Anti-igfbp5 nanobody and preparation method, application and product thereof

Abstract

The application provides an anti-IGFBP5 nanobody and a preparation method, application and product thereof, and belongs to the technical field of biotechnology.The anti-IGFBP5 nanobody provided by the application contains a CDR region shown in SEQ ID NO:1-3, or one or more amino acid sequences with a sequence similarity of at least 90% with SEQ ID NO.1-3 respectively, the anti-IGFBP5 nanobody prepared by the application has strong binding capacity and can inhibit tumor cell growth.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an anti-IGFBP5 nanobody, its preparation method, application, and product. Background Technology

[0002] Insulin-like growth factors (IGFs) are important regulators of growth hormone in the body. IGFs play a crucial role in regulating cell proliferation and apoptosis, and regulate the proliferation, differentiation, and metastasis of tumor cells. Currently, seven insulin-like growth factor binding proteins (IGFBPs), named IGFBP1-IGFBP7, have been identified in human tissues. IGFBP5, as one of these proteins, can bind to insulin-like growth factors (IGFs) and regulate IGF activity, thereby participating in various cellular processes, including cell growth, apoptosis, and other physiological or pathological processes. It is one of the IGFBPs that has a significant impact on IGF activity and plays a vital role in regulating the IGF pathway.

[0003] IGFBP5 plays a crucial role in the progression of epithelial cancers, including but not limited to ovarian cancer, endometrioid breast cancer, prostate cancer, pancreatic cancer, gastric cancer, melanoma, and glioblastoma. A study evaluating the PI3K inhibition mechanism in breast cancer showed that the histone H3K27me3 demethylase KDM6B functions through IGFBP5, conferring resistance to PI3K inhibition and the ability to evade apoptosis. [1] .

[0004] Furthermore, a cross-sectional case-control study found a positive correlation between circulating IGFBP5 levels and coronary heart disease. [2] IGFBP5 is upregulated in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF), and exogenous IGFBP5 also stimulates IPF lung fibroblasts to secrete ECM components. [3] Notably, IGFBP5 exhibits a high affinity for IGF-1 throughout its full length, thus inhibiting the activation of the IGF-1 / 1R pathway. Once enzymes in the tumor microenvironment cleave IGFBP5, its affinity for IGF-1 decreases sharply, thereby activating the IGF-1 / 1R pathway and leading to abnormal tumor cell proliferation. Therefore, effective methods are needed to prevent IGFBP5 cleavage.

[0005] Monoclonal antibodies have become an effective treatment for various cancers. However, traditional monoclonal antibodies typically have a large molecular weight (around 150 kDa), resulting in relatively poor tissue penetration after administration and high immunogenicity, which limits their targeting function against specific tumors. Nanobodies, derived from camelids and containing only heavy chains, can function without light chains. Nanobodies have a smaller molecular weight, typically 12-16 kDa, while retaining antigen-binding activity and not requiring intrachain disulfide bonds, making them increasingly promising in immunotherapy. Nanobodies are easily expressed in various expression systems, such as prokaryotic (e.g., *E. coli*), eukaryotic (e.g., yeast), and mammalian cell (e.g., CHO, HEK-293 cells), and are easy to purify. Nanobodies targeting IGFBP5 can inhibit the abnormal activation of tumor proliferation pathways by blocking the recognition site and spatially hindering enzymatic cleavage, thereby suppressing specific enzyme recognition and cleavage of IGFBP5.

[0006] In conclusion, the development of anti-IGFBP5 nanobodies is of great significance in medical research and clinical treatment, and can provide new strategies and tools for the diagnosis, treatment and prevention of related diseases.

[0007] [1]Wang W, Lim KG, Feng M, et al. KDM6B Counteracts EZH2-MediatedSuppression of IGFBP5 to Confer Resistance to PI3K / AKT Inhibitor Treatment inBreast Cancer. Mol Cancer Ther. 2018;17(9):1973-1983. doi:10.1158 / 1535-7163.MCT-17-0802

[0008] [2]Fischer F, Schulte H, Mohan S, et al. Associations of insulin-likegrowth factors, insμLin-like growth factor binding proteins and acid-labilesubunit with coronary heart disease. Clin Endocrinol (Oxf). 2004;61(5):595-602. doi:10.1111 / j.1365-2265.2004.02136.x

[0009] [3]Pilewski JM, Liu L, Henry AC, Knauer AV, Feghali-Bostwick CA.insulin-like growth factor binding proteins 3 and 5 are overexpressed inidiopathic pulmonary fibrosis and contribute to extracellular matrixdeposition. Am J Pathol. 2005;166(2):399-407. doi:10.1016 / S0002-9440(10)62263-8 Summary of the Invention

[0010] To address the above problems, this invention provides an anti-IGFBP5 nanobody, its preparation method, application, and product.

[0011] On the one hand, the present invention provides an anti-IGFBP5 nanobody.

[0012] Specifically, the complementarity-determining region of the nanobody includes: any one or more of CDR1, CDR2 and CDR3 shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; or any one or more amino acid sequences that have at least 90% sequence similarity to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively.

[0013] Preferably, the complementarity-determining regions of the nanobody are CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3.

[0014] SEQ ID NO: 1: AASGFVFSMYYMS;

[0015] SEQ ID NO:2: DIDPDGGST;

[0016] SEQ ID NO:3:KAENSWSRRTPFGA.

[0017] Specifically, the nanobody further includes a framework region FR, which includes any one or more of FR1, FR2, FR3 and FR4 as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8; or any one or more amino acid sequences that have at least 90% sequence similarity to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 respectively.

[0018] Preferably, the framework regions of the nanobody are FR1, FR2, FR3 and FR4 as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0019] SEQ ID NO:5:AVQLVESGGGLVQPGGSLRLSC;

[0020] SEQ ID NO:6:WVRQAPGKGLEWVS;

[0021] SEQ ID NO:7: YYADSVKGRITISRDNAKSTLYLQMNSLKVEDTAVYYCA.

[0022] SEQ ID NO:8: WGQGTQVTVSS.

[0023] Specifically, the nanobody contains an amino acid sequence as shown in SEQ ID NO:4, or any one or more amino acid sequences that have at least 90% sequence similarity to SEQ ID NO:4.

[0024] More specifically, the amino acid sequence of the anti-IGFBP5 nanobody is shown in SEQ ID NO:4.

[0025] SEQ ID NO:4: AVQLVESGGGLVQPGGSLRLSCAASGFVFSMYYMSWVRQAPGKGLEWVSDIDPDGGSTYYADSVKGRITISRDNAKSTLYLQMNSLKVEDTAVYYCAKAENSWSRRTPFGAWGQGTQVTVSS.

[0026] In some alternative embodiments, the nanobody is used in the form of a fusion protein, the fusion protein comprising:

[0027] The first structural domain is the anti-IGFBP5 nanobody provided by the present invention.

[0028] The second domain is a functional protein different from the anti-IGFBP5 nanobody.

[0029] Preferably, the second domain comprises: an immunoglobulin Fc region, serum albumin or a fragment thereof, a domain that binds to serum albumin, polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof; and / or a molecule that has an affinity for T cell surface molecules and / or is capable of binding to surface molecules present on T cells.

[0030] More preferably, the immunoglobulin Fc region is the human immunoglobulin Fc region; and the surface molecule is CD3.

[0031] On the other hand, the present invention provides a nucleic acid, characterized in that the nucleic acid encodes the above-mentioned anti-IGFBP5 nanobody.

[0032] In another aspect, the present invention provides an expression vector carrying the encoding gene of an anti-IGFBP5 nanobody.

[0033] In another aspect, the present invention provides a host cell comprising the aforementioned nanobody, nucleic acid and / or expression vector.

[0034] In another aspect, the present invention provides a method for preparing the above-mentioned anti-IGFBP5 nanobody.

[0035] Specifically, the method includes the following steps:

[0036] (1) Obtain alpaca peripheral blood and construct a natural blood bank;

[0037] (2) Nanobodies with high affinity for human and mouse IGFBP5 were obtained by ELISA screening;

[0038] (3) Expression of nanobodies.

[0039] In another aspect, the present invention provides the application of the above-mentioned nanobody in the preparation of formulations or kits for the diagnosis, treatment or prevention of cancer.

[0040] Specifically, the cancers include, but are not limited to: ovarian cancer, endometrioid breast cancer, prostate cancer, pancreatic cancer and gastric cancer, melanoma and / or glioblastoma.

[0041] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned anti-IGFBP5 nanobody.

[0042] Specifically, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0043] More specifically, the excipients include: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0044] Specifically, the dosage form of the composition includes: liquid dosage form, gas dosage form, solid dosage form, or semi-solid dosage form.

[0045] Specifically, the liquid dosage forms include: solvent-based, aromatic aqueous solutions, tinctures, elixirs, colloidal solutions, pastes, suspensions, or emulsifiers.

[0046] Specifically, the gaseous dosage form includes: aerosol or spray.

[0047] Specifically, the solid dosage form includes: powder, pill, tablet or film.

[0048] Specifically, the semi-solid dosage form includes ointments, suppositories, or pastes.

[0049] In another aspect, the present invention provides a kit comprising the above-mentioned anti-IGFBP5 nanobody.

[0050] Specifically, the kit also includes other reagents required for the specific detection of IGFBP5 in samples.

[0051] Compared with the prior art, the present invention has the following advantages:

[0052] 1. The anti-IGFBP5 nanobody provided by this invention has a small molecular weight, while retaining its binding activity with the antigen. It can be expressed in a variety of different expression systems and is easy to purify.

[0053] 2. The anti-IGFBP5 nanobody provided by this invention has the ability to inhibit the growth of tumor cells. Attached Figure Description

[0054] Figure 1 The results are from the ELISA affinity experiment of different concentrations of P1C9 in Example 3.

[0055] Figure 2 The results of the experiment in Example 1 on the inhibition of CT2A cell proliferation by nanobodies are shown.

[0056] Figure 3 The results of IGFBP5 digestion in each group in Experiment Example 2 are shown.

[0057] Figure 4 The results of the experiment in Example 2 on the inhibition of U251 cell proliferation by nanobodies.

[0058] Figure 5 This is a line graph showing the inhibition of tumor growth by nanobodies in Experiment Example 3.

[0059] Figure 6 This is the result of the nanobody inhibiting tumor volume in vivo in Experiment Example 3.

[0060] Figure 7 The survival status of mice in different groups in Experiment 3. Detailed Implementation

[0061] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0062] Example 1: Construction of Nanobody Library

[0063] Peripheral blood was collected from 50 alpacas, mRNA was extracted, and then cDNA encoding nanobodies was synthesized by reverse transcription. The nanobodies gene fragment was obtained by PCR amplification using the cDNA as a substrate and ligated into a phage vector. The recombinant phage plasmid was then transformed into E. coli to prepare a phage display library.

[0064] Example 2: Screening and Identification of Nanobodies

[0065] 2.1 First round of screening

[0066] 2.1.1 Remove the screening antigen from the -80℃ freezer and thaw it on ice;

[0067] 2.1.2 Coat the screening antigen onto the immunotubes (50µg / tube, coating solution is CBS, pH 9.6, 2mL / tube), and slowly rotate overnight at 4°C. At the same time, coat with 50µg 3% BSA as a control.

[0068] 2.1.3 Discard the liquid in the overnight coated immunotubes, add 2 mL of PBS buffer and wash the immunotubes 3 times at room temperature, rotating for 5 min each time;

[0069] 2.1.4 Add 2 mL of blocking buffer (3% PBSTB) and rotate to block at room temperature for 2 h;

[0070] 2.1.5 Discard the liquid in the sealed immunosorbent tube and add 2 mL of PBS buffer to wash the immunosorbent tube 3 times at room temperature, rotating for 5 min each time;

[0071] 2.1.6 Discard the washing solution in the immunotherapy tube, add 2 mL of PBS buffer, and calculate and add 300 μL of the prepared phage library according to the following formula as the first round of screening input phage library. Incubate at room temperature by rotation for 1 h:

[0072]

[0073] Where V is the volume of added phage (in μL), and Tlibrary is the phage titer;

[0074] 2.1.7 Discard the liquid in the immunoassay tube, add 2 mL of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer and wash the immunoassay tube 20 times at room temperature, rotating for 5 min each time;

[0075] 2.1.8 Discard the liquid in the immunosorbent tube, remove as much residual liquid as possible, add 1 mL of 0.25 mg / mL Trypsin solution, and elute by rotating at room temperature for 30 min;

[0076] 2.1.9 Add 10 μL of 10% AEBSF to stop elution, and transfer the solution in the immunoassay tube to a new 1.5 mL centrifuge tube. This is the first round of phage elution solution.

[0077] 2.2 First-round phage eluent titer detection

[0078] 2.2.1 The SS320 strain stored at -80℃ was streaked onto 2×YT solid medium (Tet) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 mL of 2×YT medium (Tet) and incubated overnight at 37℃.

[0079] 2.2.2 Transfer 250 μL of overnight culture to 5 mL of 2×YT liquid medium (Tet), incubate at 37°C and 250 rpm for approximately 45-60 minutes until OD500 is reached. 600 It is 0.5-0.55;

[0080] 2.2.3 Take 10 μL of the first round of phage elution buffer and serially dilute it 10-fold in a 1.5 mL centrifuge tube. Repeat this process 12 times. Specifically, take 10 μL of the first round of phage elution buffer and dilute it to 100 μL. Then take another 10 μL of the first round of phage elution buffer and dilute it to 100 μL. Repeat this process 12 times to a total of 10⁻¹². Shake well to mix.

[0081] 2.2.4 Add 90 μL of SS320 bacterial culture to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min;

[0082] 2.2.5 Take 5 μL from each dilution centrifuge tube and add it to 2×YT solid medium (Amp), and incubate overnight at 37°C upside down;

[0083] 2.2.6 The statistical panel clearly distinguishes the number of single colonies at different dilutions. The number of phage particles per milliliter of phage solution, i.e., the phage library titer, is calculated using the following formula:

[0084]

[0085] Where T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0086] 2.3 Amplification of the first round of phage elution buffer

[0087] 2.3.1 The SS320 strain, which was stored at -80℃, was streaked onto 2×YT solid medium (Tet) (Sigma, Y1003-500ML) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5mL of 2×YT medium (Tet) and incubated overnight at 37℃.

[0088] 2.3.2 Take 250 μL of overnight culture and transfer it to 5 mL of 2×YT liquid medium (Tet). Incubate at 37°C and 250 rpm for about 45 min-60 min until the OD600 value is 0.5-0.55.

[0089] 2.3.3 Add 500 μL of the phage elution buffer obtained after the first round of screening to the bacterial culture with an OD600 of 0.5-0.55 (store the remaining elution buffer at 4°C).

[0090] 2.3.4 Continue culturing at 37℃ and 250 rpm for 30 min;

[0091] 2.3.5 Spread all bacterial culture evenly onto a 150 mm circular agar plate containing 100 μg / mL Amp and 2% agarose, and incubate overnight at 37°C;

[0092] 2.3.6 Take the overnight cultured circular plate, add 6 mL of 2×YT liquid medium to the surface of the plate, gently scrape off the colonies from the circular plate with a spreader and collect the bacterial solution into a 15 mL centrifuge tube, which is the amplified bacterial sub-library. At the same time, use a spectrophotometer to measure the OD600 value of the bacterial solution, which is the OD600 value of the elution buffer bacterial library. Add glycerol to a final concentration of 20%, which is the first round bacterial library.

[0093] 2.3.7 Calculate the corresponding bacterial volume of the eluent bacterial library using the formula below, and transfer it to 100 mL.

[0094] In 2×YT liquid medium (containing 100 μg / mL Amp and Tet), to achieve an initial OD600 of 0.1:

[0095]

[0096] Where V is the volume of the transferred bacterial solution (in μL), and OD600 is the OD600 of the constructed elution bacterial library;

[0097] 2.3.8 Incubate at 37℃ and 250 rpm until the bacterial OD600 reaches 0.5-0.55;

[0098] 2.3.9 Calculate and add helper phage M13K07 according to the following formula to make the ratio of bacteria to phage 1:20. Formula:

[0099]

[0100] Where V is the volume (in mL) of helper phage added. For the auxiliary phage titer used, OD 600 OD of bacterial culture 600 value;

[0101] 2.3.10 Continue culturing at 37℃ and 250 rpm for 30 min;

[0102] 2.3.11 Add Kana to a final concentration of 50 μg / mL and IPTG to 0.2 mM, and incubate overnight at 30 °C and 250 rpm.

[0103] 2.4 First round of phage purification

[0104] 2.4.1 Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 rpm for 10 min at 4°C;

[0105] 2.4.2 Transfer the supernatant after centrifugation to a new 50mL centrifuge tube, add 1 / 4 volume of 20% PEG / 2.5M NaCl pre-cooled at 4℃, mix thoroughly, and place on ice for 30min.

[0106] 2.4.3 Centrifuge at 4000 rpm and 4℃ for 20 min, discard the supernatant, and remove as much residual liquid as possible;

[0107] 2.4.4 Add 1 mL of PBS to resuspend the precipitate, transfer the resuspended solution to a new 1.5 mL centrifuge tube, and centrifuge at 13000 rpm and 4 °C for 20 min;

[0108] 2.4.5 Transfer the supernatant after centrifugation to a new 1.5 mL centrifuge tube, add 1 / 4 volume of pre-cooled 20% PEG / 2.5 M NaCl solution, mix well and place on ice for 10 min;

[0109] 2.4.6 Centrifuge at 13000 rpm and 4℃ for 10 min, discard the supernatant, and resuspend the precipitate in 1 mL of PBS;

[0110] 2.4.7 Centrifuge at 13000 rpm, 4℃ for 2 min. Transfer the supernatant to a new 1.5 mL centrifuge tube. This is the first round of phage selection library. Aliquot 100 μL / tube. Store at -80℃ for long-term storage, or at -20℃ for short-term storage (1-2 weeks).

[0111] 2.4.8 First-round screening of phage sub-library titer detection, the method is the same as 2.2.

[0112] 2.5 Second Round of Screening

[0113] The screening method is the same as in 2.1. The input phage is 1 mL of the phage sub-library obtained from the first round of screening, which is used as the input phage library for the second round of screening to obtain the phage elution buffer for the second round of screening.

[0114] 2.6 Second round of phage eluent titer detection

[0115] The method is the same as in 2.2.

[0116] 2.7 Amplification and purification of the second round of elution buffer

[0117] The method is the same as 2.3-2.4, to obtain the second round of phage sub-libraries.

[0118] 2.8 Second-round screening of phage sub-library titer detection

[0119] The method is the same as in 2.2.

[0120] Example 3: Combination Capability Assessment

[0121] 3.1 ELISA Validation of Positive Clones

[0122] 3.1.1 The SS320 strain stored at -80℃ was streaked onto 2×YT solid medium (Tet) and incubated overnight at 37℃ (stored at 4℃ for one week). A single colony was picked from the single colony plate and transferred to 5 mL of 2×YT medium (Tet) and incubated overnight at 37℃.

[0123] 3.1.2 Transfer 250 μL of overnight culture to 5 mL of 2×YT liquid medium (Tet), incubate at 37°C and 250 rpm for approximately 45-60 minutes until OD500 is reached. 600 The value is 0.5-0.55;

[0124] 3.1.3 Take 10 μL of phage elution buffer after the second round of screening and dilute it 10-fold in a 1.5 mL centrifuge tube. Dilute it 12 times in total. That is, take 10 μL of the phage library and dilute it to 100 μL, then take 10 μL of the phage library and dilute it to 100 μL, and so on, for a total of 12 dilutions. Shake well.

[0125] 3.1.4 Add 90 μL of bacterial suspension with an OD600 value of 0.5-0.55 to each dilution centrifuge tube and mix well;

[0126] 3.1.5 Continue culturing at 37℃ and 250 rpm for 30 min;

[0127] 3.1.6 Spread the bacterial culture evenly onto a solid culture medium plate containing 100 μg / mL Amp and incubate overnight at 37°C;

[0128] 3.1.7 Randomly pick single colonies from the overnight culture medium plates and place them into sterile 96-well cell culture plates (P1-P2). Add 200 μL of 2×YT medium (containing 100 µg / mL Amp and Tet) to each well and incubate at 37°C overnight.

[0129] 3.1.8 Transfer 2 μL of the overnight culture to a new 96-well cell culture plate containing 200 μL of 2×YT liquid medium (containing 100 µg / mL Amp and Tet) per well, and incubate at 37°C for 5 h. Store the overnight culture before transfer at 4°C.

[0130] 3.1.9 Calculate and add helper phage M13K07 to each well according to the following formula to achieve a bacterial count: phage count = 1:20:

[0131]

[0132] Where V is the volume (in mL) of helper phage added. The titer of the helper phage used;

[0133] 3.1.10 Incubate at 37℃ for 30 min, add Kana to a final concentration of 50 µg / mL and 0.2 mM IPTG, and incubate at 30℃ overnight.

[0134] 3.1.11 After overnight incubation, the 96-well culture plate was centrifuged at 4000 rpm for 10 min at 4°C and stored at 4°C for later use;

[0135] 3.1.12 The screening antigen (Sino-American Biotech, 10206-H08B) was coated onto the ELISA plate (1 ng / μL, coating solution was CBS pH 9.6, 100 μL / well), and BSA was coated in parallel as a control. The coating was incubated overnight at 4°C.

[0136] 3.1.13 Discard the liquid in the overnight coated microplate, add 200 μL of PBS buffer to each well, and wash the microplate 3 times at room temperature for 10 min each time;

[0137] 3.1.14 Add 200 μL of blocking buffer (3% BSA) to each well to block the microplate and incubate at room temperature for 1 hour;

[0138] 3.1.15 Discard the blocking solution, add 200 μL of PBST (1×PBS plus 0.1% Tween 20, the same below) buffer to each well, and wash the microplate 3 times at room temperature for 10 min each time;

[0139] 3.1.16 Add 120 μL of 3% BSA to each well, followed by 80 μL of the supernatant obtained from centrifugation in step 3.1.11, and incubate at room temperature for 2 hours;

[0140] 3.1.17 Discard the liquid in the microplate, add 200 μL of PBST buffer to each well and wash 3 times, 10 min each time;

[0141] 3.1.18 Add M13 Bacteriophage Antibody (HRP) and Mouse Mab, diluted 1:40000 in blocking buffer to each well, 100 μL / well, and incubate at room temperature for 1 h;

[0142] 3.1.19 Discard the liquid in the ELISA plate, add 200 μL of PBST buffer to each well and wash 3 times, 10 min each time;

[0143] 3.1.20 Add 100 μL of TMB single-component colorimetric solution to each well, incubate in the dark for 2-3 min, then add 100 μL of 1M HCl to each well to stop the reaction. Read the OD450 value using a microplate reader, record and save the result.

[0144] 3.2 Secondary ELISA validation of positive clones

[0145] To rule out false positive results, clones initially identified as positive were subjected to a second ELISA verification, using the same method as in section 2.9.

[0146] ELISA test data can be found Figure 1 Positive monoclonal antibodies were selected based on the validation data.

[0147] ELISA analysis revealed that the expressed nanobodies exhibited high specificity and strong affinity for both human and mouse IGFBP5.

[0148] Take 5 μL of positive clone bacterial culture from the monoclonal ELISA detection plate and inoculate it into 1 mL of 2×YT medium (containing 100 µg / mL Amp and Tet). Incubate at 37°C and 250 rpm until OD600 reaches 0.8-1.0 (approximately 6-8 h). Take 0.5 mL of bacterial culture for sequencing, and store the remaining bacterial culture at 4°C.

[0149] 3.3 Sequence Analysis

[0150] The sequenced sequences were analyzed using GENtle software for sequence alignment, and the antibody sequences were translated into amino acids using GENtle software.

[0151] Among them, the P1C9 sequence is shown in SEQ ID NO.4: AVQLVESGGGLVQPGGSLRLSCAASGFVFSMYYMSWVRQAPGKGLEWVSDIDPDGGSTYYADSVKGRITISRDNAKSTLYLQMNSLKVEDTAVYYCAKAENSWSRRTPFGAWGQGTQVTVSS.

[0152] Example 4

[0153] Nanobody expression: Select the corresponding nanobody monoclonal samples from Example 3 and amplify them in 5 mL LB (Amp+) at 37°C and 220 rpm for 12-16 h. Then transfer all bacterial cultures to fresh 200 mL LB (Amp+) and amplify them at 37°C and 220 rpm for about 5 h. Add IPTG and amplify at 20°C and 200 rpm for 12-16 h. Centrifuge the bacterial cultures at 4°C and 7500 rpm for 2 min and collect the bacterial pellet.

[0154] 1. Add the bacterial precipitate to Lysis Buffer at a ratio of precipitate mass to Lysis Buffer volume of 1:10, mix thoroughly, add PMSF, and sonicate on ice (on for 4 seconds, off for 8 seconds, power 60%, total time 40 minutes).

[0155] 2. Centrifuge the disrupted bacterial cells at 4°C, 11000 rpm, and 30 min to obtain the disrupted supernatant and precipitate. Add the supernatant to His-tagged protein purification magnetic beads and incubate at 4°C for 2 h.

[0156] 3. After incubation, centrifuge at 5000 rpm for 1 min, collect the magnetic beads, add Wash Buffer, centrifuge at 4℃, 500 rpm for 10 min, and discard the supernatant. Repeat 3 times.

[0157] 4. Add Elution Buffer, incubate at 4°C for 10 min, then centrifuge at 500 rpm for 10 min at 4°C and collect the supernatant. Repeat this process three times, collecting the eluent each time.

[0158] 5. Transfer the eluent from the three elutions into a 10 kD ultrafiltration tube, centrifuge at 5000 rpm for 30 min at 4 °C, and collect the supernatant sample solution after dialysis and concentration. Perform BCA assay on the sample solution to determine protein concentration. Store the final sample solution in aliquots at -80 °C.

[0159] Experimental Example 1: Inhibition of CT2A Proliferation

[0160] CT2A cells (XYBIO, catalog number 60810497) were divided into 3 groups. After 24 hours of culture and cell adhesion, they were treated according to the methods described in Table 1. After treatment, the cells were cultured for another 48 hours, and OD was measured after adding CCK8 reagent. 450 The experimental results are as follows Figure 2 As shown.

[0161] CTA2-Luc: A cell line containing luciferous cells constructed by lentiviral transfection.

[0162] CT2A-shIGFBP5-2: A cell line in which IGFBP5 was knocked down by shRNA-1.

[0163] CT2A-shIGFBP5-3: A cell line in which IGFBP5 was knocked down by shRNA-2.

[0164] Table 1

[0165]

[0166] The experimental results are shown in Figure 2 It can be seen that the IGFBP5 nanobody P1C9 provided by the present invention has the ability to significantly inhibit the growth of CT2A cells.

[0167] Experimental Example 2: Inhibition of U251 Proliferation

[0168] U251 (XYBIO, catalog number XY-XB-1370) was divided into 3 groups. After 24 hours of culture and cell adhesion, the cells were treated according to the description in Table 2. After treatment, the cells were cultured for another 48 hours, and the OD was measured after adding CCK8 reagent. 450 The experiment was repeated twice, and the results were as follows: Figure 3 As shown.

[0169] U251 OEIGFBP5: A cell line overexpressing IGFBP5.

[0170] U251 MutantIGFBP5: A cell line with a mutation at the IGFBP5 cleavage site. After mutation, IGFBP5 can no longer be cleaved by enzymes in the tumor microenvironment. Electrophoresis images of wild-type and mutated IGFBP5 are shown below. Figure 3 .

[0171] Table 2

[0172]

[0173] The experimental results are shown in Figure 4 The experimental results show that P1C9 can significantly inhibit the growth of the U251 cell line.

[0174] Experimental Example 3

[0175] Antitumor capacity assessment: An in situ tumor-forming mouse glioma model was constructed, and the steps are as follows:

[0176] 1) Construct stable CT2A-Luc and CT2A-shIGFBP5-Luc cell lines, allowing for observation of tumor growth within the brain via in vivo imaging in small animals. Culture sufficient cells, perform cell counting, and prepare cells into 1×10⁶ cells / cells. 8 / mL of cell suspension.

[0177] 2) Prepare C57BL / 6 mice.

[0178] 3) Perform stereotactic injection to inject tumor cells into the cerebral cortex of mice.

[0179] 4) The mice were briefly anesthetized with isoflurane gas, then transferred to a stereotaxic apparatus, connected to a trachea, and kept under continuous anesthesia with a low concentration of isoflurane.

[0180] 5) Fix the mouse's head, adjust the screws, and secure the incisors in the locking holes of the dental rod inside the mask, ensuring the mouse's skull is level with the table surface; fix the ears, firmly placing the ear rod against the postorbital bone of the skull, being careful not to apply excessive pressure to avoid injuring the mouse. Gently press the mouse's head with your fingers to ensure it cannot move.

[0181] 6) Make a longitudinal incision along the midline of the mouse's skull to expose the anterior and posterior fontanelles.

[0182] 7) Positioning: Using the fontanelle as the zero point, move the position of the microinjector and adjust it to +0.5mm AP and -1.7mm ML as the target injection position. Use a marker to mark the position.

[0183] 8) Drilling: Use a 1 / 32-inch (0.88 mm) skull drill to drill a hole in the mouse skull down to the dura mater. Stop immediately when you feel a sense of loss.

[0184] 9) Rinse the microsyringe with PBS to remove air, gently mix the cells in the EP tube (cells are stored on ice), and draw 7 μL of cell suspension, ensuring no air bubbles enter the needle. Before injection, expel 1 μL of liquid to ensure the microsyringe is unobstructed.

[0185] 10) Transfer the needle of the microsyringe to the target injection site, insert the needle vertically to -3.5mm, and then slightly back up 0.3mm to -3.2mm. Inject the cells using a microsyringe pump, injecting 5μL of cell suspension. The injection should be completed in about 2 minutes.

[0186] 11) Leave the needle in place at the injection site for 10 minutes to allow the cells to settle and ensure that the cells are injected into the mouse cerebral cortex. After withdrawing the needle, wipe away any coagulated blood and brain tissue from the needle tip with an alcohol swab, and rinse again with PBS for the next round of injections.

[0187] 12) Suture the mouse's scalp. Disinfect the skin and remove the mouse. Return the mouse to its cage to recover.

[0188] 13) On day 2, in vivo imaging of mice was performed to confirm intracranial tumor cell colonization. In vivo imaging of mice was then performed every 7 days to observe intracranial tumor growth. The mice were weighed and their weight recorded every 3 days, and their survival status was observed.

[0189] Treatment with anti-IGFBP5 nanobody was administered as described in Table 3.

[0190] Table 3

[0191]

[0192] The experimental results are shown in Figure 5-7 Experimental results showed that P1C9 can significantly inhibit the in vivo growth of gliomas and effectively improve the survival rate of mice.

[0193] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-IGFBP5 nanobody, characterized in that, The complementarity determining regions of the nanobody consist of CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

3.

2. Anti-IGFBP5 Nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is as set forth in SEQ ID NO:

4.

3. A nucleic acid, characterized in that, The nucleic acid encodes the anti-IGFBP5 nanobody of any one of claims 1-2.

4. An expression vector, characterized by, The expression vector comprises the nucleic acid of claim 3.

5. A host cell, characterized in that, The host cell comprises the anti-IGFBP5 nanobody of any one of claims 1-2, the nucleic acid of claim 3 and / or the expression vector of claim 4.

6. Use of a Nanobody according to any one of claims 1-2 for the manufacture of a formulation or kit for the diagnosis, treatment or prevention of cancer, characterized in that, The cancer is glioblastoma.

7. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the anti-IGFBP5 nanobody of any one of claims 1-2.

8. A kit characterized in that, The kit comprises the anti-IGFBP5 nanobody of any one of claims 1-2.

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