Anti-IGFBP5 nano antibody as well as preparation method, application and product thereof

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an anti-IGFBP5 nano antibody and a preparation method, application and product thereof. Background Art

[0002] Insulin-like growth factor (IGFs) is an important regulator of growth hormone in the body. IGFs play an important role in regulating cell proliferation and apoptosis, and regulate the proliferation, differentiation, and metastasis of tumor cells. Currently, humans have discovered seven insulin-like growth factor binding proteins (IGFBPs) from living tissues, named IGFBP1-IGFBP7. As one of them, IGFBP5 can bind to insulin-like growth factors (IGFs) and regulate the activity of IGFs, thereby participating in various cellular processes, including cell growth, cell apoptosis, and other physiological or pathological processes. It is a protein in the IGFBPs family that has a greater impact on the activity of IGF, and plays a vital role in regulating the IGF pathway.

[0003] IGFBP5 plays an important role in the progression of epithelial cancers, including but not limited to ovarian, breast endometrioid, prostate, pancreatic and gastric cancers, as well as melanoma and glioblastoma. A study evaluating the mechanism of PI3K inhibition in breast cancer showed that the histone H3K27me3 demethylase KDM6B acts through IGFBP5 to confer resistance to PI3K inhibition and the ability to escape apoptosis [1] .

[0004] In addition, a cross-sectional case-control study found that circulating IGFBP5 levels were positively correlated with 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] In particular, IGFBP5 has a high affinity for IGF-1 when in its full length, which can prevent the activation of the IGF-1 / 1R pathway. Once the enzymes in the tumor tissue microenvironment cut IGFBP5, the affinity of IGFBP5 for IGF-1 will drop sharply, thereby activating the IGF-1 / 1R pathway and causing abnormal proliferation of tumor cells. Therefore, effective means are needed to prevent IGFBP5 from being cut.

[0005] Monoclonal antibodies have become an effective means of treating many cancers. However, traditional monoclonal antibodies are usually large in molecular weight (about 150 kDa), have relatively poor tissue permeability after administration, and have relatively high immunogenicity, which limits their targeting function for target tumors. Nanobodies are antigen-binding fragments derived from camelids that contain only heavy chains and can function without light chains. Nanobodies have a relatively small molecular weight, usually 12-16 KDa, while retaining antigen binding activity, and do not require intrachain disulfide bonds, gradually emerging in immunotherapy. Nanobodies are easy to express in a variety of different expression systems, such as prokaryotic expression systems (such as in Escherichia coli), eukaryotic expression systems (such as yeast) and mammalian cell expression systems (such as CHO, HEK-293 cells), and are easy to purify. Nanobodies targeting IGFBP5 can inhibit specific enzyme recognition and cleavage of IGFBP5 by blocking recognition sites and spatially hindering enzymatic cleavage, thereby inhibiting abnormal activation of tumor proliferation pathways.

[0006] In summary, the development of anti-IGFBP5 nanoantibodies 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 [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 [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 In order to solve the above problems, the present invention provides an anti-IGFBP5 nanobody and a preparation method, application and product thereof.

[0008] In one aspect, the present invention provides an anti-IGFBP5 nanobody.

[0009] Specifically, the complementary 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 with SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0010] Preferably, the complementary 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.

[0011] SEQ ID NO: 1: AASGFVFSMYYMS; SEQ ID NO:2: DIDPDGGST; SEQ ID NO:3:KAENSWSRRTPFGA.

[0012] Specifically, the Nanobody also 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.

[0013] Preferably, the framework region of the Nanobody is FR1, FR2, FR3 and FR4 shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0014] SEQ ID NO:5:AVQLVESGGGLVQPGGSLRLSC; SEQ ID NO:6:WVRQAPGKGLEWVS; SEQ ID NO:7: YYADSVKGRITISRDNAKSTLYLQMNSLKVEDTAVYYCA.

[0015] SEQ ID NO:8: WGQGTQVTVSS.

[0016] Specifically, the Nanobody contains an amino acid sequence as shown in SEQ ID NO: 4, or any one or more amino acid sequences having at least 90% sequence similarity to SEQ ID NO.4.

[0017] More specifically, the amino acid sequence of the anti-IGFBP5 nanobody is shown in SEQ ID NO:4.

[0018] SEQ ID NO:4: AVQLVESGGGLVQPGGSLRLSCAASGFVFSMYYMSWVRQAPGKGLEWVSDIDPDGGSTYYADSVKGRITISRDNAKSTLYLQMNSLKVEDTAVYYCAKAENSWSRRTPFGAWGQGTQVTVSS.

[0019] In certain optional embodiments, the Nanobody is used in the form of a fusion protein, and the fusion protein comprises: A first domain, wherein the first domain is an anti-IGFBP5 nanobody provided by the present invention; The second domain is a functional protein different from the anti-IGFBP5 nanobody.

[0020] Preferably, the second domain comprises: an immunoglobulin Fc region, serum albumin or a fragment thereof, a domain that binds serum albumin, polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof; and / or a molecule that has affinity for a T cell surface molecule and / or is capable of binding to a surface molecule present on a T cell. Further preferably, the immunoglobulin Fc region is a human immunoglobulin Fc region; and the surface molecule is CD3.

[0021] On the other hand, the present invention provides a nucleic acid, characterized in that the nucleic acid encodes the above-mentioned anti-IGFBP5 nanobody.

[0022] In yet another aspect, the present invention provides an expression vector carrying a gene encoding an anti-IGFBP5 nanobody.

[0023] In yet another aspect, the present invention provides a host cell comprising the above-mentioned nanobody, nucleic acid and / or expression vector.

[0024] In another aspect, the present invention provides a method for preparing the above-mentioned anti-IGFBP5 nanobody.

[0025] Specifically, the method comprises the following steps: (1) Obtaining peripheral blood from alpacas and constructing a natural blood bank; (2) ELISA screening obtained nanobodies with high affinity for human and mouse IGFBP5; (3) Expression of nanobodies.

[0026] In yet another aspect, the present invention provides the use of the above-mentioned Nanobody in the preparation of a preparation or kit for diagnosing, treating or preventing cancer.

[0027] Specifically, the cancer includes, but is not limited to, ovarian cancer, breast endometrioid cancer, prostate cancer, pancreatic cancer and gastric cancer, melanoma and / or glioblastoma.

[0028] In yet another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned anti-IGFBP5 nanobody.

[0029] Specifically, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0030] More specifically, the excipients include: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, aromatics, solubilizers, emulsifiers, solubilizers or osmotic pressure regulators.

[0031] Specifically, the dosage form of the composition includes: liquid dosage form, gas dosage form, solid dosage form or semi-solid dosage form.

[0032] Specifically, the liquid dosage forms include: solvent type, aromatic water, tincture, elixir, colloidal solution, slurry, suspension or emulsion.

[0033] Specifically, the gas dosage form includes: aerosol or spray.

[0034] Specifically, the solid dosage form includes: powder, pill, tablet or film.

[0035] Specifically, the semisolid dosage form includes: ointment, suppository or paste.

[0036] In yet another aspect, the present invention provides a kit comprising the above-mentioned anti-IGFBP5 nanobody.

[0037] Specifically, the kit also includes other reagents required for specific detection of IGFBP5 in a sample.

[0038] Compared with the prior art, the present invention has the following advantages: 1. The anti-IGFBP5 nanoantibody provided by the present invention has a small molecular weight, while retaining the binding activity with the antigen, can be expressed in a variety of different expression systems, and is easy to purify.

[0039] 2. The anti-IGFBP5 nanobody provided by the present invention has the ability to inhibit the growth of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 These are the results of the ELISA affinity experiment of different concentrations of P1C9 in Example 3.

[0041] Figure 2 This is the experimental result of the nanoantibody inhibiting the proliferation of CT2A cells in Experimental Example 1.

[0042] Figure 3 These are the enzyme digestion results of IGFBP5 in each group in Experimental Example 2.

[0043] Figure 4 This is the experimental result of the nanoantibody inhibiting the proliferation of U251 cells in Experimental Example 2.

[0044] Figure 5 This is a line graph showing the inhibition of tumor growth by nanoantibodies in Experimental Example 3.

[0045] Figure 6 This is the result of the nanoantibody inhibiting tumor volume in vivo in Experimental Example 3.

[0046] Figure 7 This is the survival status of mice in different groups in Experimental Example 3. DETAILED DESCRIPTION

[0047] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0048] Example 1 Construction of Nanobody Library After collecting peripheral blood from 50 alpacas, mRNA was extracted and reverse transcribed to synthesize cDNA that can encode nanobodies. Using cDNA as a substrate, PCR amplification was performed to obtain nanobody gene fragments, which were connected to phage vectors, and the recombinant phage plasmids were transformed into Escherichia coli to prepare phage display libraries.

[0049] Example 2 Nanobody Screening and Identification 2.1 First round of screening 2.1.1 Take out the screening antigen from the -80℃ freezer and place it on ice to thaw; 2.1.2 Coat the screening antigen onto the immunotube (50 µg / tube, coating solution is CBS, pH 9.6, 2 mL / tube), rotate slowly at 4°C overnight, and coat 50 µg 3% BSA in parallel as a control; 2.1.3 Discard the liquid in the overnight coated immunotube, add 2 mL PBS buffer and wash the immunotube three times at room temperature, rotating for 5 min each time; 2.1.4 Add 2 mL of blocking solution (3% PBSTB) and rotate at room temperature for 2 hours; 2.1.5 Discard the liquid in the sealed immunotube and add 2 mL of PBS buffer to wash the immunotube 3 times at room temperature, rotating for 5 min each time; 2.1.6 Discard the washing solution in the immunotube, add 2 mL of PBS buffer, calculate according to the following formula and add 300 μL of the prepared phage library as the first round of screening input phage library, and incubate at room temperature for 1 hour with rotation:

[0050] Wherein, V is the volume of phage added (in μL), Tlibrary is the phage titer; 2.1.7 Discard the liquid in the immunotube, add 2 mL PBST (1×PBS plus 0.1% Tween20, the same below) buffer and wash the immunotube 20 times at room temperature, rotating for 5 minutes each time; 2.1.8 Discard the liquid in the immunotube, remove the residual liquid as much as possible, add 1mL of 0.25mg / mL Trypsin solution, and rotate and elute at room temperature for 30min; 2.1.9 Add 10 μL 10% AEBSF to stop elution and transfer the solution in the immunotube to a new 1.5 mL centrifuge tube, which is the phage elution solution for the first round of screening.

[0051] 2.2 First round of phage eluate titer detection 2.2.1 Streak the SS320 strain stored at -80℃ on 2×YT solid medium (Tet) for single colony, culture at 37℃ overnight (store at 4℃ for one week), pick a single colony from the single colony plate to 5mL 2×YT medium (Tet), and culture at 37℃ overnight; 2.2.2 Transfer 250 μL of overnight culture solution to 5 mL of 2×YT liquid medium (Tet) and culture at 37°C, 250 rpm for about 45 min-60 min until OD reaches 600 0.5-0.55; 2.2.3 Take 10 μL of the first round of phage eluate and dilute it 10 times in a 1.5 mL centrifuge tube, for a total of 12 dilutions, that is, take 10 μL of the first round of phage eluate and dilute it to 100 μL, then take 10 μL from it and dilute it to 100 μL, and so on, for a total of 12 dilutions to 10-12, and shake to mix; 2.2.4 Add 90 μL of SS320 bacterial solution to each dilution centrifuge tube, mix well and incubate at 37°C for 30 min; 2.2.5 Take 5 μL from each dilution centrifuge tube and add it dropwise to 2×YT solid medium (Amp), and invert and culture at 37°C overnight; 2.2.6 Count the number of single colonies on the plate at the dilution that can clearly distinguish single colonies, and calculate the number of phagemids per milliliter of phage solution, i.e., the phage library titer, according to the following formula:

[0052] Wherein, 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.

[0053] 2.3 Amplification of the first round of phage eluate 2.3.1 Pre-streaking single colonies of SS320 strain stored at -80℃ on 2×YT solid medium (Tet) (Sigma, Y1003-500ML), cultured at 37℃ overnight (stored at 4℃ for one week), picking a single colony from the single colony plate onto 5mL 2×YT medium (Tet), cultured at 37℃ overnight; 2.3.2 Transfer 250 μL of overnight culture to 5 mL of 2×YT liquid medium (Tet) and culture at 37°C, 250 rpm for about 45 min-60 min until the OD600 value reaches 0.5-0.55; 2.3.3 Add 500 μL of the phage eluate obtained after the first round of screening to the bacterial solution with an OD600 of 0.5-0.55 (the remaining eluate is stored at 4°C); 2.3.4 Continue to incubate at 37°C, 250 rpm for 30 min; 2.3.5 Spread the entire bacterial solution evenly onto a 150 mm circular culture medium plate containing 100 μg / mL Amp and 2% agarose, and culture at 37°C overnight; 2.3.6 Take the circular plate with overnight culture, add 6 mL of 2×YT liquid culture medium to the surface of the culture plate, gently scrape the colonies on the circular plate with a spreading stick and collect the bacterial liquid 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 liquid, which is the OD600 value of the eluate bacterial library. Add glycerol with a final concentration of 20%, which is the first round of bacterial library.

[0054] 2.3.7 Calculate the amount of bacterial solution from the eluate bacterial library according to the following formula and transfer to 100 mL 2×YT liquid medium (containing 100 μg / mL Amp and Tet) to make the initial OD600 0.1:

[0055] Wherein, V is the volume of the transferred bacterial solution (in μL), and OD600 is the OD600 of the constructed eluate bacterial library; 2.3.8 Incubate at 37°C, 250 rpm until the OD600 of the bacterial solution reaches 0.5-0.55; 2.3.9 Calculate and add helper phage M13K07 according to the following formula so that the number of bacteria: number of phages = 1:20, formula:

[0056] Where V is the volume of helper phage added (in mL), is the helper phage titer used, OD 600 is the OD of bacterial solution 600 value; 2.3.10 Continue to incubate at 37°C, 250 rpm for 30 min; 2.3.11 Add Kana and 0.2 mM IPTG at a final concentration of 50 μg / mL respectively, and culture overnight at 30°C and 250 rpm.

[0057] 2.4 First round of phage purification 2.4.1 Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 rpm, 4°C for 10 min; 2.4.2 Transfer the supernatant after centrifugation to a new 50 mL centrifuge tube, add 1 / 4 volume of 4°C pre-cooled 20% PEG / 2.5M NaCl, mix thoroughly and place on ice for 30 min; 2.4.3 Centrifuge at 4000 rpm, 4°C for 20 min, discard the supernatant, and remove as much residual liquid as possible; 2.4.4 Add 1 mL 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; 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; 2.4.6 Centrifuge at 13000 rpm, 4°C for 10 min, discard the supernatant, and add 1 mL of PBS to resuspend the precipitate; 2.4.7 Centrifuge at 13000rpm, 4℃ for 2min, transfer the supernatant to a new 1.5mL centrifuge tube, which is the first round of screening of phage sub-library, 100μL / tube, long-term storage at -80℃, short-term storage (1-2 weeks) at -20℃ 2.4.8 The titer detection of the first round of screening phage sub-library is the same as 2.2.

[0058] 2.5 Second round of screening The screening method is the same as 2.1. The input phage is 1 mL of the phage sub-library obtained in the first round of screening, which is used as the input phage library in the second round of screening to obtain the phage eluate for the second round of screening.

[0059] 2.6 Second round of phage eluate titer detection The method is the same as 2.2.

[0060] 2.7 Amplification and purification of the second round of eluate The method is the same as 2.3-2.4 to obtain the second round of screening phage sub-library.

[0061] 2.8 Second round of screening of phage sub-library titer detection The method is the same as 2.2.

[0062] Example 3 Binding Capacity Assessment 3.1 ELISA verification of positive clones 3.1.1 Streak the SS320 strain stored at -80℃ on 2×YT solid medium (Tet) for single colonies, culture at 37℃ overnight (store at 4℃ for one week), pick a single colony from the single colony plate and transfer it to 5mL 2×YT medium (Tet), culture at 37℃ overnight; 3.1.2 Transfer 250 μL of overnight culture solution to 5 mL of 2×YT liquid medium (Tet) and culture at 37°C, 250 rpm for about 45 min-60 min until OD reaches 600 The value is 0.5-0.55; 3.1.3 Take 10 μL of the phage eluate after the second round of screening and dilute it 10 times in a 1.5 mL centrifuge tube, for a total of 12 dilutions, that is, take 10 μL of the phage library and dilute it to 100 μL, then take 10 μL from it and dilute it to 100 μL, and so on, for a total of 12 dilutions, and shake to mix; 3.1.4 Add 90 μL of bacterial solution with OD600 value of 0.5-0.55 to each dilution centrifuge tube and mix well; 3.1.5 Continue to incubate at 37°C, 250 rpm for 30 min; 3.1.6 Spread the bacterial solution evenly on a solid culture medium plate containing 100 μg / mL Amp and culture at 37°C overnight; 3.1.7 Randomly pick monoclonal colonies from the culture medium plate after overnight culture and place them in a sterile 96-well cell culture plate (P1-P2). Add 200 μL 2×YT culture medium (containing 100 μg / mL Amp and Tet) to each well and culture at 37°C overnight. 3.1.8 Take 2 μL of the overnight culture solution and transfer it to a new 96-well cell culture plate with 200 μL of 2×YT liquid medium (containing 100 μg / mL Amp and Tet) per well. Incubate at 37°C for 5 h. Store the overnight culture solution at 4°C before transfer. 3.1.9 Calculate according to the following formula and add helper phage M13K07 to each well so that the number of bacteria: number of phages = 1:20:

[0063] Where V is the volume of helper phage added (in mL), is the helper phage titer used; 3.1.10 Incubate at 37℃ for 30 min, add Kana at a final concentration of 50µg / mL and 0.2mM IPTG, and culture at 30℃ overnight; 3.1.11 Centrifuge the 96-well culture plate after overnight culture at 4°C, 4000 rpm for 10 min and store at 4°C for later use; 3.1.12 Coat the screening antigen (Sino Biological, 10206-H08B) on the ELISA plate (1 ng / μL, coating solution is CBS pH 9.6, 100 μL / well), and coat BSA in parallel as a control, overnight at 4°C; 3.1.13 Discard the liquid in the overnight coated ELISA plate, add 200 μL PBS buffer to each well, and wash the ELISA plate three times at room temperature, each time for 10 minutes; 3.1.14 Add 200 μL of blocking solution (3% BSA) to each well to block the ELISA plate and block at room temperature for 1 hour; 3.1.15 Discard the blocking solution, add 200 μL PBST (1×PBS plus 0.1% Tween20, the same below) buffer to each well, and wash the ELISA plate 3 times at room temperature, each time for 10 min; 3.1.16 Add 120 μL 3% BSA to each well and then add 80 μL supernatant after centrifugation in step 3.1.11, and incubate at room temperature for 2 hours; 3.1.17 Discard the liquid in the ELISA plate and add 200 μL PBST buffer to each well and wash three times, each time for 10 min; 3.1.18 Add M13 Bacteriophage Antibody (HRP), Mouse Mab to each well, dilute 1:40000 in blocking solution, 100 μL / well, incubate at room temperature for 1 hour; 3.1.19 Discard the liquid in the ELISA plate and add 200 μL PBST buffer to each well and wash three times, each time for 10 min; 3.1.20 Add 100 μL TMB single-component colorimetric solution to each well, develop for 2-3 min in the dark, add 100 μL 1M HCl to each well to terminate the reaction, read the OD450 value with a microplate reader, record and save.

[0064] 3.2 Secondary verification of positive clones by ELISA In order to exclude false positive results, clones initially identified as positive were subjected to secondary ELISA verification using the same method as 2.9.

[0065] ELISA test data see Figure 1 , positive monoclones were selected based on the validation data.

[0066] ELISA detection revealed that the expressed nanoantibodies had high specificity and strong affinity for both human and mouse IGFBP5.

[0067] Take 5 μL of the positive clone bacterial solution from the monoclonal ELISA test plate and inoculate it into 1 mL of 2×YT medium (containing 100 μg / mL Amp and Tet), culture at 37°C, 250 rpm until OD600 reaches 0.8-1.0 (about 6-8 hours), take 0.5 mL of the bacterial solution for sequencing, and store the rest of the bacterial solution at 4°C.

[0068] 3.3 Sequence analysis The sequenced sequences were compared and analyzed using GENtle software, and the antibody sequences were translated into amino acids using GENtle software.

[0069] Among them, the P1C9 sequence is shown in SEQ ID NO.4: AVQLVESGGGLVQPGGSLRLSCAASGFVFSMYYMSWVRQAPGKGLEWVSDIDPDGGSTYYADSVKGRITISRDNAKSTLYLQMNSLKVEDTAVYYCAKAENSWSRRTPFGAWGQGTQVTVSS.

[0070] Example 4 Expression of nanobodies: Pick the corresponding nanobody monoclonal clone in Example 3 and amplify it in 5 mL LB (Amp+) at 37°C and 220 rpm for 12-16 h. Then transfer all the bacterial solution to fresh 200 mL LB (Amp+) at 37°C and 220 rpm for about 5 h, add IPTG at 20°C and 200 rpm for 12-16 h). Centrifuge the bacterial solution at 4°C and 7500 rpm for 2 min, and collect the bacterial precipitate.

[0071] 1. Add Lysis Buffer to the bacterial precipitate in the ratio of precipitate mass to Lysis Buffer volume = 1:10, mix thoroughly, add PMSF, and ultrasonically disrupt on ice (on 4s, off 8s, power 60%, total time 40min).

[0072] 2. Centrifuge the disrupted cells at 4°C, 11000 rpm, 30 min to obtain the disrupted supernatant and disrupted precipitate. Add the supernatant to the His-tagged protein purification magnetic beads and incubate at 4°C for 2 h.

[0073] 3. After incubation, centrifuge at 5000 rpm for 1 min, collect the magnetic beads, add Wash Buffer, centrifuge at 4°C, 500 rpm for 10 min, and discard the supernatant. Repeat 3 times.

[0074] 4. Add Elution Buffer, incubate at 4℃ for 10min, centrifuge at 4℃, 500rpm, 10min, and collect the supernatant. Repeat 3 times and collect the eluate three times.

[0075] 5. Place the three eluates into an ultrafiltration tube with a pore size of 10 kD, centrifuge at 4°C, 5000 rpm, 30 min, collect the dialyzed and concentrated sample solution on the upper layer. Perform BCA to determine the protein concentration of the sample solution. Store the final sample solution in a tube at -80°C.

[0076] Experimental Example 1 Inhibition of CT2A proliferation CT2A cells (XYBIO, catalog number 60810497) were divided into three groups. After culturing for 24 h, the cells were treated according to Table 1. After treatment, the cells were cultured for 48 h. After adding CCK8 reagent, the OD 450 The experimental results are as follows Figure 2 shown.

[0077] CTA2-Luc: A luciferous cell line constructed by lentiviral transfection.

[0078] CT2A-shIGFBP5-2: A cell line in which shRNA-1 was introduced to knock down IGFBP5.

[0079] CT2A-shIGFBP5-3: A cell line in which shRNA-2 was introduced to knock down IGFBP5.

[0080] Table 1

[0081] 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.

[0082] Experimental Example 2 Inhibition of U251 proliferation U251 (XYBIO, catalog number XY-XB-1370) was divided into 3 groups. After 24 h of culture, the cells adhered to the wall and were treated accordingly according to Table 2. After treatment, the cells were cultured for 48 h. After adding CCK8 reagent, the OD was measured. 450 The experiment was repeated twice, and the results are as follows Figure 3 shown.

[0083] U251 OEIGFBP5: a cell line overexpressing IGFBP5.

[0084] U251 MutantIGFBP5: A cell line with a mutation in the IGFBP5 cleavage site. After the mutation, IGFBP5 can no longer be cleaved by enzymes in the tumor microenvironment. The electrophoresis of wild-type and mutant IGFBP5 is shown in Figure 3 .

[0085] Table 2

[0086] The experimental results are shown in Figure 4 The experimental results showed that P1C9 could significantly inhibit the growth of U251 cell line.

[0087] Experimental Example 3 Evaluation of anti-tumor ability: The steps of constructing an orthotopic mouse glioma model are as follows: 1) Construct CT2A-Luc and CT2A-shIGFBP5-Luc stable cell lines, so that the growth of tumors in the brain can be observed through small animal live imaging. Culture enough cells, count the cells, and make the cells into 1×10 8 / mL of cell suspension.

[0088] 2) Prepare C57BL / 6 mice.

[0089] 3) Perform stereotaxic brain injection to inject tumor cells into the cerebral cortex of mice.

[0090] 4) The mouse was anesthetized for a short time by isoflurane gas anesthesia, and the mouse was transferred to a stereotaxic apparatus, and the trachea was connected to maintain continuous anesthesia with low concentration of isoflurane.

[0091] 5) Fix the mouse's skull, adjust the screws, fix the incisors in the lockhole of the tooth bar in the mask, and level the mouse's skull with the table; fix the ears, and firmly place the ear bars against the post-orbital bone of the skull. Do not squeeze too hard to avoid injuring the mouse. Gently press the mouse's head with your fingers to make sure the head cannot move.

[0092] 6) Make a longitudinal incision on the midline of the mouse's skull and cut the skin to expose the anterior and posterior bregma.

[0093] 7) Positioning: With bregma as the zero point, move the position of the microinjector and adjust to +0.5 mm AP, -1.7 mm ML as the target injection position, and use a marker to mark the position.

[0094] 8) Drilling: Use a 1 / 32 inch (0.88 mm) cranial drill to drill a hole in the mouse skull to the position of the dura mater, and stop immediately when you feel a hole.

[0095] 9) Rinse the microsyringe with PBS, remove air, gently mix the cells in the EP tube (cells are kept on ice), and draw 7 μL of cell suspension to ensure that no bubbles enter the needle. Before injection, drain 1 μL of liquid to ensure that the microsyringe is unobstructed.

[0096] 10) Move the micro-syringe needle to the target injection point, insert the needle vertically into -3.5mm, and slightly move upward 0.3mm to -3.2mm. Inject cells, inject 5μL of cell suspension through the micro-syringe pump, and the injection is completed in about 2 minutes.

[0097] 11) Leave the needle at the injection site for 10 minutes to allow the cells to settle at the injection site for 10 minutes to ensure that the cells are injected into the mouse cerebral cortex. After withdrawing the needle, wipe off the coagulated blood and brain tissue on the needle tip with an alcohol cotton ball, and rinse again with PBS for the next round of injection.

[0098] 12) Suture the mouse's scalp. Disinfect the skin and remove the mouse. Return the mouse to its cage to recover.

[0099] 13) Perform live imaging of mice on day 2 to confirm that tumor cells have colonized in the brain, and then perform live imaging of mice every 7 days to observe the growth of tumors in the brain. Weigh and record every 3 days, and observe the survival status of the mice.

[0100] Anti-IGFBP5 nanobody treatment was given as described in Table 3.

[0101] Table 3

[0102] The experimental results are shown in Figure 5-7 The experimental results show that P1C9 can significantly inhibit the growth of glioma in vivo and effectively improve the survival rate of mice.

[0103] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-IGFBP5 nanobody, characterized in that: The complementary 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 having at least 90% sequence similarity with SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

2. The anti-IGFBP5 nanobody according to claim 1, characterized in that The nanobody contains an amino acid sequence as shown in SEQ ID NO: 4, or any one or more amino acid sequences having at least 90% sequence similarity to SEQ ID NO.

4.

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

4. An expression vector, characterized in that: 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 according to any one of claims 1-2, the nucleic acid according to claim 3 and / or the expression vector according to claim 4.

6. The method for preparing the anti-IGFBP5 nanobody according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Obtaining peripheral blood from alpacas and constructing a natural blood bank; (2) ELISA screening obtained nanobodies with high affinity for human and mouse IGFBP5; (3) Expression of nanobodies.

7. Use of the Nanobody according to any one of claims 1 to 2 in the preparation of a preparation or kit for diagnosing, treating or preventing cancer.

8. The use according to claim 7, characterized in that: The cancers include: ovarian cancer, breast endometrioid cancer, prostate cancer, pancreatic cancer and gastric cancer, melanoma and / or glioblastoma.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-IGFBP5 nanobody according to any one of claims 1-2.

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

Images