A nanobody or antigen-binding fragment against the pan-lysyl oxidase family and its application

Abstract

The application belongs to the field of medicine and biology, and relates to an anti-pan-lysyl oxidase family nanobody or antigen binding fragment and application. The anti-pan-lysyl oxidase family nanobody or antigen binding fragment comprises three complementarity determining regions CDR1, CDR2 and CDR3; wherein the amino acid sequence of CDR1 is a sequence shown in one of SEQ ID NO:1 to SEQ ID NO:8, the amino acid sequence of CDR2 is a sequence shown in one of SEQ ID NO:9 to SEQ ID NO:17, and the amino acid sequence of CDR3 is a sequence shown in one of SEQ ID NO:18 to SEQ ID NO:26. The application uses a flow washing method to screen a library, and finally obtains an anti-human / mouse anti-pan-lysyl oxidase family nanobody gene sequence. The anti-pan-lysyl oxidase family nanobody of the application has small molecular weight, high affinity and good specificity. The anti-pan-lysyl oxidase family nanobody has good prospects for development as a diagnostic fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biology, specifically relating to an anti-pan-lysyl oxidase family nanobody or antigen-binding fragment and its application. Background Technology

[0002] The lysyl oxidase (LOX) family, comprising LOX and four LOX-like proteins (LOXL1-4), promotes covalent cross-linking between newly formed collagen proteins, primarily involved in extracellular matrix remodeling and cross-linking of collagen and elastic fibers. All members of the lysyl oxidase family share a conserved catalytic domain, suggesting similar functions in collagen cross-linking.

[0003] The LOX family promotes the oxidative deamination of lysine residues, thereby cross-linking collagen and elastin, affecting collagen fiber stiffness, and participating in extracellular matrix remodeling in tumors and cardiovascular-related diseases. Changes in its expression and activity can lead to various diseases and are considered biomarkers for the progression of various diseases, such as liver, lung, and heart fibrosis, scleroderma, and cancer. For example, in tumors, LOX overexpression can induce epithelial-mesenchymal transition. LOXL2 can drive liver cancer cell proliferation, migration, invasion, and cell survival. LOXL4 promotes cancer cell death by activating impaired wild-type p53. LOX family members can function as extracellular matrix enzymes or as intracellular proteins involved in signal transduction or epigenetic regulation. Therefore, the LOX family can serve as potential targets for the diagnosis and treatment of tumors and fibrosis-related diseases. Currently, several pan-LOX family targeted inhibitors are under investigation, including PXS-5505, which can inhibit the activity of LOX family enzymes in tissues and has entered phase I / II clinical trials for the treatment of myelofibrosis.

[0004] Nanobodies are the smallest known antigen-binding fragments, only one-tenth the size of monoclonal antibodies, and possess structural stability and binding activity comparable to proto-heavy-chain antibodies. Compared to traditional antibodies, nanobodies have several unique advantages, such as excellent tissue penetration, rapid clearance, ease of production and modification, low immunogenicity, and high stability, making them a promising new type of antibody molecule. Nanobodies combine the advantages of small-molecule peptides and traditional antibodies, showing broad application prospects and clinical value in targeted therapy and precision diagnostics; however, very few antibodies have yet entered clinical trials.

[0005] There are no reports or clinical applications of nanobodies targeting pan-LOX family targets, so there is an urgent need in this field to develop new and effective specific nanobodies targeting pan-LOX family targets. Summary of the Invention

[0006] definition

[0007] Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art as would be understood by those skilled in the art. Furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as would be understood by those skilled in the art.

[0008] Unless otherwise stated, the interchangeable terms “antibody” or “immunoglobulin” used herein, whether referring to heavy-chain antibodies or conventional four-chain antibodies, are used generally to include full-length antibodies, their individual chains, and all their portions, domains, or fragments (including, but not limited to, antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). Furthermore, the term “sequence” as used herein (e.g., in the terms “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence,” etc.) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding said amino acid sequence, unless a more specific interpretation is required herein.

[0009] The purpose of this invention is to provide an anti-human / mouse nanobody that blocks the cross-linking of pan-LOX family fibers, and to provide the coding sequence of the nanobody, as well as the preparation method and application of the nanobody.

[0010] To achieve the above objectives, a first aspect of the present invention provides a broad-spectrum anti-pan-lysyl oxidase (LOX) family nanobody or antigen-binding fragment comprising three complementarity-determining regions CDR1, CDR2, and CDR3; wherein,

[0011] The amino acid sequence of CDR1 is one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4;

[0012] The amino acid sequence of CDR2 is the sequence shown in one of SEQ ID NO: 5 to SEQ ID NO: 8;

[0013] The amino acid sequence of CDR3 is shown in one of SEQ ID NO: 9 to SEQ ID NO: 12.

[0014] In this invention, "having the same function" means that it can bind to pan-LOX family proteins, which include LOX proteins and LOX-like proteins 1-4. "Being able to bind to pan-LOX family proteins" means that it has the ability to bind to each protein in the family.

[0015] The location of the CDR in the antibody or nanobody sequence can be determined by those skilled in the art using existing techniques. Typically, the CDR can be identified by sequencing the DNA of the antibody or nanobody, and the resulting sequence can then be analyzed using a specialized database, such as the international ImMunoGeneTics database or IMGT.

[0016] In the sequence provided by this invention, CDR is drawn according to IMGT (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results).

[0017] According to a preferred embodiment of the present invention, the anti-pan-LOX family nanobody or antigen-binding fragment has any of the following CDR sequence characteristics:

[0018] (1) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9, respectively;

[0019] (2) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10, respectively;

[0020] (3) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 11, respectively;

[0021] (4) The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO:4, SEQ ID NO:8 and SEQ ID NO:12, respectively.

[0022] According to the present invention, in addition to the aforementioned complementarity-determining regions, the anti-pan-LOX family nanobody or antigen-binding fragment further comprises four frame regions FR1, FR2, FR3, and FR4 alternately arranged with the three complementarity-determining regions, wherein the amino acid sequence of FR1 is the sequence shown in any one of SEQ ID NO: 13 to SEQ ID NO: 16; the amino acid sequence of FR2 is the sequence shown in any one of SEQ ID NO: 17 to SEQ ID NO: 20; the amino acid sequence of FR3 is the sequence shown in any one of SEQ ID NO: 21 to SEQ ID NO: 24; and the amino acid sequence of FR4 is the sequence shown in any one of SEQ ID NO: 25 to SEQ ID NO: 26.

[0023] According to a preferred embodiment of the present invention, the anti-pan-LOX family nanobody or antigen-binding fragment has any of the following FR sequence characteristics:

[0024] (a) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, and SEQ ID NO: 25, respectively;

[0025] (b) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 22, and SEQ ID NO: 26, respectively;

[0026] (c) The amino acid sequences of FR1, FR2, FR3, and FR4 are the sequences shown in SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 23, and SEQ ID NO: 25, respectively;

[0027] (d) The amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

[0028] This invention includes all sequences that satisfy the above sequence characteristics. Specifically, preferably, the nanobody or antigen-binding fragment comprises one of the following sequences:

[0029] (i) An amino acid sequence as shown in any one of SEQ ID NO: 27 to SEQ ID NO: 30;

[0030] (ii) An amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence shown in any one of SEQ ID NO: 27 to SEQ ID NO: 30 and having the same function;

[0031] (iii) An amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted in the amino acid sequence shown in any one of SEQ ID NO: 27 to SEQ ID NO: 30, and retains the function of the amino acid sequence shown in any one of SEQ ID NO: 27 to SEQ ID NO: 30.

[0032] 317-ph-1-D12: AVQLVDSGGGLVQAGDSLRLSCAASGVAFTRSNMG WFRQVPGKEREFVAAISWSGGGKYYSDPVRGRFTISRDNAKGTVYLQMDNLKPEDTAVYYCAASGRVATFGEYDYWGQGTQVTVSS (SEQ IDNO: 27).

[0033] 318-3-E10: QLQLVESGGGFVQAGGSLRLSCAASRRIFSINAMGWFRQ APGKEREFVAAVTSGGSTHYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRGWHYGMDYWGKGTLVTVSS (SEQ ID NO: 28).

[0034] 318-ph-2-C06: QVKLEESGGGLVQAGGSPRLSCAVSGSIFSLFAMGWY RQAPGKQRELVAAVSSIGTTNYADSVKGRFTISRDNAKNTLYLRMNSLKPEDTAVYYCAADRGRGSWYRAQLDYWGQGTQVTVSS (SEQ IDNO: 29).

[0035] 317-ph-3-C04:AVQLVESGGGLVQPGGSLRLSCAASGSIFSTDVMNW YRQVPGKRRGLVARITDDDATAYVDSVLGRFTISKDNAKNTVYLQMNSLKPEDTAVYYCNVRSRVRWGLITDYWGQGTQVTVSS (SEQ ID NO: 30).

[0036] A second aspect of the present invention provides a nucleic acid molecule or carrier, said nucleic acid molecule encoding the above-described anti-pan-LOX family nanobody or antigen-binding fragment; said carrier comprising said nucleic acid molecule.

[0037] A third aspect of the present invention provides a host cell containing the nucleic acid molecule or carrier described in the second aspect. The host cell includes, but is not limited to, bacterial cells, fungal cells, animal cells, plant cells, or progeny cells of these cells.

[0038] The anti-pan-LOX family nanobody of the present invention can be obtained by the following methods:

[0039] (1) Yeast display library was prepared by immunizing alpacas with human / mouse LOXL2 eukaryotic protein.

[0040] (2) Use human / mouse pan-LOX family protein affinity screening of yeast display libraries;

[0041] (3) ELISA identification of positive clones;

[0042] (4) Expression and purification of pan-LOX family nanobodies.

[0043] According to one specific embodiment, the present invention first immunized two adult healthy alpacas with the LOXL2 protein. After three consecutive immunizations, peripheral blood was extracted from the alpacas to separate peripheral blood lymphocytes, and an immunotherapy library of pan-LOX family single-domain heavy chain antibodies was constructed. Then, magnetic sorting and flow cytometry were used to screen the immunotherapy library with human / mouse pan-LOX family proteins to obtain single-domain heavy chain antibodies targeting the pan-LOX family, thereby obtaining a highly efficient nanobody strain.

[0044] A fourth aspect of the present invention provides a method for engineered production of anti-pan-LOX family nanobodies, comprising the steps of: (a) culturing the host cells under conditions suitable for nanobodies production to obtain a culture containing the anti-pan-LOX family nanobodies; (b) isolating and / or recovering the anti-pan-LOX family nanobodies from the culture; and optionally, (c) purifying and / or modifying the anti-pan-LOX family nanobodies obtained in step (b).

[0045] A fifth aspect of the present invention provides an antibody-drug conjugate comprising the aforementioned anti-pan-LOX family nanobody or antigen-binding fragment, a linker, and an effector; preferably, the effector comprises at least one of a radionuclide, a cytotoxic agent, a fluorescent group, an enzyme that catalyzes substrate color development, a chemiluminescent reagent, and a nanoparticle-based label. The radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide, preferably a diagnostic radionuclide. 18 F, 32 P, 33 P, 45 Ti、 47 Sc、 52 Fe、 59 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 75 Sc、 77 As、 86 Y、 90 Y、 89 Sr、 89 Zr、 94 Tc, 94 Tc, 99m Tc,99 Mo、 105 Pd, 105 Rh、 111 Ag、 111 ln、 123 I, 124 I, 125 I, 131 I, 142 Pr, 143 Pr, 149 Pm, 153 Sm、 154"1581 Gd, 161 Tb, 166 Dy、 166 Ho、 169 Er、 175 Lu、 177 Lu、 186 Re、 188 Re、 189 Re、 194 lr、 198 Au、 199 Au、 211 At、 211 Pb, 212 Bi、 212 Pb, 213 Bi、 223 Ra and 225 At least one of Ac; the therapeutic radionuclide is preferably... 32 P, 47 Sc、 57 Co、 89 Sr、 90 Y、 103 Pd, 106 Ru、 124 I, 125 I, 131 I, 131 Cs、 137 Cs、 177 Lu、 192 Ir、 212 Bihe 225 At least one of Ac.

[0046] A sixth aspect of the present invention provides a pharmaceutical composition comprising the above-described anti-pan-LOX family nanobody or antigen-binding fragment, or the above-described antibody-drug conjugate.

[0047] The above-mentioned pharmaceutical composition may also include one or more pharmaceutical excipients.

[0048] The seventh aspect of the present invention provides the following uses of the aforementioned anti-pan-LOX family nanobodies or antigen-binding fragments:

[0049] (i) Use in the preparation of reagents for detecting pan-LOX family-mediated diseases; said reagent is preferably a kit for detecting pan-LOX family-mediated diseases; or

[0050] (ii) Use in the preparation of medicaments for the diagnosis and / or treatment of pan-LOX family-mediated diseases; specifically, as a multimodal imaging agent for diagnosis, or as a fibrosis inhibitor;

[0051] The diseases mediated by the pan-LOX family are preferably cancer, connective tissue diseases, or cardiovascular diseases; the cardiovascular diseases are preferably at least one of atherosclerosis, heart failure, and myocardial ischemia; the cancers are preferably at least one of head and neck squamous cell carcinoma, breast cancer, lung cancer, colorectal cancer, gastric cancer, cervical cancer, liver cancer, esophageal cancer, pancreatic cancer, oral squamous cell carcinoma, and renal cell carcinoma; the connective tissue diseases are preferably at least one of cutis laxity and fibrotic diseases.

[0052] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0053] (1) In this invention, human / mouse LOXL2 eukaryotic protein is used to immunize alpacas, and the VHH gene sequence is amplified from alpaca peripheral blood lymphocytes to construct a nanobody phage display library. Through four rounds of phage library screening and single-domain antibody affinity detection with LOX family proteins, the gene sequence of anti-human / mouse pan-LOX nanobody is finally obtained.

[0054] (2) The phage display library constructed in this invention has a large capacity and the diversity of nanobodies obtained by screening is high.

[0055] (3) Compared with conventional antibodies, the anti-pan-LOX nanobody of the present invention has a small molecular weight (about 13kDa), high affinity, and good specificity. It has good prospects for development as a diagnostic tool for fibrosis and provides an alternative for anti-fibrosis treatment.

[0056] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0057] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0058] Figure 1 This is an RNA electrophoresis image. M: DNA marker; 1 is 317# triple-immune RNA, and 2 is 318# triple-immune RNA.

[0059] Figure 2 The image shows the electrophoresis results of VHH's first-round amplification; 1 and 2 are the amplification results of 317# and 318#, respectively.

[0060] Figure 3 The image shows the electrophoresis results of the second round of VHH amplification; 1 and 2 are the amplification results of 317# and 318#, respectively.

[0061] Figure 4 The amino acid alignment sequences for library diversity sequencing are shown.

[0062] Figures 5-1 to 5-8 The results of phage ELISA monoclonal assay are shown.

[0063] Figures 6-1 to 6-3 The results of the LOX family affinity test for candidate antibodies in the ELISA assay are shown, in which... Figure 6-1 and 6-2 The results show the affinity of the candidate antibody for LOXL2. Figure 6-3 Results showing the affinity of candidate antibodies for LOX, LOXL1, LOXL3, and LOXL4.

[0064] Figure 7 The EC50 of nanobody 318-ph-2-C06 against various antigens of the LOX family is shown. Detailed Implementation

[0065] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0066] Example 1: Preparation of LOXL2 recombinant protein

[0067] (1) The sequence information of LOXL2 (UniProtKB: Q9Y4K0-1) was retrieved from the UniProt database, optimized according to the original human codon preference, and a His tag was added to the C-terminus. The LOXL2 protein was subcloned into the pcDNA3.4 vector to construct a eukaryotic expression vector. After Sanger sequencing confirmed that the sequence was correct, plasmid extraction was performed. The amino acid sequence of the constructed human LOXL2 protein is shown in SEQ ID NO: 31, and the amino acid sequence of the mouse LOXL2 protein is shown in SEQ ID NO: 32.

[0068] (2) After plasmid extraction, the constructed LOXL2 protein eukaryotic expression vector was transiently transfected into HEK 293 cells and cultured for 5-7 days. The supernatant of the culture medium was collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube and purified using Ni packing material.

[0069] (3) SDS-PAGE was used to detect the purity of the target protein, and the purity was >90%.

[0070] Example 2: Construction of a single-domain heavy chain antibody immune library targeting human / mouse LOXL2

[0071] 1. Alpacas were immunized with human LOXL2 protein (317# and 318# respectively) every two weeks for a total of three consecutive immunizations. During the third immunization, mouse LOXL2 protein was also used to immunize the alpacas.

[0072] 2. After three immunizations, serum titers were detected using indirect ELISA.

[0073] Experimental Procedure: Dilute the antigen to 2 μg / mL with 0.05M carbonate buffer (pH 9.6), add 100 μL / well, and coat overnight at 4 °C. Discard the coating solution, wash three times with PBST, add 300 µL of 5% skim milk to each well, and block at 37 °C for 1 h. Wash three times with PBST, add 100 μL / well of serum diluent (serial dilution starting from 1:2000), and incubate at 37 °C for 45 min. Wash five times with PBST, add 100 μL / well of Goat anti-Alpaca IgG (H+L) HRP (Cat#: S001H, diluted 1:1W with PBS), and incubate at 37 °C for 45 min. Wash five times with PBST. Add TMB chromogenic solution for color development, 100 μL / well, and incubate at 37 °C for 5 min. Add stop solution to stop the reaction, 50 μL / well, and measure the optical density at 450 nm.

[0074] Experimental results:

[0075] Table 1. Serum titer detection for three types of immunity.

[0076]

[0077] After three vaccinations, the alpaca valence reached 1:64K (OD450>0.2). The valence of both alpacas was normal, and subsequent database construction and screening can proceed.

[0078] 3. After the third immunization, 50 mL of peripheral blood was collected. PBMCs were isolated according to the instructions for using the lymphocyte separation medium. Total RNA was then extracted, and cDNA was reverse transcribed to construct a phage library for screening. Using a single-domain antibody cloning primer combination, the VHH sequence was amplified from the cDNA sample and subcloned into the phage display vector pDisplay. This was then electrotransformed into SS320 competent cells to construct a single-domain antibody yeast display library. Specifically,

[0079] (1) Total RNA was extracted from PBMCs using Takara's RNAiso Plus reagent. 1 μg of RNA was electrophoresed to determine RNA purity. Figure 1As shown, the results indicate that the RNA purity is good. RNA was reverse transcribed into cDNA using the PrimeScript™ II 1st StrandcDNA Synthesis Kit. Specifically,

[0080] Prepare the following reaction mixture Mix1 in a 200 μL PCR tube: 8 μL Oligo dT Primer (50 μM), 8 μL dNTP Mixture (10 mM each), 20 μg total RNA sample, and RNase-free water (to 80 μL).

[0081] After holding at 65°C for 5 minutes, cool rapidly on ice.

[0082] Prepare the following reaction solution in the PCR tubes described above: 80 μL of the denatured reaction solution, 32 μL of 5×PrimeScript II Buffer, 4 μL of RNase Inhibitor (40 M / μL), 8 μL of PrimeScript II RTase (200 M / μL), and 36 μL of RNase-Free water.

[0083] After mixing by pipetting, dispense 80 μL / tube and place in a PCR instrument at 42°C for 1 hour, followed by heat inactivation at 70°C for 15 minutes. Finally, store the cDNA samples on ice or at -20°C for long-term storage.

[0084] (2) The VHH chain was amplified using PCR, which included two rounds of PCR.

[0085] Table 2 Nested PCR one-round reaction system

[0086]

[0087] Table 3. Reaction conditions for nested PCR in two rounds

[0088]

[0089] After the first round of nested PCR amplification was completed, all PCR amplification products were collected and subjected to 2% agarose gel electrophoresis. Figure 2 ), and tapped the rubber to recover approximately 750bp of VHH fragments.

[0090] Then, a second round of nested PCR was performed to amplify the target gene fragment, followed by 2% agarose gel electrophoresis. The results are as follows: Figure 3 As shown, the approximately 500 bp VHH fragment was further purified and recovered using a DNA product purification kit. The purified VHH fragment was stored at -80°C or below.

[0091] (3) The phage display vector pDisplay was linearized, and the enzyme digestion system was as follows: 140 μg pYDisplay, 200 μL 10×CutSmart® Buffer, 1200 U (60 μL) SfiI, and sterile water (to 2000 μL).

[0092] (3-1) Digest the pYDisplay vector with SfiI enzyme, aliquot 100 μL / tube, and digest overnight at 50°C;

[0093] (3-2) The pYDisplay vector fragment was separated using a 1% agarose gel, a 5000 bp fragment was excised and recovered from the gel, and the concentration was determined using NanoDrop.

[0094] (3-3) Aliquot 200 μL of the recovered pYDisplay enzyme digestion product into each 1.5 mL centrifuge tube, add 1 / 10 volume (20 μL) of 3 M sodium acetate and 1 μg / μL of glycogen, mix by pipetting and aspiration, add 880 μL of anhydrous ethanol, mix by inversion, freeze at -80°C for 2 hours to precipitate the product, resuspend the precipitated product with 60 μL of ddH2O to control the final product concentration at 1000-2000 ng / μL for subsequent electroporation library construction.

[0095] (4) Electroporation to construct a phage display library

[0096] (4-1) Add the pre-cooled library construction and ligation product to the electroporation competent cells and place them on ice for 1 minute. Add 300 μL of DNA / competent cell mixture to each electroporation cuvette and place the cuvette on ice. Perform electroporation at 2500 V for 5 ms. Immediately after electroporation, add SOC medium equilibrated to room temperature to 200 mL to resuspend the cells. Incubate at 37°C on a shaker for 1 hour. After centrifugation, resuspend the cells in 20 mL of 2YT (containing ampicillin) and incubate overnight.

[0097] (4-2) Take 15 mL of bacterial culture for phage rescue directly, and add an equal volume of 50% glycerol to the remaining 5 mL of electroporation product. Mix well and store at -80°C. Separately, take 20 μL of bacterial culture, add 980 μL of 2YT medium for dilution, take 100 μL of the diluted product, add 900 μL of 2YT medium for a second dilution, and take 100 μL to spread evenly on LB agar plates containing ampicillin. Incubate overnight at 37°C.

[0098] (4-3) On the second day, remove the plate and calculate the number of clones that each ligation can produce. Calculate the library capacity: Phage library capacity = Dilution factor 10000 (200mL, take 20μL) × Dilution factor 10 (20μL + 980μL, take 100μL) × Dilution factor 10 (100μL + 900μL, take 100μL) × Ampicillin plate clone count. At the same time, pick 20 single clones from the plate and put them into 2YT medium containing ampicillin. Incubate at 37°C with shaking for about 6-8 hours. Send the bacterial culture for sequencing (sequencing universal primer M13R) and calculate the library diversity.

[0099] (4-4) Dilute 15 mL of overnight cultured bacterial product with 2 YT to adjust the OD600 to approximately 0.25. Add ampicillin to a final concentration of 100 μg / mL and incubate in a constant temperature shaker at 37°C and 225 rpm until the OD600 reaches 0.6. Add M13KO7, shake well, and incubate at 37°C for 30 minutes, then incubate at 37°C and 225 rpm for 1 hour. M13KO7 volume = 10 × volume × OD600 × 5 × 10 8 / M13KO7 titer. After centrifuging the bacterial culture at 6000 rpm for 10 minutes, resuspend it in 200 mL of 2YT-AK medium and incubate overnight at 25°C and 200 rpm; centrifuge the bacterial culture at 10000 rpm for 15 minutes; discard the precipitate, transfer the supernatant to a new centrifuge tube, add 1 / 5 volume of PEG / NaCl to the tube, mix well, and incubate at 4°C for 2 hours;

[0100] (4-5) Centrifuge the precipitated phage supernatant at 10,000 rpm, 4°C for 30 minutes; discard the supernatant, and resuspend the precipitate (phage) in 1 mL sterile PBS for each 50 mL centrifuge tube; transfer the resuspended phage to a 1.5 mL EP tube, centrifuge at 12,000 x g, 4°C for 5 minutes; transfer the supernatant to a new 1.5 mL EP tube, add 250 μL of PEG / NaCl to each tube, mix well, incubate at 4°C for 10 minutes, centrifuge at 12,000 x g for 10 minutes, discard the supernatant, resuspend in 1 mL PBS, centrifuge at 12,000 x g for 5 minutes, discard the precipitate, transfer the supernatant to a new 1.5 mL EP tube, centrifuge at 12,000 x g for 5 minutes, and transfer the supernatant to a new 1.5 mL EP tube to obtain the original phage library. Take 10 μL of the precipitate and add it to 90 μL of 2YT medium, labeled as 10⁻¹. Dilute it 10-fold to 10⁻⁹. Take 20 μL of each of the three dilutions (10⁻⁷, 10⁻⁸, and 10⁻⁹) and add them to 200 μL of ER2738 with an OD600 of 0.5. Mix well and place in a 37°C water bath for 10 minutes. Spread 100 μL onto one LB-AMP solid plate and incubate overnight at 37°C. Count the spots the next day to determine the titer.

[0101] (4-6) Titer calculation: Select plates with a number of spots between 30 and 300, take the average value of two plates, multiply the number of spots by the dilution factor and then multiply by 100 to obtain the titer.

[0102] (4-7) Phage library diversity analysis: Randomly selected single clones were sequenced to analyze the diversity of the constructed phage display library. All sequences were antibody sequences with no identical sequences, indicating good library diversity. Alpaca #317 library size: 1.058 × 10⁻⁶. 9 317# Alpaca storage capacity: 1.217 × 10 9 Constructing a phage library, such as Figure 4 As shown.

[0103] Example 3: Screening, identification, and sequencing of anti-pan-LOX family nanobodies

[0104] 1. Alternating panning of solid-phase proteins in a phage library

[0105] (1) In a clean bench, add 500 μL of CBS solution to a 1.5 mL sterile EP tube, add 25 μg of LOXL2-His antigen to the CBS (the volume added is calculated based on the antigen concentration) to a final concentration of 50 μg / mL, invert the mixture to form a protein coating solution, add 120 μL of the protein mixture to a 96-well solid-phase plate, and coat 4 wells overnight at 4°C. Use a pipette to add 1.2 mL of 3% MPBS to a 1.5 mL sterile EP tube and incubate overnight at 4°C.

[0106] (2) On the second day, use a 200 μL pipette to remove the LOXL2-His protein solution from the 96-well plate. Replace the pipette tip and add 200 μL of sterile PBS solution to the LOXL2-His protein wells. Let it stand for 1 minute. Use a 200 μL pipette tip to remove the PBS solution from the wells. This is the first cleaning. Repeat the cleaning of the protein wells once with sterile PBS solution. Then add 200 μL of 3% MPBS buffer to each protein well. Place the 96-well plate in a sterile workbench at room temperature for 1 h to block it.

[0107] (3) Take 150 μL of the phage from the previous round of amplification, add it to a 1.5 mL sterile EP tube containing 350 μL of 1% PBSA, remove the MPBS solution of the protein, and transfer the phage solution to the well coated with LOXL2 protein. Incubate at room temperature with shaking for 1 h.

[0108] (4) Remove the phage from the LOXL2 protein wells, replace the pipette tip and add 200 μL of sterile 0.05% PBST solution to the LOXL2-His protein wells. Let stand for 1 minute, replace the pipette tip and use 200 μL of pipette tip to remove the 0.05% PBST solution from the wells. This is one wash. Wash 6 times with 0.05% PBST. Add 200 μL of sterile PBS solution to the LOXL2-His protein wells and let stand for 1 minute. Replace the pipette tip and use 200 μL of pipette tip to remove the PBS solution from the wells. This is one wash. Wash 4 times with PBS. At the same time, wash the pre-blocked 1.5 ml EP tube twice with 1 mL of PBS.

[0109] (5) Add 100 µL of pH 2.2 Gly-HCl elution buffer to each well, and incubate at 37°C with shaking for 8 minutes to elute the specifically bound phages; use a 200 μL pipette tip to transfer the elution buffer to a 1.5 mL sealed sterile centrifuge tube, and quickly aspirate 15 μL of Tris-HCl buffer to neutralize the elution buffer. This is one elution; repeat this step to elute the protein wells again, and store the eluted product at 4°C.

[0110] (6) The elution product is used for subsequent Phage infection.

[0111] Repeat the above steps four times to perform four rounds of selection.

[0112] The following table shows the panning results for the LOX family phage display libraries. Table 4: Calculation results of enrichment factors for phage library #317. Table 5: Calculation results of enrichment factors for phage library #318. The results show that the recovery rate increases with each panning round, indicating that the target phages are specifically enriched.

[0113] Table 4. Calculation results of enrichment factors for phage library #317

[0114]

[0115] Table 5. Calculation results of enrichment factors for phage library #318

[0116]

[0117] 2. Phage ELISA Single Clonal Detection

[0118] (1) Dilute the antigen protein with sterile CBS to a final concentration of 1 µg / mL. (1) Take a new 96-well microplate, add 100 μL / well, and coat at 4°C for 2 hours; (2) Remove the antigen coating solution and wash 5 times with PBST (containing 0.05% Tween 20); (3) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours; (4) Remove the blocking buffer and wash the plate 5 times with PBST; (5) Add transfection supernatant (100 μL / well) to each well and incubate at room temperature for 1 hour, with PBS as the control well; (6) Remove the liquid in the well and wash 5 times with PBST; (7) Add 100 μL / well of HRP-Protein A antibody (1:10000 dilution) and incubate at room temperature for 1 hour; (8) Remove the liquid in the well and wash the plate 5 times with PBST; (9) Add 100 μL / well of TMB chromogenic solution; (10) Incubate at room temperature in the dark for 10-15 minutes; Add 50 μL / well stop solution; read the OD450 in the well using an ELISA reader.

[0119] The results of solid-phase screening of proteins from the phage display library are as follows: Figures 5-1 to 5-8 As shown. Figures 5-1 to 5-4 The results are for alpaca #1, #3, #4, and #5. Figures 5-5 to 5-8 The results are for alpaca plates #2, #6, #7, and #8 (318#); AD is the parallel test group, EF is the positive control (immune serum 1:1K), and GH is the negative control (PBS control).

[0120] Example 4: Affinity test of candidate anti-LOX family nanobodies with LOXL2

[0121] 1) Antibody affinity was determined using a ForteBio OCTET R2 instrument. Biotin-Human TSLP (R127A, R130A)-C-His was immobilized using an SA sensor at a concentration of 5 μg / mL for 60 s.

[0122] 2) The buffer solution was PBST (PBS + 0.02% tween 20), and the candidate antibodies were diluted to 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, 0.3125 nM, and 0 nM.

[0123] 3) Affinity test: Equilibration 60 s, binding 180 s, dissociation 180 s, detection temperature 25°C. ℃.

[0124] 4) Dynamic characterization analysis was performed using the ForteBio OCTET R2 system.

[0125] Test results as follows Figure 6-1 and 6-2 As shown, 317-ph-1-D12, 318-3-E10, 318-ph-2-C06, and 317-ph-3-C04 all exhibit good affinity for LOXL2.

[0126] Example 5: Binding of candidate anti-LOX family nanobodies to various LOX family antigens

[0127] The binding characteristics of anti-pan-LOX family nanobodies were determined using a non-competitive ELISA method: LOX, LOXL1, LOXL2, LOXL3, and LOXL4 His tags were coated onto ELISA plates at a concentration of 2 μg / mL as antigen proteins. (50 mM NaHCO3, pH=9.6) 100 μL / well, incubated overnight at 4 ℃. Washed three times with PBST, and blocked with 300 μL / well of 5% milk. Incubated at 37 ℃ for 1 h; washed once with PBST, and each protein to be tested was serially diluted 1:5 from 200 nM (5% milk dilution); 100 μL of each diluted protein solution was incubated with the ELISA plates coated with the respective antigen proteins at 37 ℃ for 1 h. Washed five times with PBST, and the corresponding secondary antibody (IgG-HRP 1:10K) diluted with blocking buffer was added. Wash the ELISA plate incubated with secondary antibody five times with PBST, add 100 μL of TMB single-component chromogenic solution to each well, and incubate at 37°C for 7 min. Stop the reaction by adding 50 μL / well of 1M HCl. OD 450 Readings. Absorbance values ​​reflect the binding affinity of the anti-pan-LOX family nanobodies to various LOX family antigens. Detection results are as follows: Figure 6-3 As shown, the following candidate antibodies bind strongly to each antigen of the LOX family: 317-ph-1-D12, 318-3-E10, 318-ph-2-C06, and 317-ph-3-C04.

[0128] according to Figure 6-3As shown, the nanobody 318-ph-2-C06 exhibited the strongest binding affinity to various antigens of the LOX family. It was selected for further analysis to determine the binding curves between this nanobody and the target antigens, and the EC50 value was calculated. The detection results are as follows: Figure 7 As shown, the EC50 values ​​of nanobody 318-ph-2-C06 for each antigen of the LOX family are relatively small, indicating that the nanobody has a high affinity for each antigen of the LOX family and is the optimal broad-spectrum anti-pan-LOX family nanobody.

[0129] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A nanobody or antigen-binding fragment thereof against the family of prolyl oxidases with a broad spectrum of activity, characterized in that, The nanobody or the antigen-binding fragment thereof has the CDR sequence characteristics of any one of the following: (1) the amino acid sequences of CDR1, CDR2 and CDR3 are respectively the sequences shown in SEQ ID NO: 1, SEQ ID NO: 5 and SEQ ID NO: 9; (2) the amino acid sequences of CDR1, CDR2 and CDR3 are respectively the sequences shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10; (3) the amino acid sequences of CDR1, CDR2 and CDR3 are respectively the sequences shown in SEQ ID NO: 3, SEQ ID NO: 7 and SEQ ID NO: 11; (4) the amino acid sequences of CDR1, CDR2 and CDR3 are respectively the sequences shown in SEQ ID NO: 4, SEQ ID NO: 8 and SEQ ID NO:

12.

2. The Nanobody or antigen-binding fragment thereof according to claim 1, characterized in that, It also comprises four framework regions FR1, FR2, FR3 and FR4 alternately arranged with three complementarity determining regions, wherein, the amino acid sequence of FR1 is the sequence shown in one of SEQ ID NO: 13 to SEQ ID NO: 16; the amino acid sequence of FR2 is the sequence shown in one of SEQ ID NO: 17 to SEQ ID NO: 20; the amino acid sequence of FR3 is the sequence shown in one of SEQ ID NO: 21 to SEQ ID NO: 24; the amino acid sequence of FR4 is the sequence shown in one of SEQ ID NO: 25 to SEQ ID NO:

26.

3. The Nanobody or antigen-binding fragment thereof according to claim 1, characterized in that, The nanobody or the antigen-binding fragment thereof comprises one of the following sequences: (i) the amino acid sequence shown in one of SEQ ID NO: 27 to SEQ ID NO: 30; (ii) an amino acid sequence that is at least 80% identical to the amino acid sequence shown in one of SEQ ID NO: 27 to SEQ ID NO: 30 and has the same function.

4. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the nanobody or the antigen-binding fragment thereof is an amino acid sequence that is at least 85% identical to the amino acid sequence shown in one of SEQ ID NO: 27 to SEQ ID NO:

30.

5. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the nanobody or the antigen-binding fragment thereof is an amino acid sequence that is at least 90% identical to the amino acid sequence shown in one of SEQ ID NO: 27 to SEQ ID NO:

30.

6. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the nanobody or the antigen-binding fragment thereof is an amino acid sequence that is at least 91% identical to the amino acid sequence shown in one of SEQ ID NO: 27 to SEQ ID NO:

30.

7. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the nanobody or the antigen-binding fragment thereof is an amino acid sequence that is at least 92% identical to the amino acid sequence shown in one of SEQ ID NO: 27 to SEQ ID NO:

30.

8. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the nanobody or the antigen-binding fragment thereof is an amino acid sequence that is at least 93% identical to the amino acid sequence shown in one of SEQ ID NO: 27 to SEQ ID NO:

30.

9. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the Nanobody or antigen-binding fragment thereof is an amino acid sequence that is at least 94% identical to the amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO:

30.

10. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the Nanobody or antigen-binding fragment thereof is an amino acid sequence that is at least 95% identical to the amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO:

30.

11. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the Nanobody or antigen-binding fragment thereof is an amino acid sequence that is at least 96% identical to the amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO:

30.

12. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the Nanobody or antigen-binding fragment thereof is an amino acid sequence that is at least 97% identical to the amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO:

30.

13. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the Nanobody or antigen-binding fragment thereof is an amino acid sequence that is at least 98% identical to the amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO:

30.

14. The Nanobody or antigen-binding fragment thereof according to claim 3, characterized in that, The sequence of the Nanobody or antigen-binding fragment thereof is an amino acid sequence that is at least 99% identical to the amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO:

30.

15. The Nanobody or antigen-binding fragment thereof according to any one of claims 1-14, wherein, The lysyl oxidase family of enzymes comprises LOX proteins and LOX-like proteins 1-4.

16. A nucleic acid molecule or vector, characterized in that, The nucleic acid molecule encodes a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 15; the vector comprises the nucleic acid molecule.

17. A host cell, characterized in that, It comprises the nucleic acid molecule or vector according to claim 16.

18. A method of producing anti-pan-lysmoxydase family nanobodies, characterized in that, comprising the steps of: (a) culturing the host cell according to claim 17 under conditions suitable for production of a Nanobody, thereby obtaining a culture comprising said anti-lysyl oxidase family of enzymes Nanobody; (b) isolating and / or recovering said anti-lysyl oxidase family of enzymes Nanobody from the culture; and optionally (c) purifying and / or modifying the anti-lysyl oxidase family of enzymes Nanobody obtained in step (b).

19. A pharmaceutical composition comprising, It comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 15.

20. Use of a Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 15: (i) in the manufacture of a reagent for detecting a lysyl oxidase family of enzymes mediated disease; or (ii) in the manufacture of a medicament for treating a lysyl oxidase family of enzymes mediated disease; The lysyl oxidase family of enzymes mediated disease is myelofibrosis.

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