Monoclonal antibody specifically targeting human-derived slc46a2 extracellular epitope and use thereof

Abstract

The present application relates to the technical field of antibodies, in particular to a monoclonal antibody specifically targeting a human SLC46A2 extracellular epitope and application thereof. The present application expresses and purifies human SLC46A2 protein through a eukaryotic expression system; then uses the purified human SLC46A2 protein as an immunogen to immunize mice to produce antibodies; finally, a monoclonal antibody AB7 specifically recognizing and binding to human SLC46A2 protein is obtained through flow enrichment and screening, and the epitope recognized and bound by the antibody is identified; the complex structure of human SLC46A2-Fab7 is analyzed through cryo-EM to further determine the binding epitope of the antibody. It has been verified that the monoclonal antibody AB7 provided by the present application can specifically recognize and bind to the amino acid residues of human SLC46A2 protein, has high affinity, and can be used as a molecular tool for the detection of human SLC46A2 protein, and has high sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to a monoclonal antibody that specifically targets the extracellular surface of human SLC46A2 cells and its applications. Background Technology

[0002] SLC46A2 (solute carrier family 46 member 2), also known as the thymic stromal cotransporter (TSCOT), is a highly conserved 12-transmembrane transporter within the major promoter superfamily (MFS). It is specifically and highly expressed in human tissues such as the thymus, skin, and lungs, and is localized to the plasma membrane and acidic organelles such as endosomes and lysosomes. Functional studies have shown that SLC46A2 is the first identified protein in primary human monocytes and monocyte-derived macrophages to introduce 2'3'-cyclic guanosine monophosphate (2'3'-cGAMP) and other cyclic dinucleotides (CDNs), thereby activating the STING signaling pathway, which has antiviral and antitumor activities. Simultaneously, it activates the NOD1 signaling pathway in keratinocytes by mediating the uptake of diaminopimelic acid (DAP)-type cell wall peptides, thereby inducing psoriatic inflammation.

[0003] Given the crucial role of SLC46A2 in tumor immunology and inflammatory diseases, developing high-affinity and high-specificity SLC46A2 antibodies is essential. These antibodies could serve not only as molecular detection tools for protein expression detection and functional studies but also as potential biological agents targeting SLC46A2. Currently, there is a lack of monoclonal antibodies that specifically target human SLC46A2 and clearly define its binding epitope. This leads to two main limitations: firstly, research tools are restricted, as the expression, distribution, and dynamic changes of endogenous SLC46A2 protein cannot be detected with high sensitivity and specificity, severely hindering research on its functional mechanisms and pathological characteristics; secondly, the development of related drugs is hampered, as the lack of SLC46A2-targeting antibodies means that potential drug development lacks key lead molecules.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a monoclonal antibody AB7 that specifically targets the extracellular surface of human SLC46A2 cells and its applications.

[0006] Specifically, the technical solution of the present invention is as follows: In a first aspect, the present invention provides an antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain CDR1 of the antibody or the antigen-binding fragment thereof is shown in SEQ ID NO.7, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.9; and the amino acid sequence of the light chain CDR1 of the antibody or the antigen-binding fragment thereof is shown in SEQ ID NO.10, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.11, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.12.

[0007] Preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO.4 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO.6 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.6.

[0008] Preferably, the antibody or its antigen-binding fragment includes at least one of monoclonal antibody, Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, and single-chain antibody.

[0009] In a second aspect, the present invention provides bispecific or multispecific antibodies comprising the antibody or its antigen-binding fragment.

[0010] Thirdly, the present invention provides a nucleic acid molecule that encodes the antibody or its antigen-binding fragment.

[0011] Fourthly, the present invention provides a biomaterial comprising the nucleic acid molecule, wherein the biomaterial is an expression cassette, a vector, or a host cell.

[0012] Fifthly, the present invention provides an antibody conjugate obtained by conjugating the antibody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody, to a label or protein; wherein the label is selected from one or more of chemiluminescent dye labeling, enzyme labeling, biotin labeling, fluorescent dye labeling, colloidal gold labeling, and radioactive labeling.

[0013] In a sixth aspect, the present invention provides a method for preparing the antibody or its antigen-binding fragment, the method comprising: culturing host cells capable of expressing the antibody or its antigen-binding fragment, and separating the antibody or its antigen-binding fragment.

[0014] In a seventh aspect, the present invention provides any of the following applications of the antibody or its antigen-binding fragment, or the bispecific antibody or multispecific antibody, or the nucleic acid molecule, or the biological material, or the antibody conjugate: (1) Use in the preparation of products for detecting the presence or level of human SLC46A2 protein or cells expressing human SLC46A2 protein in a sample; (2) Application in detecting the presence or level of human SLC46A2 protein in a sample; (3) Application in detecting the presence or level of cells expressing human SLC46A2 protein in a sample.

[0015] Eighthly, the present invention provides a reagent or pharmaceutical composition for detecting human SLC46A2 protein, wherein the reagent or pharmaceutical composition for detecting human SLC46A2 protein comprises the antibody or its antigen-binding fragment, or comprises the bispecific antibody or multispecific antibody, or comprises the antibody-drug conjugate.

[0016] Beneficial effects: This invention provides a monoclonal antibody AB7 that specifically targets the epitopes of human SLC46A2 and its applications. The invention involves expressing and purifying human SLC46A2 protein using a eukaryotic expression system; then using the purified human SLC46A2 protein as an immunogen to immunize mice to generate antibodies; finally, flow cytometry enrichment and screening yields the monoclonal antibody AB7 that specifically recognizes and binds to human SLC46A2 protein, and the epitope to which the antibody binds is identified; the structure of the human SLC46A2-FAB7 complex is resolved by cryo-electron microscopy to further clarify the antibody's binding epitope. Verification shows that the monoclonal antibody provided by this invention can specifically recognize and bind to amino acid residues of human SLC46A2 protein, exhibiting high affinity. It can be used as a molecular tool for the detection of human SLC46A2 protein, demonstrating high sensitivity and specificity, and has advantages in the detection of human SLC46A2 protein. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be described below.

[0018] Figure 1 The results show the affinity assay results between antibody 7 in Example 3 and human SLC46A2.

[0019] Figure 2 The results are from the flow cytometry analysis of antibody 7 in Example 3.

[0020] Figure 3The results of Western blot detection of human SLC46A2 protein using antibody 7 in Example 3 are shown.

[0021] Figure 4 The cryo-electron microscopy structure of the human SLC46A2-Fab7 complex in Example 3 is shown. Detailed Implementation

[0022] This invention provides a monoclonal antibody that specifically targets a specific epitope of the human SLC46A2 protein. On the one hand, it can serve as a highly sensitive and specific detection tool to aid in the study of the function and mechanism of SLC46A2; on the other hand, it can serve as a potential biological agent for the development of subsequent therapeutic drugs.

[0023] This invention first uses an insect cell expression system to express and purify human SLC46A2 protein (its amino acid sequence is shown in SEQ ID NO.1). After removing the N-terminal and C-terminal irregular sequences, a truncated amino acid sequence from positions 11-469 is expressed and purified as an immunogen to immunize mice and generate antibodies. Mouse spleen cells are then fused with myeloma cells. After successful fusion, hybridoma clones that specifically secrete anti-human SLC46A2 antibodies are screened by flow cytometry. Subsequently, positive monoclonal hybridoma cells secreting anti-human SLC46A2 antibodies are obtained through multiple subcloning processes. Finally, these monoclonal antibodies are screened by flow cytometry and Western blot using 293T cells expressing human SLC46A2 protein and human-mouse SLC46A2 chimeras, respectively. Ultimately, monoclonal antibodies specifically binding to human SLC46A2 are screened, and the corresponding antibody binding epitopes and functions are identified.

[0024] The amino acid sequence (SEQ ID NO.1) of the human SLC46A2 protein is shown below: MSPEVTCPRRGHLPRFHPRTWVEPVVASSQVAASLYDAGLLLVVKASYGTGGSSNHSASPSPRGALEDQQQRAISNFYIIYNLVVGLSPLLSAYGLGWLSDRYHRKISICMSLLGFLLSRLGLLLKVLLDWPVEVLYGAAALNGLFGGFSAFWSGVMALGSLGSSEGRRSVRLILIDLMLGLAGFCGSMASGHLFKQMAGHSGQGLILTACSVSCASFALLYSLLVLKVPESV AKPSQELPAVDTVSGTVGTYRTLDPDQLDQQYAVGHPPSPGKAKPHKTTIALLFVGAIIYDLAVVGTVDVIPLFVLREPLGWNQVQVGYGMAAGYTIFITSFLGVLVFSRCFRDTTMIMIG MVSFGSGALLLAFVKETYMFYIARAVMLFALIPVTTIRSAMSKLIKGSSYGKVFVILQLSLALTGVVTSTLYNKIYQLTMDMFVGSCFALSSFLSFLAIIPISIVAYKQVPLSPYGDIIEK.

[0025] The amino acid sequence (SEQ ID NO.2) of the mouse SLC46A2 protein is shown below: MGPGGTCPWSSRLSGFRVRTWIEPVVASTQVAGSLYDAGLLLVVKESFKSEAGGSSNYSANQSLVEYQEDQQQKAISNFNIIYNLVLGLTPLLSAYGLGWLSDRYHRKISICTAMLGFLL SRIGLLLKVMLDWPVEVMYGAAALNGLCGSFSAYWSGVMALGSLGCSEGRRSVRLILIDLVLGLAGFSGSMASGHLFKQIVGHSAQGLLLTACSVGCAAFALFYSLFVLKVPESKPNKVH PTVDTVSGMMGTYRTLDPDQQDKQNVPRNPRTPGKGKSSQREVVALLFVGAIIYDLAAVGTVDVMALFVLKEPLHWNQVQLGYGMASGYIIFITSFLGVLVFSRCFRDTTMIIIGMLSFG SGALLLAFVKETYMFYIARAIMLFALIPITTIRSAMSKLIKDSSYGKIFVILQLCLTLTGVVTSTIYNKIYQLTLDKFIGTCFVLSSFLSFLAIVPIGVVAYKQVPRSQQGECAEKQRS.

[0026] In a more specific embodiment, the monoclonal antibody No. 7 prepared in this invention can specifically recognize and bind to amino acid residues 58-67 of the human SLC46A2 protein, forming a stable rigid complex with the human SLC46A2 protein, increasing the mass of the water-soluble region, and helping to resolve the structure of SLC46A2. Furthermore, the monoclonal antibody No. 7 can be used as a molecular tool for detecting the expression and distribution of the human SLC46A2 protein using Western blot, flow cytometry, and other methods.

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0028] The endpoints and any values ​​of the ranges disclosed in this specification are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0029] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "specific implementation," or "some specific implementations," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0030] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0031] Example 1 This example demonstrates the expression and purification of human SLC46A2 protein. The specific steps are as follows: (1) Gene synthesis and vector construction.

[0032] Synthetic human Slc46a2 The gene (Gene ID: 57864) was modified by deleting sequences at its N-terminus and C-terminus that differed significantly from those in the mouse protein. An 11×His tag was fused to the C-terminus of amino acids 11-469 via a tve restriction enzyme site, and the resulting plasmid was constructed into the pFastBac insect cell expression vector. The constructed plasmid was transformed into *E. coli* competent cells TOP10, amplified, and extracted. Sequencing confirmed the sequence was correct, yielding the target plasmid vector pFastBac-SLC46A2-tev-His. This plasmid vector was then transformed into *E. coli* DH10Bac strain and plated on blue-white screening plates. After colony formation, white colonies were selected for propagation and culture, and baculovirus (Bacmid) was extracted.

[0033] (2) Protein expression and purification.

[0034] Mix 1 μg Bacmid and 6 μL Cellfectin II reagent separately with 100 μL Grace medium, then combine the two components and incubate at room temperature for 30 minutes. Next, add 800 μL Grace medium to the mixture and pour it into pre-coated six-well plates (5 × 10⁶ cells per well). 5(100 sf9 cells). After incubation at 27°C for 5 hours, the transfection medium was aspirated and replaced with fresh insect culture medium containing 10% FBS. After culturing for another 72 hours, the supernatant was collected by centrifugation at 2500 rpm to obtain P1 virus. Then, P1 virus was added to 10 mL of sf9 cells at a ratio of 5%, and after vortexing and culturing for 72 hours, the supernatant was collected by centrifugation to obtain P2 virus. P2 virus was further inoculated into 50 mL of sf9 cells, and after vortexing for 72 hours, the supernatant was collected by centrifugation to obtain P3 virus. P3 virus was inoculated into 600 mL of 2×10⁻⁶ cells. 6 In sf9 cells at a concentration of cells / mL, after culturing for 72 hours, the cells were collected by centrifugation and the cell pellet was fully resuspended in 20 mL of solution 1 (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol, 10% glycerol).

[0035] The resuspended cells were sonicated and then incubated on ice for 2 h after adding a final concentration of 1% (w / v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace) and 0.1% (w / v) Cholesteryl Hemisuccinate TrisSalt (CHS, Anatrace). The cells were then centrifuged at 18,000 rpm for 30 min at 4°C, and the precipitate was discarded. The supernatant was added to a pre-equilibrated nickel column. After all the supernatant had been loaded, the column was washed with 5 column volumes of Solution 1 (additional 0.05% DDM + 0.005% CHS). The column was then washed with 5 column volumes of Solution 2 (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 40 mM imidazole, 5 mM β-mercaptoethanol, 10% glycerol, 0.05% DDM + 0.005% CHS). Finally, the protein was eluted with solution 3 (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 400 mM imidazole, 10% glycerol, 0.05% DDM + 0.005% CHS). The eluted protein solution was then added with 2 mM EDTA, 10 mM DTT, and tevase, and digested overnight. Subsequently, the protein was concentrated to 500 μL by centrifugation in a 50 kDa tube. A Superdex 200 gel chromatography column was equilibrated with solution 4 (20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.01% GDN), and the concentrated protein solution was loaded for further separation and purification. After SDS-PAGE gel chromatography, the target protein sample with acceptable purity was concentrated to approximately 1 mg / ml, flash-frozen in liquid nitrogen, and stored at –80°C for later use.

[0036] Example 2 This example demonstrates the preparation of a monoclonal antibody, and the specific steps are as follows: (1) Immunize BALB / c mice.

[0037] The purified human SLC46A2 protein sample was diluted to 100 μg / mL with solution 4 and mixed with 150 μg / mL CpG1826 (Invitrogen) at a 1:1 volume ratio to obtain suspension 1. Suspension 1 was then mixed with complete Freund's adjuvant at a 1:1 volume ratio to form suspension 2; suspension 1 was then mixed with incomplete Freund's adjuvant at a 1:1 volume ratio to form suspension 3. For the initial immunization, 0.2 mL of suspension 2 was injected subcutaneously at multiple sites into 6-week-old BALB / c mice. On days 30 and 60 post-immunization, 0.2 mL of suspension 3 was injected twice more in the same manner. On day 90, a booster immunization was performed by intraperitoneal injection of human SLC46A2 protein diluted to 25 μg / mL with 0.2 mL PBS.

[0038] (2) Hybridoma cell fusion and screening.

[0039] Preparation of feeder cells: One day before fusion, Balb / c mice were euthanized and disinfected. A pre-cooled 0.34M sucrose solution was injected intraperitoneally. After massage, the peritoneal lavage fluid was collected, centrifuged, and the precipitate was resuspended in HAT medium to a density of 1×10⁻⁶. 5 Cells / mL, spread in 96-well plates for later use.

[0040] Cell fusion: Spleens of mice after booster immunization were harvested, and single-cell suspensions of spleen cells were prepared and counted for later use. Myeloma cells in logarithmic growth phase were collected, washed, and mixed with spleen cells in a specific ratio. After centrifugation and discarding the supernatant, 1 ml of preheated 50% PEG was added dropwise in a 37°C water bath to initiate fusion. Subsequently, preheated RPMI 1640 medium was slowly added to terminate fusion. After centrifugation and discarding the supernatant, the cells were resuspended in HAT medium and seeded into 96-well plates containing feeder cells for culture.

[0041] Screening for anti-human SLC46A2 antibodies: 7-10 days after fusion, hybridoma cell supernatant was collected and added to a 96-well plate containing 293T cells expressing human SLC46A2. The plates were incubated at 4°C for 30 minutes, washed, and then incubated with PE-labeled goat anti-mouse IgG secondary antibody at 4°C in the dark for 30 minutes, followed by another wash. Flow cytometry was then used to detect the positive threshold based on negative and positive controls, screening for hybridoma clones that specifically secrete anti-human SLC46A2 antibodies.

[0042] Hybridoma cell subcloning: Positive clone wells were selected, and cell state and cell cluster size were observed under a microscope. Subsequently, positive monoclonal hybridoma cells were selected for the second and third subcloning.

[0043] Retrieving antibody genes from hybridoma cells: mRNA carrying antibody gene information was extracted from monoclonal hybridoma cells, then converted into cDNA via reverse transcription, and finally the antibody gene was amplified. The heavy chain variable region nucleic acid sequence (SEQ ID NO.3) of antibody 7 is as follows: GAAGTGAAGCTCGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTCTCAGTTCCTATACCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGACTGCAGTGGGTCGCTTCCATTGGTCGTGGTGGTACCGCCTACTAT TCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAAAGAGCAGTCTGAGGTCTGAGGACACGGCCATCTATTACTGTACAAGAGAAAAGGATGGTAACTACTTCTCTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCG.

[0044] The heavy chain variable region protein sequence (SEQ ID NO.4) of antibody 7 is as follows: EVKLVESGGGLVQPGGSLKLSCAASGFTLSSYTMSWVRQTPEKRLQWVASIGRGGTAYYSDSVKGRFTISRDNARNILYLQKSSLRSEDTAIYYCTREKDGNYFSDFWGQGTTLTVSS.

[0045] The heavy chain CDR1 of antibody 7: GFTLSSYT (SEQ ID NO.7).

[0046] The heavy chain CDR2 of antibody 7: IGRGGTA (SEQ ID NO.8).

[0047] The heavy chain CDR3 of antibody 7: TREKDGNYFSDF (SEQ ID NO.9).

[0048] The light chain variable region nucleic acid sequence (SEQ ID NO.5) of antibody 7 is as follows: GACATTTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTATTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGG GCATCCACTCGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAGCAATATTTTACCTATTCCACGTTCGGTACTGGGACCAAGCTGGAGCTGAAA.

[0049] The light chain variable region protein sequence (SEQ ID NO.6) of antibody 7 is as follows: DIVMSQSPSSLAVSVGEKVIMSCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISNVKAEDLAVYYCQQYFTYSTFGGTKLELK.

[0050] The light chain CDR1 of antibody 7: QSLLYSSNQKNY (SEQ ID NO.10).

[0051] The light chain CDR2 of antibody 7: WAS (SEQ ID NO.11).

[0052] The light chain CDR3 of antibody 7: QQYFTYST (SEQ ID NO.12).

[0053] The CDR analysis method is IMGT.

[0054] (3) Preparation of ascites.

[0055] NCG mice were intraperitoneally injected with 500 μL of liquid paraffin on day 0. From day 9 to 11, monoclonal hybridoma cells against human SLC46A2 in the logarithmic growth phase were collected, centrifuged at 1500 r / min for 5 min, washed 1-2 times, resuspended in PBS, and counted. The cell density was adjusted to 1-2 × 10⁻⁶ cells / mL. 6Cells / mL were injected intraperitoneally into each mouse with 500 μL of cell suspension. From day 19 to 23 post-inoculation, ascites fluid was collected based on the abdominal distension of the mice. The ascites fluid was centrifuged at 12000 r / min for 10 min, and the upper pale yellow ascites supernatant was collected, aliquoted, and frozen at -80℃ to obtain the monoclonal antibody against human SLC46A2.

[0056] (4) Expression and purification of the target antibody.

[0057] The ascites fluid was collected, diluted 10-fold with PBS, and filtered. After filtration, the sample was loaded onto a PBS-equilibrated Protein A column, washed with 5 column volumes of PBS, and then eluted with 0.1 M Glycine (pH 2.7), followed by neutralization with 2 M Tris-HCl at pH 8.0. The eluted sample was concentrated and replaced with PBS solution.

[0058] Example 3 This embodiment describes the identification and analysis of antibody binding ability and binding epitopes. The specific steps are as follows: (1) The affinity of the antibody was determined using a ForteBio Octet Red96 biomembrane interferometer.

[0059] First, human SLC46A2 protein was diluted to five concentration gradients (120 nM, 40 nM, 13.3 nM, 4.44 nM, 1.48 nM) using Solution 4 (20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.01% GDN). The antibody to be tested was then diluted to a concentration of 25 μg / ml. After setting up the biomembrane interferometer (ForteBio Octet Red96), detection was performed using Octet ProG Biosensors. The final data were processed to obtain affinity (KD), with antibody 7 showing an affinity (KD) of 1.704 × 10⁻⁶. -08 M ( Figure 1 ).

[0060] (2) Flow cytometry identification of antibody-binding epitopes.

[0061] To identify the Ab7 binding epitope, a series of chimeric human SLC46A2 variants, T1 (amino acids 233-276 replaced with amino acids 235-276 of the mouse protein) and T2 (amino acids 56-62 replaced with amino acids 58-64 of the mouse protein, and amino acids 233-276 replaced with amino acids 235-276 of the mouse protein), were designed and fused with GFP, and constructed into the pTT3 plasmid vector. After sequencing verification, the constructed plasmids were transfected into 293T cells. Twenty-four hours later, the cells were resuspended and fixed in PBS containing 2% PFA for 20 minutes. The cell pellet was then collected by centrifugation at 1500 rpm and resuspended in permeabilization buffer. After centrifugation at 1500 rpm, the pellet was collected, and ascites fluid diluted 1:1000 with permeabilization buffer was added as the primary antibody, followed by incubation for 30 minutes. After centrifugation, the supernatant was discarded, and the samples were resuspended in permeabilization buffer and washed to remove excess unbound primary antibody. The samples were then resuspended in a 1:1000 dilution of goat anti-mouse IgG secondary antibody diluted in permeabilization buffer and incubated for 30 minutes. Centrifugation was then performed again, and excess unbound secondary antibody was washed away with PBS. Finally, the cells were resuspended in PBS for flow cytometry analysis. Flow cytometry analysis first used samples with negative mouse serum as the primary antibody as a negative control to screen for a batch of monoclonal antibodies that could significantly bind to human SLC46A2. Next, cells expressing SLC46A2 chimeric proteins T1 or T2, which replaced the mouse protein sequence, were further screened. The results showed that antibody 7 could specifically bind to the WT and T1 of SLC46A2, but hardly bound to T2. This experiment demonstrates that antibody 7 recognizes and binds to amino acid residues 58-64 of the human SLC46A2 protein (…). Figure 2 Furthermore, this experiment demonstrates that antibody 7 can be used for flow cytometry detection of human SLC46A2 protein.

[0062] (3) Western blot identification of antibody-binding epitopes Furthermore, the detection function of the monoclonal antibody was further analyzed by Western blot. The previously purified human SLC46A2 protein was subjected to SDS-PAGE and then transferred to an NC membrane. After blocking with blocking buffer for 1 hour, the purified antibody 7 was diluted to 50 μg / mL with antibody dilution buffer and incubated overnight at 4°C with the NC membrane. After thorough washing with PBST, the membrane was incubated at room temperature for 1 hour with HRP-labeled goat anti-mouse secondary antibody. After further washing with PBST, development was performed. The results showed that antibody 7 exhibited a significant positive band (…). Figure 3 The antibody 7 contains the full-length (~45 kDa) and N-terminal domain (~23 kDa) of SLC46A2. This indicates that antibody 7 can be used for Western blot detection of human SLC46A2 protein.

[0063] (4) Structural analysis of the complex of antibody 7 and SLC46A2.

[0064] In addition, the in vitro purified antibody 7 was purified by papain digestion to obtain the Fab fragment. Subsequently, Fab7 was incubated with human SLC46A2, and the SLC46A2-Fab7 complex was separated and purified using a Superdex 200 gel chromatography column. Cryo-electron microscopy samples of the complex were prepared, and approximately 3000 images were collected. The complex structure was reconstructed using CrypSPARC calculations to obtain a resolution of 3.57 Å. Figure 4 Cryo-electron microscopy revealed that SLC46A2 exhibits an open extracellular conformation, and antibody 7 primarily interacts with amino acids 58-67 of SLC46A2. This indicates that antibody 7 can be used for structural analysis of the human SLC46A2 protein.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or its antigen-binding fragment targeting SLC46A2, characterized in that, The amino acid sequences of the heavy chain CDR1 of the antibody or its antigen-binding fragment are shown in SEQ ID NO.7, the amino acid sequences of the heavy chain CDR2 are shown in SEQ ID NO.8, and the amino acid sequences of the heavy chain CDR3 are shown in SEQ ID NO.9; the amino acid sequences of the light chain CDR1 of the antibody or its antigen-binding fragment are shown in SEQ ID NO.10, the amino acid sequences of the light chain CDR2 are shown in SEQ ID NO.11, and the amino acid sequences of the light chain CDR3 are shown in SEQ ID NO.

12.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.4; the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO.

6.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment includes at least one of monoclonal antibodies and Fab.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid molecule of claim 4, wherein the biomaterial is an expression cassette, a vector, or a host cell.

6. The method for preparing the antibody or its antigen-binding fragment according to any one of claims 1 to 3, characterized in that, The method includes: culturing host cells capable of expressing the antibody or its antigen-binding fragment, and then isolating the antibody or its antigen-binding fragment.

7. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, or the biomaterial according to claim 5 in the preparation of a product for detecting the presence or level of human SLC46A2 protein or cells expressing human SLC46A2 protein in a sample.

8. A reagent for detecting human SLC46A2 protein, characterized in that, The reagent for detecting human SLC46A2 protein comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

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