Anti-LOXL2 nano antibody or antigen binding fragment and application

Abstract

The invention belongs to the field of medical biology, and relates to an anti-LOXL2 nano antibody or antigen binding fragment and application. The anti-LOXL2 nano antibody or the antigen binding fragment comprises three complementary determining regions (CDR1, CDR2 and CDR3); wherein the amino acid sequence of the CDR1 is a sequence as shown in one of SEQ ID NO: 1 to SEQ ID NO: 3, the amino acid sequence of the CDR2 is a sequence as shown in one of SEQ ID NO: 5 to SEQ ID NO: 8, and the amino acid sequence of the CDR3 is a sequence as shown in one of SEQ ID NO: 10 to SEQ ID NO: 13. The anti-LOXL2 nano antibody disclosed by the invention is small in molecular weight, high in affinity and relatively good in specificity. The method has a good prospect of being developed to diagnose fibrosis.

Description

[0001] This application is a divisional application of the invention patent application filed on May 21, 2025, with application number 202510658983.6 and invention title "An anti-LOXL2 nanobody or antigen-binding fragment and its application". Technical Field

[0002] This invention belongs to the field of pharmaceutical biology, specifically relating to an anti-LOXL2 nanobody or antigen-binding fragment and its application. Background Technology

[0003] Early fibrosis is reversible and curable, making early diagnosis and assessment of its severity crucial. Identifying fibrosis-related biomarkers, targeting molecular-level changes during the fibrotic process, is of significant clinical value for early diagnosis. Furthermore, while substantial evidence exists regarding the pathological and molecular biological basis of fibrosis, further in-depth research is needed to identify anti-fibrotic targets. Relevant intervention targets include the classic TGF-β pathway, and novel molecular targets such as peroxisome proliferator-activated receptor (PPAR), angiotensin receptor, endothelin receptor, farnesoid X receptor, and cannabinoid receptor. Numerous antibody drugs targeting different targets have been approved for clinical use, but their efficacy requires further validation. There is an urgent need to discover new therapeutic targets and develop new, safer, and more effective treatment methods.

[0004] LOXL2, a member of the LOX family, possesses a conserved catalytic domain that 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 diseases. Changes in its expression and activity can lead to various diseases, and it is considered a biomarker for the progression of various diseases, such as liver, lung, and cardiac fibrosis, scleroderma, and cancer. Therefore, LOXL2 can serve as a potential target for the diagnosis and treatment of tumors and fibrosis-related diseases. Currently, several targeted inhibitory drugs for LOXL2 are under development, including GB2064, which, as a selective, mechanism-based small molecule inhibitor, has entered Phase II clinical trials.

[0005] 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 offer several unique advantages, such as excellent tissue penetration, rapid clearance, ease of production and modification, high stability, and low immunogenicity, 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 diagnosis; however, very few antibodies have entered clinical trials. Traditional monoclonal antibodies have complex preparation processes, high production costs, large molecular weights, and poor tissue penetration.

[0006] There are no reports or clinical applications of nanobodies targeting LOXL2, so there is an urgent need in this field to develop new and effective specific nanobodies targeting LOXL2. Summary of the Invention

[0007] definition

[0008] 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.

[0009] 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.

[0010] The purpose of this invention is to provide an anti-human / mouse nanobody that can block the cross-linking of LOXL2 fibers, and to provide the coding sequence of the nanobody, as well as the preparation method and application of the nanobody.

[0011] To achieve the above objectives, a first aspect of the present invention provides an anti-LOXL2 nanobody or antigen-binding fragment comprising three complementarity-determining regions CDR1, CDR2, and CDR3; wherein... The amino acid sequence of CDR1 is one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 3; The amino acid sequence of CDR2 is the sequence shown in one of SEQ ID NO: 5 to SEQ ID NO: 8; The amino acid sequence of CDR3 is one of the sequences shown in SEQ ID NO: 10 to SEQ ID NO: 13.

[0012] In this invention, "having the same function" means being able to bind to the LOXL2 protein.

[0013] 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.

[0014] In the sequence provided by this invention, CDR is drawn according to IMGT (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results).

[0015] According to a preferred embodiment of the present invention, the anti-LOXL2 nanobody or antigen-binding fragment has any of the following CDR sequence characteristics: (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: 10, respectively; (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: 11, respectively; (3) The amino acid sequences of CDR1, CDR2, and CDR3 are the sequences shown in SEQ ID NO: 1, SEQ ID NO: 7, and SEQ ID NO: 12, respectively; (4) The amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 3, SEQ ID NO: 8 and SEQ ID NO: 13, respectively.

[0016] According to the present invention, in addition to the aforementioned complementarity-determining regions, the anti-LOXL2 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 one of SEQ ID NO: 15 to SEQ ID NO: 19; The amino acid sequence of FR2 is the sequence shown in one of SEQ ID NO: 20 to SEQ ID NO: 24; The amino acid sequence of FR3 is shown in one of SEQ ID NO: 25 to SEQ ID NO: 29; The amino acid sequence of FR4 is shown in one of SEQ ID NO: 30 to SEQ ID NO: 31.

[0017] According to a preferred embodiment of the present invention, the anti-LOXL2 nanobody or antigen-binding fragment has any of the following FR sequence characteristics: (a) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 25, and SEQ ID NO: 30, respectively; (b) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, and SEQ ID NO: 30, respectively; (c) The amino acid sequences of FR1, FR2, FR3, and FR4 are the sequences shown in SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 27, and SEQ ID NO: 30, respectively; (d) The amino acid sequences of FR1, FR2, FR3, and FR4 are shown in SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 28, and SEQ ID NO: 30, respectively; (e) The amino acid sequences of FR1, FR2, FR3 and FR4 are shown in SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 29 and SEQ ID NO: 31, respectively.

[0018] This invention includes all sequences that satisfy the above sequence characteristics. Specifically, preferably, the nanobody or antigen-binding fragment comprises one or more of the following sequences: (i) An amino acid sequence as shown in any one of SEQ ID NO: 32 to SEQ ID NO: 35; (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: 32 to SEQ ID NO: 35 and having the same function; (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: 32 to SEQ ID NO: 35, and retains the function of the amino acid sequence shown in any one of SEQ ID NO: 32 to SEQ ID NO: 35.

[0019] 317-ph-1-B12:AVQLVDSGGGLVQAGGSLRLSCAASGRTFSSYAMGW FRQAPGKEREFVAGISWIASSTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAARNRDYDLSFGSWGQGTQVTVSS (SEQ ID NO: 32).

[0020] 318-ph-2-D06: AVQLVESGGGLVQAGDSLRLSCAASGRTFSSRTMGW FRQAPGKEREFVAIVAWNGAYTYYTNSVKGRFTISRDNAKSTGWLQMNSLKPEDTAVYYCAAHGRIAVVSTDPGDFGSWGQGTQVTVSS (SEQ ID NO: 33).

[0021] 318-ph-2-E07: PVQLVDSGGGLVQAGDSLRLSCAASGRTFSSYAMGW FRQVPGKEREFVAGINWSGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAARNYYSDYDFGAWGQGTQVTVSS (SEQ ID NO: 34).

[0022] 317-ph-5-E1: AVQLVDSGGGLVQAGGTLRLSCLASGHTFSSRTMGWF RQAPGKEREFVATITWDGAYIYYTNSVKGRFTISRDNAKSTVWLQMNSLKPEDTAVYYCAAHGRIAVVSTDPGAFGSWGQGTQVTVSS (SEQ IDNO: 35).

[0023] 318-ph-6-A09: QVKLEESGGGSVQPGGSLRLSCAASGSIFHINVMGWF RQAPGKQRELVAIISTGGAINYADSVKDRFTISRDNARNAVYLQMNSLKSEDTAVYYCNLGQGIRGDYWGPGTQVTVSS (SEQ ID NO: 36).

[0024] A second aspect of the present invention provides a nucleic acid molecule that encodes the above-described anti-LOXL2 nanobody or antigen-binding fragment.

[0025] A third aspect of the present invention provides a carrier comprising the nucleic acid molecule described in the second aspect.

[0026] A fourth aspect of the present invention provides a host cell containing the vector described in the third aspect or the nucleic acid molecule described in the second aspect.

[0027] The host cells include, but are not limited to, bacterial cells, fungal cells, animal cells, plant cells, or their progeny cells.

[0028] The anti-LOXL2 nanobody of the present invention can be obtained by the following methods: (1) Yeast display library was prepared by immunizing alpacas with human / mouse LOXL2 eukaryotic protein.

[0029] (2) Yeast display library was screened using human / mouse LOXL2 protein affinity; (3) ELISA identification of positive clones; (4) Expression and purification of LOXL2 nanobodies.

[0030] According to one specific embodiment, the present invention first immunized two healthy adult alpacas with the LOXL2 protein. After three consecutive immunizations, peripheral blood was extracted from the alpacas to separate peripheral blood lymphocytes, and a LOXL2-specific single-domain heavy chain antibody immune library was constructed. Then, magnetic sorting and flow cytometry were used to screen the immune library with human / mouse LOXL2 protein to obtain single-domain heavy chain antibodies against human LOXL2, thereby obtaining a highly efficient nanobody strain.

[0031] A fifth aspect of the present invention provides a method for engineered production of anti-LOXL2 nanobodies, comprising the following steps: (a) The host cells are cultured under conditions suitable for the production of nanobodies to obtain a culture containing the anti-LOXL2 nanobodies; (b) Isolating and / or recovering the anti-LOXL2 nanobody from the culture; and optionally... (c) Purification and / or modification of the anti-LOXL2 nanobody obtained in step (b).

[0032] A sixth aspect of the present invention provides an antibody-drug conjugate comprising the aforementioned anti-LOXL2 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.

[0033] A seventh aspect of the present invention provides a pharmaceutical composition comprising the above-described anti-LOXL2 nanobody or antigen-binding fragment, or the above-described antibody-drug conjugate.

[0034] The above-mentioned pharmaceutical composition also includes one or more pharmaceutical excipients.

[0035] The eighth aspect of the present invention provides the following uses of the anti-LOXL2 nanobody or antigen-binding fragment: (i) Use in the preparation of reagents for detecting LOXL2-mediated diseases in humans; said reagents are preferably kits for detecting LOXL2-mediated diseases; or (ii) Use in the preparation of medicaments for the diagnosis and / or treatment of LOXL2-mediated diseases; specifically, as a multimodal imaging agent for diagnosis, or as a fibrosis inhibitor; The diseases mediated by LOXL2 are preferably cancer, connective tissue diseases, or cardiovascular diseases; The cancer is preferably at least one of the following: 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 disease is preferably at least one of skin laxity and fibrosis; The cardiovascular disease is preferably at least one of atherosclerosis and myocardial ischemia.

[0036] Compared with the prior art, the technical solution of the present invention has the following advantages: (1) In this invention, alpaca were immunized with human / mouse LOXL2 eukaryotic protein, and the VHH gene sequence was amplified from alpaca peripheral blood lymphocytes to construct a yeast display library of nanobodies. The library was screened by flow cytometry to finally obtain the anti-human / mouse LOXL2 specific nanobodies gene sequence.

[0037] (2) The yeast display library constructed in this invention has a large capacity and the diversity of nanobodies obtained by screening is high.

[0038] (3) Compared with conventional antibodies, the anti-LOXL2 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.

[0039] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0040] 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.

[0041] Figure 1 This is an RNA electrophoresis image, M: DNA marker; where 1 is 317# triple-immune RNA and 2 is 318# triple-immune RNA.

[0042] Figure 2 The images are electrophoresis results of VHH amplification in one round; 1 represents the amplification result of 317# and 2 represents the amplification result of 318#.

[0043] Figure 3 These are electrophoresis images of VHH amplification in two rounds; where 1 is the amplification result of 317# and 2 is the amplification result of 318#.

[0044] Figure 4 The amino acid alignment sequence of LOXL2 library construction diversity sequencing is shown.

[0045] Figures 5 to 9 The sorting results of the LOXL2 yeast display library are shown.

[0046] Figure 5 A: NC group; B: Original library: FITC-V5; C: 1MACS: Primary antibody: Bio-Human-LOXL2-His, Secondary antibody: SA-APC+FITC-V5; D: 1MACS: Primary antibody: Bio-Mouse-LOXL2-His, Secondary antibody: SA-APC+FITC-V5.

[0047] Figure 6A: NC group; B: 1MACS: Primary antibody: Bio-Mouse-LOXL2-His, Secondary antibody: SA-APC+FITC-V5.

[0048] Figure 7 A: NC group; B: Original library: FITC-V5; C: 1MACS: Primary antibody: Bio-Human-LOXL2-His, Secondary antibody: SA-APC+FITC-V5; D: 1MACS: Primary antibody: Bio-Mouse-LOXL2-His, Secondary antibody: SA-APC+FITC-V5.

[0049] Figure 8 A: NC group; B: 1MACS: Primary antibody: Bio-Mouse-LOXL2-His, Secondary antibody: SA-APC+FITC-V5.

[0050] Figure 9 A: NC group; B: 1M+1F: Primary antibody: Bio-Human-LOXL2-His, Secondary antibody: SA-APC+FITC-V5; C: 1M+1F: Primary antibody: Bio-Mouse-LOXL2-His, Secondary antibody: SA-APC+FITC-V5.

[0051] Figures 10 to 14 The results of flow cytometry detection of yeast monoclonal antibodies are shown.

[0052] Figure 15 The results of the LOXL2 His affinity test for the candidate antibody were shown in the ELISA test.

[0053] Figure 16 The results of the affinity test for the candidate antibody are shown.

[0054] Figure 17 The results of affinity tests for LOXL2 with LOXL2 are shown for the LOXL2-specific candidate antibodies 317-ph-1-B12, 318-ph-2-D06, 318-ph-2-E07, 317-ph-5-E1, and 318-ph-6-A09. Detailed Implementation

[0055] 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.

[0056] Example 1: Preparation of LOXL2 recombinant protein

[0057] (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: 37, and the amino acid sequence of the mouse LOXL2 protein is shown in SEQ ID NO: 38.

[0058] (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.

[0059] (3) SDS-PAGE was used to detect the purity of the target protein, and the purity was >90%.

[0060] Example 2: Construction of a single-domain heavy chain antibody immune library targeting human / mouse LOXL2

[0061] 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.

[0062] 2. After three immunizations, serum titers were detected using indirect ELISA.

[0063] 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.

[0064] Experimental results: Table 1. Serum titer detection for three types of immunity.

[0065] As shown in the table above, after three vaccinations, the alpaca titer reached 1:64K (OD450>0.2). The titers of the two alpacas are normal, and subsequent database construction and screening can proceed.

[0066] 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, (1) Total RNA was extracted from PBMCs using Takara's RNAiso Plus reagent. 1 μg of RNA was electrophoresed to determine RNA purity. Figure 1 As shown, the results indicate that the RNA purity is good. RNA was reverse transcribed into cDNA using the PrimeScript™ II 1st StrandcDNA Synthesis Kit.

[0067] (2) The VHH chain was amplified using PCR, which consisted of two rounds of PCR. After the first round of nested PCR amplification, 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.

[0068] 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 below -20°C.

[0069] Table 2 Nested PCR one-round reaction system

[0070] Table 3. Reaction conditions for nested PCR in two rounds

[0071] (3) The yeast display vector pYDisplay was linearized, and the enzyme digestion system was as follows:

[0072] (3-1) Digest the pYDisplay vector with SfiI enzyme, aliquot 100 μL / tube, and digest overnight at 50 °C; (3-2) The pYDisplay vector fragment was separated using a 1% agarose gel, a 5kb fragment was cut off and recovered from the gel, and the concentration was determined by NanoDrop. (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.

[0073] (4) Construction of yeast display library by electroconversion

[0074] (4-1) Streak the competent yeast strains frozen at -80 °C onto YPD solid medium plates and activate them at 30 °C for 3-5 days; (4-2) Inoculate single colony yeast competent cells into 50 mL of YPD medium and incubate at 250 rpm and 30 °C for 1-2 days; (4-3) Preparation of competent yeast strains. The linearized vector fragment and PCR product were mixed and added to an electroporation cuvette and electroporated; the electroporated competent yeast strains were then transfected into culture flasks and incubated at 220 rpm and 30 °C for 1 h. (4-4) Take 20 μL of resuspension, dilute it 5000 times with SDCAA, take 100 μL, spread it on an SDCAA plate, incubate for 2-3 days, calculate the library volume, and continue to incubate the remaining bacterial culture for 24 h; (4-5) Preservation of bacteria: Collect the remaining bacterial culture into a 50 mL centrifuge tube, centrifuge at 3000×g for 5 min, discard the supernatant, add 10 mL SDCAA to resuspend, mix with 50% glycerol: resuspension = 1:1, and freeze at -80 °C. (4-6) The frozen bacterial culture was plated on selective plates, single colonies were isolated and verified, and 56 positive clones were sent for sequencing. After removing the bimodal stop codons, the remaining 43 clones showed differentially expressed antibody sequences, indicating good library diversity. Figure 4 As shown.

[0075] Example 3: Screening, identification, and sequencing of anti-LOXL2 nanobodies

[0076] 1. Pretreatment of streptavidin magnetic beads and yeast cells

[0077] (1) Take 5-10 mL (approximately 2 × 10⁻⁶) of yeast cultured in SGCAA medium. 8Add yeast cells to a 50 mL centrifuge tube, centrifuge at 3000×g for 5 minutes, and simultaneously dilute sterile 5% PBSA (PBS+5% BSA) to 0.5% PBSA.

[0078] (2) Discard the supernatant after centrifugation of yeast, resuspend it in 1 mL of 0.5% PBSA, add it to a 1.5 mL centrifuge tube, centrifuge at 3000×g for 5 minutes, and discard the supernatant. Wash again with 0.5% PBSA.

[0079] (3) Prepare three 1.5 mL centrifuge tubes (two centrifuge tubes for negative panning of empty magnetic beads and one centrifuge tube for positive panning of magnetic beads and antigen binding), and add 1 mL of 0.5% PBSA to each centrifuge tube.

[0080] (4) Resuspend the aliquoted streptavidin magnetic beads thoroughly by pipetting, and add 10 μL of magnetic beads to a prepared 1.5 mL centrifuge tube. Place these centrifuge tubes in a bag, fix them on a rotary mixer, and incubate at 4°C for 5 minutes.

[0081] (5) Place the centrifuge tube on a magnetic rack for 5 minutes, then use a pipette to remove the supernatant, add 1 mL of 0.5% PBSA, and wash again (incubate at 4°C for 5 minutes).

[0082] 2. Biotin-LOXL2 Magnetic Separator

[0083] (1) In the positively coated magnetic bead tube, Biotin-LOXL2 protein was diluted with 0.5% PBSA to 50 μg / mL, with a total volume of 100 μL. After thorough mixing with a 200 μL pipette, the mixture was placed in a bag and incubated at a constant speed of 4°C for 60 minutes. Then, 1 mL of 0.5% PBSA was added, and the mixture was allowed to stand for 5 minutes. The centrifuge tube was kept on the magnetic rack, and the supernatant was removed with a pipette. Then, 1 mL of 0.5% PBSA was added, and the centrifuge tube was removed from the magnetic rack. After mixing with a pipette, the centrifuge tube was placed on the magnetic rack again, and the mixture was allowed to stand for 5 minutes. The supernatant was removed with a pipette. The above washing steps were repeated once more to obtain the positively coated magnetic beads with Biotin-LOXL2 coating.

[0084] (2) Add the positively selected magnetic beads containing Biotin-LOXL2 to the yeast cells that have completed the negative selection, put them into a bag and place them on a rotary mixer to incubate at 4°C for 60 minutes. After incubation, place them on a magnetic rack and let them stand at room temperature for 15 minutes.

[0085] (3) Keep the centrifuge tube on the magnetic rack, discard the yeast culture with a pipette, and keep the magnetic beads and the yeast cells adsorbed on the magnetic beads; remove the centrifuge tube from the magnetic rack and add 1 mL of sterile 0.5% PBSA buffer to the centrifuge tube with a pipette, gently blow the magnetic beads and the yeast cells adsorbed on the magnetic beads, and transfer them to a sterile 1.5 mL centrifuge tube, then place the 1.5 mL centrifuge tube on the magnetic rack and let it stand at room temperature for 5 minutes. Discard the supernatant with a pipette; repeat the washing steps twice. The entire operation should be performed in accordance with aseptic operation requirements.

[0086] (4) After washing, resuspend the magnetic beads and the adhered yeast cells in 1 mL of SDCAA medium. Take 20 μL of the resuspension into 180 μL of SDCAA medium and spread it on two plates, each plate having a volume of 100 μL. Take another 5 μL of the resuspension into 95 μL of SDCAA medium and spread it on one plate. In total, 3 plates are needed (different dilution ratios are convenient for calculating the library volume). Add 25 μL of SDCAA medium to the remaining resuspension and mix it by pipetting. Divide it into two equal portions (500 μL / portion). Add 500 μL of 50% sterile glycerol to one portion, mix it thoroughly by pipetting, and store it in the yeast display library at -80°C. Add the other portion to a sterile shaking tube, add 2 mL of SDCAA medium, and incubate at 30°C and 220 rpm for 16 h-32 h.

[0087] (5) After the culture is completed, transfer the bacterial solution in the shake tube to two 1.5 mL sterile centrifuge tubes, place them on a magnetic rack, and let them stand at room temperature for 15 minutes to remove the interference of the magnetic beads on subsequent experiments (such as interference with antigen binding, magnetic beads clogging the flow cytometer tubing, etc.). Keep the centrifuge tubes on the magnetic rack, use a pipette to transfer the bacterial solution to a new 1.5 mL sterile centrifuge tube, centrifuge at 3000×g for 3 minutes, discard the supernatant, resuspend the bacterial cells with 2 mL of SGCAA medium, use 1 mL for each resuspension, repeat the operation twice, and transfer the bacterial cells to the shake tube, and culture overnight at 30°C and 220 rpm.

[0088] Yeast display library sorting results are as follows Figures 5 to 9 As shown.

[0089] Figure 5The results of one round of magnetic sorting for alpaca #317 are shown: A: NC group; B: Original library: FITC-V5; C: 1 MACS: Primary antibody: Bio-Human-LOXL2-His, Secondary antibody: SA-APC+FITC-V5; D: 1 MACS: Primary antibody: Bio-Mouse-LOXL2-His, Secondary antibody: SA-APC+FITC-V5. The results indicate that after using Bio-Human-LOXL2-His protein magnetic sorting, the positive rate of Human-LOXL2 protein in the library was 1.299%, and the positive rate of Mouse-LOXL2 protein was 1.172%.

[0090] Figure 6 The results show that 317# alpaca cells were sorted using magnetic sorting in one round: A: NC group; B: 1MACS: primary antibody: Bio-Mouse-LOXL2-His, secondary antibody: SA-APC+FITC-V5. The results indicate that 10042 cells were sorted using Mouse LOXL2-His protein in one round, and yeast monoclonal flow cytometry analysis was performed.

[0091] Figure 7 The results of one round of magnetic sorting for alpaca #318 are shown: A: NC group; B: Original library: FITC-V5; C: 1 MACS: Primary antibody: Bio-Human-LOXL2-His, Secondary antibody: SA-APC+FITC-V5; D: 1 MACS: Primary antibody: Bio-Mouse-LOXL2-His, Secondary antibody: SA-APC+FITC-V5. The results indicate that after using Bio-Human-LOXL2-His protein magnetic sorting, the positive percentage of Human-LOXL2 protein in the library was 2.265%, and the positive percentage of Mouse-LOXL2 protein was 2.561%.

[0092] Figure 8 The results show that after one round of magnetic sorting of alpaca cells (number 318#), cells were sorted: A: NC group; B: 1MACS: primary antibody: Bio-Mouse-LOXL2-His, secondary antibody: SA-APC+FITC-V5. The results indicate that after one round of magnetic sorting using Mouse LOXL2-His protein, 38,414 cells were sorted, and a second round of flow sorting was then arranged.

[0093] Figure 9The results of flow cytometry sorting of alpaca #318 are shown: A: NC group; B: 1M+1F: primary antibody: Bio-Human-LOXL2-His, secondary antibody: SA-APC+FITC-V5; C: 1M+1F: primary antibody: Bio-Mouse-LOXL2-His, secondary antibody: SA-APC+FITC-V5. The results indicate that after flow cytometry sorting using Bio-Mouse-LOXL2-His protein, the positive rate of Human-LOXL2 protein in the display library was 21.69%, and the positive rate of Mouse-LOXL2 protein was 16.131%. Yeast monoclonal flow cytometry detection was then performed.

[0094] 3. Yeast monoclonal flow cytometry detection

[0095] After sorting, yeast culture was plated on SDCAA plates, and single clones were picked and cultured. After induced expression for 48 h, the culture was incubated with Biotin-LOXL2 antigen. APC-Streptavidin was used as the secondary antibody. Flow cytometry was performed after incubation. Yeast clones binding to the target antigen were lysed using 0.2% SDS (incubated at 95 °C for 10 min), centrifuged, and 0.5 μL of the supernatant was used as a template for PCR amplification and assay (the remaining culture was stored at -20 °C). Results are as follows. Figures 10 to 14 As shown, where Figure 10 The results are for alpaca #317, plate #1. Figures 11 to 14 The results are for plates #1, #2, #3, and #4 of the 318# alpaca series. In the above figures: left inset, primary antibody: Bio-Human-LOXL2-His, secondary antibody: SA-APC+FITC-V5; right inset, primary antibody: Bio-Mouse-LOXL2-His, secondary antibody: SA-APC+FITC-V5.

[0096] Example 4: Expression and purification of LOXL2 nanobodies

[0097] (1) Based on the ELISA results of the candidate antibodies, positive clones were selected for antibody expression preparation. The positive yeast clones were subjected to PCR to obtain the antibody sequence, a His tag was added to the C-terminus, and after digestion with SfiI, it was ligated into the eukaryotic expression vector pcDNA3.4-human IgG1Fc to construct the antibody expression vector. After verification by Sanger sequencing, plasmid extraction was performed.

[0098] (2) Remove the LVTransm transfection reagent and pcDNA3.4-human IgG1Fc antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting. Remove the PBS buffer and warm it to room temperature. Take 2 mL of PBS into one well of a 6-well plate, add 20 μg of antibody expression vector, mix thoroughly by pipetting, add 60 μL of LVTransm, mix immediately by pipetting, and let stand at room temperature for 10 minutes.

[0099] (3) Add the above DNA / LVTransm complex to 20 mL of 293F cells and gently shake to mix thoroughly. Incubate the cells at 37°C, 5% CO2, 130 rpm for further culture.

[0100] (4) After continuous culture for 5-7 days, the supernatant of the culture medium is collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate is transferred to a sterile centrifuge tube and purified using a Protein A column.

[0101] Example 5: Assay of the binding of LOXL2 nanobody to LOXL2 antigen

[0102] The binding characteristics of LOXL2 nanobodies were determined using a non-competitive ELISA method: ELISA plates were coated with LOXL2 His tag at a concentration of 2 μg / mL as the antigen protein. 100 μL / well (50 mM NaHCO3, pH=9.6) was incubated overnight at 4 °C. The plates were washed three times with PBST and blocked with 300 μL / well of 5% milk. The plates were incubated at 37 °C for 1 h. After washing once with PBST, each protein to be tested was serially diluted 1:5 from 200 nM (using 5% milk). 100 μL of each diluted protein solution was incubated with the ELISA plate coated with the LOXL2 His tag antigen. The plates were incubated at 37 °C for 1 h. The plates were 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 between the anti-LOXL2 nanobody and the LOXL2 antigen. Detection results are as follows: Figure 15 As shown, candidate antibodies 317-ph-1-B12, 318-ph-2-D06, 318-ph-2-E07, 317-ph-5-E1, and 318-ph-6-A09 (highlighted in red) exhibit strong binding to LOXL2. However, candidate antibodies 318-1-H12, 318-1-C06, 318-3-A03, and 318-3-D-02 show relatively poor affinity.

[0103] Example 6: Binding assay of LOXL2 nanobody to LOX family (LOX, LOXL1, LOXL3, and LOXL4) antigens

[0104] The binding characteristics of LOX, LOXL1, LOXL3, and LOXL4 nanobodies were determined using a non-competitive ELISA method, and the specificity of LOXL2 nanobodies for LOXL2 was established. ELISA plates were coated with LOX, LOXL1, LOXL3, and LOXL4 His tags as antigen proteins at a concentration of 2 μg / mL. 100 μL / well (50 mM NaHCO3, pH=9.6) was incubated overnight at 4 °C. The plates were washed three times with PBST and blocked with 300 μL / well of 5% milk. The plates were incubated at 37 °C for 1 h. After washing once with PBST, each protein was serially diluted 1:5 from 200 nM (using 5% milk). 100 μL of each diluted protein solution was incubated with the ELISA plates coated with LOX, LOXL1, LOXL3, and LOXL4 His tags as antigens. The plates were incubated at 37 °C for 1 h. Wash five times with PBST, then add the secondary antibody (IgG-HRP 1:10K) diluted with blocking buffer accordingly. Wash the ELISA plate incubated with the 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-LOXL2 nanobody to antigens LOX, LOXL1, LOXL3, and LOXL4. Detection results are as follows: Figure 16 As shown, the candidate antibodies 317-ph-1-B12, 318-ph-2-D06, 318-ph-2-E07, 317-ph-5-E1, and 318-ph-6-A09, which have good affinity for LOXL2, bind poorly to LOX, LOXL1, LOXL3, and LOXL4, indicating that these LOXL2 nanobodies have specificity for LOXL2.

[0105] Example 7: Affinity Test of LOXL2 Nanobody

[0106] 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.

[0107] 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.

[0108] 3) Affinity test: Equilibration 60 s, binding 180 s, dissociation 180 s, detection temperature 25°C. ℃.

[0109] 4) Dynamic characterization analysis was performed using the ForteBio OCTET R2 system.

[0110] Test results as follows Figure 17 As shown in the figure, it can be seen that the above-mentioned LOXL2-specific candidate antibodies 317-ph-1-B12, 318-ph-2-D06, 318-ph-2-E07, 317-ph-5-E1 and 318-ph-6-A09 all have good affinity for LOXL2.

[0111] 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. An anti-LOXL2 nanobody or antigen-binding fragment, characterized in that, It contains three complementary determinant regions, CDR1, CDR2, and CDR3; among them, The amino acid sequence of CDR1 is one of the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 3; The amino acid sequence of CDR2 is the sequence shown in one of SEQ ID NO: 5 to SEQ ID NO: 8; The amino acid sequence of CDR3 is one of the sequences shown in SEQ ID NO: 10 to SEQ ID NO:

13.

2. The anti-LOXL2 nanobody or antigen-binding fragment according to claim 1, characterized in that, It also includes four frame regions FR1, FR2, FR3, and FR4, which are alternately set with the three complementary determinant regions. The amino acid sequence of FR1 is the sequence shown in one of SEQ ID NO: 15 to SEQ ID NO: 19; The amino acid sequence of FR2 is the sequence shown in one of SEQ ID NO: 20 to SEQ ID NO: 24; The amino acid sequence of FR3 is shown in one of SEQ ID NO: 25 to SEQ ID NO: 29; The amino acid sequence of FR4 is shown in one of SEQ ID NO: 30 to SEQ ID NO:

31.

3. The anti-LOXL2 nanobody or antigen-binding fragment according to claim 1, characterized in that, The nanobody or antigen-binding fragment comprises one of the following sequences: (i) An amino acid sequence as shown in any one of SEQ ID NO: 32 to SEQ ID NO: 35; (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: 32 to SEQ ID NO: 35 and having the same function; (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: 32 to SEQ ID NO: 35, and retains the function of the amino acid sequence shown in any one of SEQ ID NO: 32 to SEQ ID NO:

35.

4. A nucleic acid molecule, characterized in that, Its encoding is the anti-LOXL2 nanobody or antigen-binding fragment as described in any one of claims 1-3.

5. A carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It contains the vector as described in claim 5 or the nucleic acid molecule as described in claim 4; the host cell is preferably a bacterial cell, fungal cell, animal cell, plant cell or a descendant cell of these cells.

7. A method for generating anti-LOXL2 nanobodies, characterized in that, Includes the following steps: (a) Under conditions suitable for the production of nanobodies, the host cells as described in claim 6 are cultured to obtain a culture containing the anti-LOXL2 nanobodies; (b) Isolating and / or recovering the anti-LOXL2 nanobody from the culture; and optionally... (c) Purification and / or modification of the anti-LOXL2 nanobody obtained in step (b).

8. An antibody-drug conjugate, characterized in that, The invention comprises the anti-LOXL2 nanobody or antigen-binding fragment, linker, and effector as described in any one of claims 1-3; 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 is 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.

9. A pharmaceutical composition, characterized in that, Contains an anti-LOXL2 nanobody or antigen-binding fragment as described in any one of claims 1-3, or an antibody-drug conjugate as described in claim 8.

10. The following uses of the anti-LOXL2 nanobody or antigen-binding fragment according to any one of claims 1-3, or the antibody-drug conjugate according to claim 8: (i) Use in the preparation of reagents for detecting LOXL2-mediated diseases; said reagents are preferably kits for detecting LOXL2-mediated diseases; or (ii) Use in the preparation of medicaments for the treatment of LOXL2-mediated diseases; The diseases mediated by LOXL2 are preferably cancer, connective tissue diseases, or cardiovascular diseases; The cancer is preferably at least one of the following: 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 disease is preferably at least one of skin laxity and fibrosis; The cardiovascular disease is preferably at least one of atherosclerosis and myocardial ischemia.

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