Targeted ALOX5 nano antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a targeted ALOX5 nano antibody. The nano antibody comprises an HCDR1, an HCDR2 and an HCDR3, wherein the amino acid sequences of the HCDR1, the HCDR2 and the HCDR3 are shown as SEQ ID NO.1-3; or an amino acid sequence having at least one amino acid difference compared with SEQ ID NO.1-3. Compared with the prior art, the targeted ALOX5 nano antibody provided by the invention has higher affinity and excellent treatment effect, and has better application in the medicine industry, especially the macromolecular medicine industry.

Description

[0001] The present invention claims priority to Chinese patent application No. 202410737781.6 filed on June 7, 2024, entitled “A nanobody targeting ALOX5 and its application”, the entire contents of which are incorporated by reference in the application. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to an ALOX5-targeted nanoantibody and an application thereof. Background Art

[0003] Nanobodies are a type of natural antibody that exists in large quantities in the serum of camelids and sharks. Compared with conventional monoclonal antibodies, nanobodies lack light chains, and the CDR3 region of their heavy chain variable region VHH is longer, making them the smallest unit known to bind to antigens. Their special structural features and properties give them some characteristics that other conventional antibodies or antibody fragments do not have.

[0004] Similar to the VH folding structure of conventional monoclonal antibodies, the crystal and water-soluble structures of nano antibodies are composed of two β sheets. The CDR3 region of VHH is relatively long, and the average length of the CDR3 region of human and mouse antibody VH is 9-12 amino acids, and the CDR3 region of VHH is 16-18 amino acids. The expansion of the variable region can form a richer antigen binding conformation, which makes up for the deficiency of decreased binding force caused by the loss of light chain to a certain extent, so that the nano antibody itself has a stronger antigen binding ability. The CDR3 region of the nano antibody can form a special convex loop, most of which is folded on FR2, where the hydrophobic residues are protected and can avoid contact with the external water environment. The cysteine ​​in the convex loop forms a disulfide bond with the cysteine ​​in the CDR1 (or FR2) region to stabilize its structure. The convex loop structure can bind to the cleft or cavity of the enzyme, so it can be a good inhibitor of the enzyme, an agonist or antagonist of the receptor.

[0005] ALOX5 (Arachidonate 5-Lipoxygenase) is an important enzyme that participates in the 5-lipoxygenation reaction in the arachidonic acid metabolic pathway to produce various biologically active leukotrienes. The activity of ALOX5 is closely related to the development of various inflammatory diseases and allergic diseases.

[0006] Currently, most antibodies targeting ALOX5 are monoclonal antibodies or polyclonal antibodies, and there are no nanoantibodies targeting ALOX5. Summary of the invention

[0007] In view of the above shortcomings, the present invention provides a nano antibody targeting ALOX5. The nano antibody comprises: HCDR1, HCDR2 and HCDR3 of the amino acid sequence as shown in SEQ ID NO.1-3; or an amino acid sequence having at least one amino acid difference compared with SEQ ID NO.1-3. The nano antibody targeting ALOX5 provided by the present invention has higher affinity than the prior art, excellent therapeutic effect, and has better application in the pharmaceutical industry, especially the macromolecular drug industry.

[0008] In order to achieve the above technical objectives, the present invention hereby proposes the following technical solutions:

[0009] In a first aspect, the present invention provides a nanobody targeting ALOX5, wherein the nanobody comprises:

[0010] (1) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO. 1-3; or

[0011] (2) An amino acid sequence having at least one amino acid difference compared to SEQ ID NO. 1-3.

[0012] In some embodiments, the amino acid difference is achieved by at least one of amino acid addition, deletion, modification and / or substitution.

[0013] SEQ ID NO. 1: EASRLTFSIYAMG.

[0014] SEQ ID NO. 2: GHSSSGGNT.

[0015] SEQ ID NO. 3: ASLRGYSRVVPTTGGEYPY.

[0016] In some embodiments, the Nanobody comprises:

[0017] 1) FR1, FR2, FR3 and FR4 of the amino acid sequence as shown in SEQ ID NO.4-7; or

[0018] 2) An amino acid sequence having at least one amino acid difference compared to SEQ ID NO. 4-7.

[0019] In some embodiments, the amino acid difference is achieved by at least one of amino acid addition, deletion, modification and / or substitution.

[0020] SEQ ID NO.4:AVQLVESGGGLVQAGGSLRLSC.

[0021] SEQ ID NO. 5: WFRQAPGKEREFVA.

[0022] SEQ ID NO. 6: YYKDSVEGFTISRDNAKNTLYLQMNSLKPEDTAVYYCA.

[0023] SEQ ID NO. 7: WGQGTQVTVSS.

[0024] In some embodiments, the nanobody has an amino acid sequence as shown in SEQ ID NO.8. SEQ ID NO.8:

[0025] AVQLVESGGGLVQAGGSLRLSCEASRLTFSIYAMGWFRQAPGKEREFVA GHSSSGGNTYYKDSVERGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAASLR GYSRVVPTTGGEYPYWGQGTQVTVSS.

[0026] In some embodiments, the Nanobody further comprises a biologically active protein or a functional fragment thereof that assists its expression and / or secretion, or prolongs its half-life in vivo.

[0027] In some embodiments, the biologically active protein or its functional fragment is selected from at least one of an immunoglobulin Fc domain, serum albumin, an albumin binding polypeptide, prealbumin, a carboxyl-terminal peptide, an elastin-like polypeptide, a His tag, a GST tag, an MBP tag, a FLAG tag, and a SUMO tag.

[0028] In some preferred embodiments, the biologically active protein or a functional fragment thereof is a human immunoglobulin Fc domain.

[0029] In some preferred embodiments, the biologically active protein or a functional fragment thereof is the Fc domain of human IgG, such as the Fc domain of human IgG1, IgG2, IgG3, or IgG4.

[0030] In a second aspect, the present invention provides an antibody preparation comprising any of the aforementioned nanobodies and at least one pharmaceutically acceptable carrier.

[0031] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to: surfactants, solubilizers, stabilizers, preservatives and the like.

[0032] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned nanobody.

[0033] In a fourth aspect, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0034] In some embodiments, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

[0035] In a fifth aspect, the present invention provides a host cell comprising the above-mentioned expression vector.

[0036] Aspect 6. The present invention provides a pharmaceutical composition, comprising the above-mentioned rice antibody, antibody preparation, nucleic acid molecule, expression vector and / or host cell.

[0037] In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0038] In some embodiments, the pharmaceutically acceptable excipient is selected from a solvent, a diluent, a disintegrant, a precipitation inhibitor, a surfactant, a glidant, a binder, a lubricant, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative, a stabilizer, a hydrating agent, an emulsification accelerator, a buffer, an absorbent, a colorant, a flavoring agent, a sweetener, an ion exchanger, a release agent, a coating agent, a flavoring agent or an antioxidant.

[0039] In a seventh aspect, the present invention provides the use of the above-mentioned nanobodies, antibody preparations, nucleic acid molecules, host cells or pharmaceutical compositions in the preparation of drugs for preventing and / or treating tumors.

[0040] In some embodiments, the tumor comprises glioma, prostate cancer, ovarian cancer, lung cancer, colorectal cancer, liver cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, gastric cancer, cervical cancer.

[0041] In some embodiments, the tumor is a glioma.

[0042] The beneficial effects of the present invention are:

[0043] Compared with the prior art, the ALOX5-targeted nanoantibody provided by the present invention has higher affinity and excellent therapeutic effect, and has better application in the pharmaceutical industry, especially the macromolecular drug industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Blue and white screening of pFastBac-Alox5 antigen protein recombinant expression plasmid.

[0045] Figure 2 Coomassie Brilliant Blue staining for ALOX5 protein.

[0046] Figure 3 Nanobody (KD = 4.35 × 10-8 M) Biolayer interferometry analysis.

[0047] Figure 4 5-HETE levels in the culture medium of GL261 and CT2A cells stimulated with AAV-ALOX5-Nb or AAV-Cont-Nb (n=6); * in the figure indicates significant difference (p<0.05).

[0048] Figure 5 In vivo bioluminescence imaging of glioma-bearing mice at the indicated time points.

[0049] Figure 6 The figure shows the survival curve of mice (n=10); * in the figure indicates significant difference (p<0.05).

[0050] Figure 7 is the level of 5-HETE in tumor (n=6); * in the figure indicates significant difference (p<0.05).

[0051] Figure 8 Immunofluorescence staining of CD206+M2-GAMs in tumors.

[0052] Fig. 9 This is a bar graph of F4 / 80+CD11b+ flow cytometry analysis; * in the figure indicates significant difference (p<0.05).

[0053] Fig.10 This is a flow cytometry analysis of F4 / 80+CD11b+.

[0054] Fig.11 Figure 3 is a bar graph of CD86+ and CD206+ flow cytometry analysis; Figure A is a bar graph of CD86+ flow cytometry analysis; Figure B is a bar graph of CD206+ flow cytometry analysis; * represents a significant difference (p<0.05).

[0055] Fig.12 This is a CD86+ flow cytometry analysis diagram.

[0056] Fig.13 This is a CD206+ flow cytometry analysis diagram. DETAILED DESCRIPTION

[0057] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa. Unless the context clearly indicates otherwise, the expressions "a" and "an" used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents known to those skilled in the art, etc.

[0058] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0059] To make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning Experiment Guide (Fourth Edition)" (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience.

[0060] Example 1 ALOX5 antigen protein expression

[0061] 1.1 ALOX5 antigen protein expression

[0062] The ALOX5 protein sequence (P09917) was obtained from the Uniprot database and the expression sequence was constructed into the pfastbac vector (operated by General Bio (Anhui) Co., Ltd.). The white spot clones (such as Figure 1 As shown), in vitro culture, using the bacmid small-scale extraction kit (purchased from Biyuntian, catalog number D0031) to extract bacmid, PCR identification and confirmation, bacmid liposome packaging transfection method (LipoInsect TMTransfection reagent, purchased from Bio-Tech, catalog number C0551) was used to transfect insect cells sf9 (ATCC, CRL-1711). The insect cell culture and expression process were performed according to the Bac-to- The Baculovirus Expression System was used. During the expression process, the cells were observed to be lysed under a microscope, indicating that the baculovirus infection was successful and the expression process was normal. Finally, 200 mL of the expressed cells were collected for purification.

[0063] 1.2 ALOX5 antigen protein purification

[0064] The reagents and components used for ALOX5 antigen protein purification are shown in Table 1.

[0065] Table 1 Reagents and components used for ALOX5 antigen protein purification

[0066]

[0067] The expressed insect cells sf9 were collected by centrifugation at 4000 rpm. The bacteria were resuspended in Lysis buffer and ultrasonically disrupted under the following conditions: 300 W, work 3s, off 5s, time 30 min. The ultrasonic sample was centrifuged at 4°C and 18000 rpm for 15 min. The supernatant obtained above was purified by Ni affinity chromatography to obtain ALOX5 antigen protein. The specific steps are as follows:

[0068] a) Add 1 mL NiResinFF (GenScript, L00465) and incubate at 4°C for 1 h;

[0069] b) After incubation, the sample was loaded onto the column and flowed through the nickel column at a rate of 0.5 mL / min;

[0070] c) After the flow-through is completed, rinse the column with 150 mL Wash buffer;

[0071] d) After washing away the impurities, elute the target protein with 10 mL of Luteobuffer;

[0072] e) Take samples step by step and perform SDS-PAGE to detect the purification results of the target protein. The results are shown in Figure 2 .

[0073] Example 2 Preparation of Nanobodies Targeting ALOX5

[0074] 2.1 First round of screening

[0075] (1) Take out the ALOX5 antigen protein for screening from the -80°C freezer and place it on ice to thaw;

[0076] (2) The ALOX5 antigen protein was coated on the immunotube (50 μg / tube, the coating solution was CBS, pH 9.6, 2 mL / tube), and slowly rotated at 4°C overnight. At the same time, 50 μg 3% BSA was coated in parallel as a control;

[0077] (3) Discard the liquid in the overnight coated immunotube, add 2 mL of PBS buffer and wash the immunotube three times at room temperature, rotating for 5 min each time;

[0078] (4) Add 2 mL of blocking solution (3% PBSTB) and rotate at room temperature for 2 h;

[0079] (5) Discard the liquid in the sealed immunotube and add 2 mL of PBS buffer to wash the immunotube three times at room temperature, rotating for 5 min each time;

[0080] (6) Discard the washing solution in the immunotube, add 2 mL of PBS buffer, calculate according to the following formula and add 50 μL of the prepared phage library as the first round of screening input phage library, and incubate at room temperature for 1 h with rotation:

[0081]

[0082] Where, V is the volume of phage added (in μL), T library is the phage titer;

[0083] (7) Discard the liquid in the immunotube, add 2 mL of PBST (1×PBS plus 0.1% Tween20) buffer and wash the immunotube 20 times at room temperature, rotating for 5 min each time;

[0084] (8) Discard the liquid in the immunotube, remove as much residual liquid as possible, add 1 mL of 0.25 mg / mL Trypsin solution, and rotate and elute at room temperature for 30 min;

[0085] (9) Add 10 μL of 10% AEBSF to terminate 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.

[0086] 2.2 First round of phage eluate titer detection

[0087] (1) The SS320 strain (purchased from Antibody Design Labs, catalog number PC002) stored in a -80°C freezer was streaked with single colonies on 2×YT solid medium (containing 100 μg / mL Tet) and cultured overnight at 37°C (stored at 4°C for one week). A single colony was picked from the single colony plate and transferred to 5 mL of 2×YT medium (containing 100 μg / mL Tet) and cultured overnight at 37°C;

[0088] (2) Transfer 250 μL of overnight culture to 5 mL of 2×YT liquid medium (containing 100 μg / mL Tet) and culture at 37°C, 250 rpm for about 45 min-60 min until OD 600 0.5-0.55;

[0089] (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 , oscillate to mix;

[0090] (4) Add 90 μL of SS320 bacterial solution to each dilution centrifuge tube, mix well and incubate at 37°C for 30 min;

[0091] (5) Take 5 μL from each dilution centrifuge tube and add it dropwise to 2×YT solid medium (containing 100 μg / mL Amp) and culture it upside down at 37°C overnight;

[0092] (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:

[0093] T (pfu / mL) = N × D × 400

[0094] Wherein, T is the phage titer (unit: pfu / mL), D is the dilution multiple, and N is the number of single colonies at the corresponding dilution multiple.

[0095] 2.3 Amplification of the first round of phage eluate

[0096] (1) The SS320 strain stored at -80°C was streaked with single colonies on 2×YT solid medium (containing 100 μg / mL Tet) and cultured overnight at 37°C (stored at 4°C for one week). A single colony was picked from the single colony plate and transferred to 5 mL of 2×YT medium (containing 100 μg / mL Tet) and cultured overnight at 37°C.

[0097] (2) Transfer 250 μL of overnight culture to 5 mL of 2×YT liquid medium (containing 100 μg / mL Tet) and culture at 37°C, 250 rpm for about 45 min-60 min until OD 600 The value is 0.5-0.55;

[0098] (3) Add 500 μL of phage eluate obtained after the first round of screening until the OD 600The bacterial solution is 0.5-0.55 (the remaining eluate is stored at 4°C);

[0099] (4) Continue culturing at 37°C, 250 rpm for 30 min;

[0100] (5) Spread the entire bacterial solution evenly on a 150 mm circular culture medium plate containing 100 μg / mL Amp and 2% agarose and culture overnight at 37°C

[0101] (6) Take the circular plate cultured overnight, 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 spreader stick 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 OD value of the bacterial solution. 600 The value is the OD of the eluate bacterial library 600 value, and add glycerol with a final concentration of 20%, which is the first round of bacterial library.

[0102] (7) The amount of bacterial solution of the eluate bacterial library was calculated according to the following formula and transferred to 100 mL of 2×YT liquid culture medium (containing 100 μg / mL Amp and 100 μg / mL Tet) to make the initial OD 600 is 0.1:

[0103]

[0104] Where, V is the volume of the inoculated bacterial solution (in μL), OD 600 OD of the constructed eluate bacterial library 600 ;

[0105] (8) Cultivate at 37°C, 250 rpm until the bacterial solution OD reaches 0. 600 Reach 0.5-0.55;

[0106] (9) Calculate and add helper phage M13K07 according to the following formula so that the number of bacteria: number of phages = 1:20:

[0107]

[0108] Where V is the volume of helper phage added (in mL), T helper-phage is the helper phage titer used, OD 600 is the OD of bacterial solution 600 value;

[0109] (10) Continue culturing at 37°C, 250 rpm for 30 min.

[0110] (11) Kana and 0.2 mM IPTG were added to the cells at a final concentration of 50 μg / mL and cultured overnight at 30°C and 250 rpm.

[0111] 2.4 First round of phage purification

[0112] (1) Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 rpm and 4°C for 10 min;

[0113] (2) Transfer the supernatant after centrifugation to a new 50 mL centrifuge tube, add 1 / 4 volume of 4°C precooled 20% PEG / 2.5 M NaCl, mix thoroughly, and place on ice for 30 min;

[0114] (3) Centrifuge at 4000 rpm and 4°C for 20 min, discard the supernatant, and remove as much residual liquid as possible;

[0115] (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 13,000 rpm and 4°C for 20 min;

[0116] (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;

[0117] (6) Centrifuge at 13000 rpm, 4°C for 10 min, discard the supernatant, and add 1 mL of PBS to resuspend the precipitate;

[0118] (7) Centrifuge at 13000 rpm and 4°C for 2 min. Transfer the supernatant to a new 1.5 mL centrifuge tube for the first round of screening of the phage sub-library. Aliquot 100 μL / tube and store at -80°C for long-term storage or at -20°C for short-term storage (1-2 weeks).

[0119] (9) Titer detection of the first round of phage sub-library screening, the method is the same as "2.2 Titer detection of the first round of phage eluate".

[0120] 2.5 Second round of screening

[0121] The screening method is the same as "2.1 First Round Screening". 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 for the second round of screening to obtain the phage eluate for the second round of screening.

[0122] 2.6 Second round of phage eluate titer detection

[0123] The method is the same as “2.2 First round of phage eluate titer detection”.

[0124] 2.7 Amplification and purification of the second round of eluate

[0125] The method is the same as “2.3 Amplification of the first round of phage eluate” and “2.4 First round of phage purification”.

[0126] 2.8 Second round of screening of phage sub-library titer detection

[0127] The method is the same as “2.2 First round of phage eluate titer detection”.

[0128] 2.9 Monoclonal ELISA detection

[0129] (1) The SS320 strain stored at -80°C was streaked with single colonies on 2×YT solid medium (containing 100 μg / mL Tet) and cultured overnight at 37°C (stored at 4°C for one week). A single colony was picked from the single colony plate and transferred to 5 mL of 2×YT medium (containing 100 μg / mL Tet) and cultured overnight at 37°C.

[0130] (2) Transfer 250 μL of overnight culture to 5 mL of 2×YT liquid medium (containing 100 μg / mL Tet) and culture at 37°C, 250 rpm for about 45 min-60 min until OD 600 The value is 0.5-0.55;

[0131] (3) Take 10 μL of the phage eluate after the third 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;

[0132] (4) Add 90 μL OD 600 The bacterial solution with a value of 0.5-0.55 is mixed evenly;

[0133] (5) Continue culturing at 37°C, 250 rpm for 30 min;

[0134] (6) Spread the bacterial solution evenly on a solid culture medium plate containing 100 μg / mL Amp and culture at 37°C overnight;

[0135] (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 of 2×YT medium (containing 100 μg / mL Amp and 100 μg / mL Tet) to each well and culture at 37°C overnight.

[0136] (8) Take 2 μL of the overnight cultured bacterial 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 100 μg / mL Tet) per well. Incubate at 37°C for 5 h. Store the overnight cultured bacterial solution at 4°C before transfer.

[0137] (9) Calculate according to the following formula and add helper phage M13K07 to each well so that the number of bacteria: number of phage = 1:20;

[0138]

[0139] Where V is the volume of helper phage added (in mL), T helper-phage is the helper phage titer used;

[0140] (10) Incubate at 37°C for 30 min, add Kana at a final concentration of 50 μg / mL and 0.2 mM IPTG, and incubate at 30°C overnight;

[0141] (11) After overnight culture, the 96-well culture plate was centrifuged at 4°C, 4000 rpm for 10 min and stored at 4°C for later use;

[0142] (12) The screening antigen was coated on an ELISA plate (1 ng / μL, coating solution was CBS, pH 9.6, 100 μL / well), and BSA was coated in parallel as a control at 4°C overnight;

[0143] (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 min;

[0144] (14) Add 200 μL of blocking solution (3% BSA) to each well to block the ELISA plate and block at room temperature for 1 h;

[0145] (15) Discard the blocking solution, add 200 μL PBST (1× PBS plus 0.1% Tween 20) buffer to each well, and wash the ELISA plate three times at room temperature, each time for 10 min;

[0146] (16) Add 120 μL of 3% BSA and (after adding 0.02% Tween20) to each well and then add 80 μL of the supernatant after centrifugation in step (11) and incubate at room temperature for 2 h;

[0147] (17) Discard the liquid in the ELISA plate and add 200 μL PBST buffer to each well and wash three times for 10 min each time;

[0148] (18) Add M13 Bacteriophage Antibody (HRP) and Mouse Mab to each well, diluted 1:40,000 in blocking buffer, 100 μL / well, and incubate at room temperature for 1 h;

[0149] (19) Discard the liquid in the ELISA plate and add 200 μL PBST buffer to each well and wash three times for 10 min each time;

[0150] (20) Add 100 μL of TMB single-component colorimetric solution to each well and color for 2-3 min in the dark. Add 100 μL of 1 M HCl to each well to terminate the colorimetric reaction. Read the OD value using a microplate reader. 450 value, record and save.

[0151] 2.10 Secondary verification of positive clones by ELISA

[0152] 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 Monoclonal ELISA Detection”.

[0153] 2.11 Sequencing of positive clones

[0154] Select positive monoclones based on ELISA test data and secondary validation data. Take 5 μL of positive clones from the monoclonal ELISA test plate and inoculate into 1 mL of 2×YT medium (containing 100 μg / mL Amp and 100 μg / mL Tet), and culture at 37°C, 250 rpm until OD 600 When the concentration reaches 0.8-1.0 (about 6-8h), 0.5mL of bacterial solution was taken for sequencing and the rest of the bacterial solution was stored at 4℃.

[0155] 2.12 Sequence analysis

[0156] The sequenced sequences were compared and analyzed using GENtle software, and the antibody sequences were translated into amino acids using GENtle software.

[0157] 2.13 Results of three rounds of screening

[0158] The results of three rounds of screening are shown in Table 2. The results showed that after three rounds of screening, ELISA monoclonal verification was performed.

[0159] Table 2 Three rounds of screening

[0160] Screening rounds Input valence Screening titer Enrichment Round 1 <![CDATA[1×10 12 pfu / mL]]> <![CDATA[2×10 8 pfu / mL]]> <![CDATA[2×10 -4 ]]> Round 2 <![CDATA[2×10 12 pfu / mL]]> <![CDATA[4×10 8 pfu / mL]]> <![CDATA[2×10 -4 ]]> Round 3 <![CDATA[7×10 12 pfu / mL]]> <![CDATA[2.8×10 10 pfu / mL]]> <![CDATA[4×10 -3 ]]>

[0161] Example 3 Affinity and specificity of ALOX5 nanobody

[0162] The DNA sequence of nanobodies (Nbs) is shown in SEQ ID NO.9:

[0163] GCCGTGCAACTGGTTGAGAGCGGCGGTGGCCTGGTGCAAGCGGGTGGTAGCCTGCGCCTGAGCTGCGAAGCGAGCCGCCTGACCTTTAGCATTTATGCGATGGGCTGGTTTCGCCAAGCGCCGGGCAAAGAACGCGAATTTGTGGCGGGCCATAGCAGTAGCGGCGGCAACACCTATTATAAAGATAGCGT GGAAGGCCGCTTTACCATTAGCCGCGATAACGCGAAAAACACCCTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTATTGCGCGGCGAGCCTGCGCGGCTATAGCCGCGTGGTGCCGACCACCGGCGGCGAATATCCGTATTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGC.

[0164] The DNA sequence of Nbs was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. and subcloned into the pET22b plasmid to obtain a recombinant plasmid. Place 100 μL of competent BL21 Escherichia coli strain (purchased from Thermo Fisher, catalog number EC0114) on ice for 30 minutes, add 2 μL of recombinant plasmid and flick the bottom of the tube to mix, and continue to place on ice for 20 minutes. Transfer the Ep tube to 42°C for heat shock for 45 seconds and quickly transfer to ice for 5 minutes. Add 900 μL of LB liquid culture medium without Amp, 200 rpm, and culture at 37°C for 1 hour. Transfer all bacteria to an LB plate containing 100 μg / mL Amp and culture overnight at 37°C.

[0165] Pick the above cultured monoclonal bacteria into LB medium containing 100 μg / mL ampicillin and culture at 37°C until OD 600 Reach 0.6-0.8. Induce protein expression with 0.25 mM isopropyl β-D-1-thiogalactopyranoside at 16°C for 20 hours. Use ultrasonic lysis method to extract protein, use Vibra cell TM probe in ice with alternating cycle for 15-30 minutes (3 second pulse every 8 seconds). Purify protein using Ni-NTA affinity chromatography.

[0166] BLI: OctetRED 96 system (ForteBio, Shanghai) was used to detect the affinity and specificity of ALOX5 nanobody by BLI. The detection process included five steps: baseline (2 min), loading (2 min), washing (3 min), binding (5 min) and dissociation (5 min). The baseline solution (200 μL, pH 7.5, 50 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl 2 ), loading solution (i.e., baseline solution), washing solution (i.e., baseline solution), binding solution (i.e., baseline solution), and dissociation solution (i.e., baseline solution) were added to the corresponding wells of a 96-well microplate. The reaction data were normalized using Octet Data Analysis Software CFR Part 11 Version 6.x.

[0167] Nanobodies were detected by BLI. The results of BLI detection are shown in Figure 3 The results showed that the nanobody could effectively bind to the ALOX5 antigen with an affinity of 4.35×10 -8 M.

[0168] Example 4 Application of ALOX5-targeted nanoantibodies in the treatment of gliomas

[0169] 4.1 Preparation of AAV-ALOX5-Nb and AAV-Cont-Nb

[0170] The genes expressing Nb and Renilla luciferase were recombined into the AAV9 vector. The AAV9-Nb expression vector was synthesized by VectorBuilder, Inc., headquartered in Illinois, USA. The virus packaging was obtained by the three-plasmid transfection method, and all related operations were completed by VectorBuilder, Inc. AAV-ALOX5-Nb and AAV-Cont-Nb were synthesized by VectorBuilder, Inc. The vector sequence is shown in SEQ ID NO.10, and the bold underlined part is the nanobody coding sequence:

[0171]

[0172]

[0173]

[0174]

[0175] 4.2 In vitro activity of AAV-ALOX5-Nb

[0176] The culture conditions of GL261 cells (purchased from ATCC, catalog number CRL-2611) and CT2A cells (purchased from Millipore, catalog number SCC194) were: DMEM+10% FBS+1% P / S.

[0177] 5 μL, 1×10 12 After GL261 and CT2A cells were treated with GC / mL of AAV-ALOX5-Nb and AAV-Cont-Nb for 48 h, the culture medium was replaced with DMEM+10% FBS+1% P / S for a further 24 h, and the 5-HETE level in the culture medium was determined by HPLC-MS.

[0178] The results of the test are shown in Figure 4 The results showed that AAV-ALOX5-Nb could significantly downregulate 5-HETE levels in the culture medium of GL261 and CT2A cells.

[0179] 4.3 Orthotopic GBM mouse model and drug administration

[0180] The present invention uses SPF-grade healthy C57BL / 6 mice of 6-8 weeks old and weighing about 20 g, half male and half female, purchased from Guangdong Gempharmatech-GD Company.

[0181] Establishment of GBM orthotopic mouse model: C57BL / 6 mice were randomly divided into two groups (AAV-ALOX5-Nb group and AAV-Cont-Nb group). 4 CT2A glioma cells were orthotopically implanted into the mouse skull. CT2A cells (purchased from Sigma-Aldrich, catalog number SCC194) were pre-infected with firefly luciferase lentivirus.

[0182] Administration: On the day of modeling, mice in the AAV-ALOX5-Nb group were intravenously injected with 100 μL of 1×10 13 GC / kg of AAV-ALOX5-Nb; the AAV-Cont-Nb group was injected with an equal volume of AAV-Cont-Nb.

[0183] (1) In vivo bioluminescence imaging and survival rate

[0184] Tumor growth was monitored by IVIS Spectrum in vivo imaging system (PerkinElmer) after intraperitoneal injection of luciferin (150 mg / kg; catalog number P1043; Promega). Figure 5 The survival curve of mice is shown in Figure 6 The results showed that AAV-ALOX5-Nb could significantly inhibit tumor growth and improve the survival rate of GBM orthotopic mice.

[0185] (2) 5-HETE levels in tumors

[0186] After the mice died, the orthotopic tumors were removed and the 5-HETE level in the tumors was detected by HPLC-MS. Figure 7 As shown, the results showed that AAV-ALOX5-Nb could significantly inhibit the level of 5-HETE in tumors.

[0187] (3) IF staining of CD206

[0188] For IF staining of CD206 on mouse tumors, antigen retrieval was performed on deaffinity and rehydrated sections in sodium citrate buffer (Catalog #G1219; Servicebio), followed by blocking with 10% fetal bovine serum (FBS) and 3% bovine serum albumin (BSA) in PBS. After incubation with anti-CD206 (1:200, Catalog #24595; Cell Signaling Technology) antibody at 4°C overnight, sections were stained with red fluorescent anti-rabbit IgG (1:500; Catalog #a-11012; ThermoFisherScientific) for 1 hour, followed by DAPI (Catalog #P36931; ThermoFisher Scientific) staining for 10 minutes at room temperature.

[0189] The staining results are shown in Figure 8 The measurement results showed that the CD206 content in the tumors of mice in the AAV-ALOX5-Nb group was significantly reduced.

[0190] (4) Flow cytometry analysis

[0191] Tumor tissues obtained from mice bearing orthotopic gliomas were separated into single-cell suspensions by filtration through a 40 μm cell strainer and stained with the indicated antibodies. The stained cells were subjected to FCM analysis using a Sony SA3800 analyzer.

[0192] F4 / 80+CD11b+ flow cytometry results are shown in Figure 9-10 The content of F4 / 80+CD11b+TAMs cells in the tumor tissues of mice in the AAV-ALOX5-Nb group was significantly decreased compared with that in the AAV-Cont-Nb group.

[0193] CD86+ and CD206+ flow cytometry results are shown in Figure 11-13 The measurement results showed that compared with the AAV-Cont-Nb group mice, the proportion of CD86+ M1 cells in TAMs of tumor tissues of the AAV-ALOX5-Nb group mice was significantly increased, while the proportion of CD206+ M2 cells was significantly decreased.

[0194] 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. A nanobody targeting ALOX5, characterized in that: The nanobody comprises: (1) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO. 1-3; or (2) An amino acid sequence having at least one amino acid difference compared to SEQ ID NO. 1-3.

2. The Nanobody according to claim 1, characterized in that The nanobody comprises: 1) FR1, FR2, FR3 and FR4 of the amino acid sequence as shown in SEQ ID NO.4-7; or 2) An amino acid sequence having at least one amino acid difference compared to SEQ ID NO. 4-7.

3. The Nanobody according to claim 1, characterized in that The nanobody has an amino acid sequence as shown in SEQ ID NO.

8.

4. An antibody preparation, characterized in that The antibody preparation comprises the Nanobody according to any one of claims 1 to 3 and at least one pharmaceutically acceptable carrier.

5. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Nanobody according to any one of claims 1 to 3.

6. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that The host cell comprises the expression vector according to claim 6.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody according to any one of claims 1 to 3, the antibody preparation according to claim 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6 or the host cell according to claim 7.

9. Use of the Nanobody according to any one of claims 1 to 3, the antibody preparation according to claim 4, the nucleic acid molecule according to claim 5, the expression vector according to claim 6, the host cell according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a drug for preventing or treating tumors.

10. The use according to claim 9, characterized in that: The tumors include glioma, prostate cancer, ovarian cancer, lung cancer, colorectal cancer, liver cancer, bladder cancer, breast cancer, pancreatic cancer, kidney cancer, gastric cancer, and cervical cancer.

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