS; 32) CDR1: DKITAEAMG, CDR2: SIGTPS, CDR3: GLDMDFGGKAANYDKEAEVPF; 33) CDR1: FTFSAEDMS, CDR2: AIIVNS, CDR3: TAYKRDGIPEDAPLGF; 34) CDR1: VTVSDEDMG, CDR2: SIEGTG, CDR3: RPIVNSISTSENMRF; 35) CDR1: VKFSSENMS, CDR2: TITVAN, CDR3: ADEEWCTPTPMRS; 36) CDR1: FSINNENMG, CDR2: GITAKN, CDR3: SLESAQTRKKQDMEF.

2. The single domain antibody according to claim 1, characterized in that: The single domain antibody further comprises four framework regions FR1, FR2, FR3, and FR4, and the amino acid sequences of the framework 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 according to 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 according to any one of claims 1 to 3.

5. A carrier, characterized in that: The vector contains the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that: The host cell contains the nucleic acid molecule according to claim 4 or the vector according to 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 comprises the steps of culturing the host cell according to claim 7, and isolating and purifying the single domain antibody or recombinant protein from the culture.

8. 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 drug targeting VEGF for treating a disease.

9. The use according to claim 8, characterized in that: The diseases include neovascular eye disease, tumors, cardiovascular disease, and rheumatoid arthritis.

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

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