A fully human anti-human sphingosine-1-phosphate receptor 3 antibody and uses thereof

Abstract

The application relates to the technical field of biological medicine, and provides a full-human anti-human sphingosine-1-phosphate receptor 3 antibody and application thereof, specifically including an amino acid sequence and a nucleic acid sequence coding the full-molecule antibody and application thereof and the like. Related experiments prove that the antibody can specifically recognize a human sphingosine-1-phosphate receptor 3 protein, an antigen epitope is an A98-A114 region, and can be used for treating obesity (OB) and metabolic dysfunction related fatty liver disease (MASLD) and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a fully human anti-human sphingosine-1-phosphate receptor 3 antibody and its application. Background Technology

[0002] Sphingosine-1-phosphate receptor 3 (S1PR3) is a G protein-coupled receptor (GPCR) encoded by the S1PR3 gene, and is one of the five members of the S1PR family (S1PR1–5). S1PR3 possesses typical GPCR characteristics: a seven-transmembrane helical domain, an extracellular N-terminus for ligand binding, and an intracellular C-terminus for coupling with G proteins. Its endogenous ligand is the sphingolipid metabolite sphingosine-1-phosphate (S1P). When S1P binds to S1PR3, the receptor primarily couples with the Gαi, Gαq, and Gα12 / 13 protein families, thereby activating multiple downstream signaling pathways. This includes: promoting cell proliferation and survival by inhibiting adenylate cyclase and activating the Ras-MAPK / ERK pathway via Gαi; activating phospholipase C (PLC) via Gαq, producing IP3 and DAG, leading to intracellular calcium mobilization and activation of protein kinase C (PKC); and activating Rho GTPase via Gα12 / 13, affecting cytoskeleton rearrangement and cell migration. S1PR3 is widely expressed in various tissues, including the cardiovascular, immune, and nervous systems, as well as the liver and adipose tissue. Its functions are complex and diverse, involving key physiological processes such as lipid homeostasis, angiogenesis, vascular tone regulation, immune cell migration, and cell proliferation and survival. In the liver, the S1P / S1PR3 axis can regulate lipid metabolism in hepatocytes, affecting fatty acid uptake and triglyceride synthesis. Knockdown of S1PR3 by siRNA can significantly inhibit lipid accumulation in hepatocytes. In addition, the S1PR3 signaling pathway is closely related to adipocyte differentiation, lipid storage, and energy homeostasis, and can also indirectly affect systemic lipid metabolism by regulating insulin sensitivity and inflammatory responses. Therefore, S1PR3 is considered a potential therapeutic target for metabolic diseases such as metabolic dysfunction-related fatty liver disease and obesity.

[0003] Although S1PR3 plays a crucial role in metabolic regulation, particularly in the pathological mechanisms of fatty liver disease associated with metabolic dysfunction, there is currently a lack of approved therapeutic drugs specifically targeting S1PR3, as well as a lack of high-sensitivity, high-specificity, high-quality antibody tools for detection and research. This situation severely restricts in-depth mechanistic research on S1PR3, accurate diagnosis of related disease states, and the development of targeted therapies.

[0004] Currently available anti-S1PR3 antibodies (such as Proteintech 55204-AP and Santa Cruz sc-16076) are all murine or rabbit polyclonal / monoclonal antibodies, primarily used for research and testing purposes. Due to the limitation of their species origin, these antibodies face inherent defects in human therapeutic applications, such as high immunogenicity and short half-life, making them unsuitable as candidate molecules for therapeutic antibody drugs. Summary of the Invention

[0005] This invention provides a fully human anti-human sphingosine-1-phosphate receptor 3 protein antibody 2E3 and its application. This antibody can specifically recognize human sphingosine-1-phosphate receptor 3 protein A98~A114 and can be used in diagnostic reagents and / or therapeutic drugs for metabolic diseases such as metabolic dysfunction-associated fatty liver disease (MASLD), obesity, and metabolic syndrome.

[0006] The antibodies of this invention are fully human antibodies. Compared with humanized chimeric antibodies, fully human antibodies directly isolated from human B cells possess natural human antibody framework regions and CDR sequences, exhibiting lower immunogenicity and a lower risk of clinical translation. In particular, antibodies screened from the autoimmune responses of patients with metabolic diseases naturally possess biological functions related to disease progression, providing a novel strategy for the development of therapeutic antibody drugs targeting S1PR3.

[0007] In a first aspect, the present invention provides a fully human anti-human sphingosine-1-phosphate receptor 3 antibody or an antigen-binding fragment thereof, comprising VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO: 1-3, VLCDR1 as shown in SEQ ID NO: 4, VLCDR2 being GNS and VLCDR3 as shown in SEQ ID NO: 5.

[0008] As an optional embodiment, the fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 6 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 7.

[0009] As an optional embodiment, the fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO: 10 and a light chain with an amino acid sequence as shown in SEQ ID NO: 11.

[0010] As an alternative, the antibody or its antigen-binding fragment can specifically bind to amino acid residues 98-114 in the extracellular region of the human sphingosine-1-phosphate receptor 3 protein, as shown in SEQ ID NO: 14.

[0011] As an option, the fully human anti-human sphingosine-1-phosphate receptor 3 antibody is a monoclonal antibody; the antigen-binding fragment of the antibody is selected from Fab, Fab', F(ab')2, Fv or scFv or any other antibody fragment that binds to the antigen but does not contain the complete antibody structure.

[0012] In a second aspect, the present invention provides a nucleic acid encoding the above-described fully human anti-human sphingosine-1-phosphate receptor 3 antibody or an antigen-binding fragment thereof.

[0013] Alternatively, the nucleic acid may comprise a nucleic acid sequence encoding the heavy chain variable region as shown in SEQ ID NO: 8 and a nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO: 9.

[0014] Alternatively, the nucleic acid may comprise a nucleic acid sequence encoding the heavy chain as shown in SEQ ID NO: 12 and a nucleic acid sequence encoding the light chain as shown in SEQ ID NO: 13.

[0015] A third aspect of the present invention provides an expression vector comprising the above-described nucleic acid.

[0016] In a fourth aspect, the present invention provides a transgenic cell line or recombinant bacteria comprising the above-described nucleic acid or the above-described expression vector.

[0017] A fifth aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described above, and a pharmaceutically acceptable carrier or excipient.

[0018] A sixth aspect of the present invention provides an immunoconjugate comprising an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the antibody described above or an antigen-binding fragment thereof; the coupling portion is selected from detectable markers, cytotoxic drugs, or combinations thereof; wherein the detectable marker is selected from fluorescent substances, chemiluminescent substances, radioisotopes, or enzymes.

[0019] In a seventh aspect, the present invention provides a detection reagent or kit for detecting human sphingosine-1-phosphate receptor 3, said detection reagent or kit comprising the antibody or antigen-binding fragment thereof described above.

[0020] An eighth aspect of the present invention provides the use of the above-described antibody or its antigen-binding fragment in the preparation of a medicament for the prevention or treatment of metabolic diseases, said metabolic diseases including metabolic dysfunction-related fatty liver disease, obesity, or metabolic syndrome.

[0021] The sequences involved in the present invention are as follows: SEQ ID NO: 1 is the amino acid sequence of the heavy chain variable region CDR1; SEQ ID NO: 2 is the amino acid sequence of the CDR2 variable region of the heavy chain; SEQ ID NO: 3 is the amino acid sequence of the CDR3 variable region of the heavy chain; SEQ ID NO: 4 is the amino acid sequence of the CDR1 variable region of the light chain; GNS is the amino acid sequence of the CDR2 variable region of the light chain; SEQ ID NO: 5 is the amino acid sequence of the CDR3 variable region of the light chain; SEQ ID NO: 6 is the amino acid sequence of the heavy chain variable region; SEQ ID NO: 7 is the amino acid sequence of the light chain variable region; SEQ ID NO: 8 is the nucleic acid sequence of the heavy chain variable region; SEQ ID NO: 9 is the nucleic acid sequence of the light chain variable region; SEQ ID NO: 10 is the heavy chain amino acid sequence; SEQ ID NO: 11 is the light chain amino acid sequence; SEQ ID NO: 12 is a heavy chain nucleic acid sequence; SEQ ID NO: 13 is a light chain nucleic acid sequence; SEQ ID NO: 14 is human sphingosine-1-phosphate receptor 3 protein A98~A114.

[0022] This invention discloses a fully human anti-human sphingosine-1-phosphate receptor 3 (S1PR3) antibody 2E3 and its applications. Specifically, it discloses the amino acid sequence, nucleic acid sequence, and complementarity-determining region (CDR) sequence encoding this full-molecule antibody, and its application in the preparation of diagnostic reagents and / or therapeutic drugs for metabolic disorders such as metabolic dysfunction-associated fatty liver disease (MASLD), obesity, and metabolic syndrome. Related experiments have confirmed that the fully human antibody of this invention can specifically recognize the A98~A114 epitope region of the human sphingosine-1-phosphate receptor 3 protein and exhibits a significant weight control effect in a high-fat diet-induced obese mouse model. Attached Figure Description

[0023] Figure 1 Screening of serum samples for fatty liver disease associated with obesity and metabolic dysfunction.

[0024] Figure 2 Flow cytometry sorting of S1PR3-specific anti-B cells; In the figure, A. Lymphocytes in PBMCs; B. Percentage of B cells in lymphocytes; C. Percentage of specific B cells that specifically bind to S1PR3 protein and do not bind to streptomycin.

[0025] Figure 3 Affinity chromatography purification diagram of anti-S1PR3 specific antibody 2E3. The anti-human sphingosine-1-phosphate receptor 3 antibody of this invention is named 2E3.

[0026] Figure 4 SDS-PAGE electrophoresis image of anti-S1PR3 specific antibody 2E3.

[0027] Figure 5 Image of ELISA detection of anti-S1PR3 specific antibody 2E3.

[0028] Figure 6 . Western Blot image of anti-S1PR3 specific antibody 2E3.

[0029] Figure 7 Affinity assay for anti-S1PR3 specific antibody 2E3.

[0030] Figure 8 Effects of anti-S1PR3 specific antibody 2E3 on Huh7 cell proliferation.

[0031] Figure 9 Effects of anti-S1PR3 specific antibody 2E3 on lipid accumulation in Huh7 cells.

[0032] Figure 10 Effect of anti-S1PR3 specific antibody 2E3 on body weight in mice with high-fat diet-induced obesity.

[0033] Figure 11 Effects of anti-S1PR3 antibody on lipid accumulation in the liver of MASLD mice induced by a high-fat diet.

[0034] Figure 12 Effects of anti-S1PR3 antibody on liver function and lipid metabolism in high-fat diet-induced MASLD mice. Detailed Implementation

[0035] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments.

[0036] Example 1: Screening of serum samples for fatty liver disease associated with obesity and metabolic dysfunction.

[0037] With informed consent, peripheral blood samples were collected from patients clinically diagnosed with obesity (OB) and metabolic dysfunction-associated fatty liver disease (MASLD) during their recovery period for serological titer screening of anti-S1PR3 antibodies. The collected blood was allowed to stand at room temperature for 1 hour before being centrifuged at 2000 rpm for 10 minutes. The milky-yellow supernatant was collected as serum and stored at -80°C.

[0038] Serum anti-S1PR3 antibody titer determination: 1) Use S1PR3 recombinant protein for coating. Dissolve the protein in a carbonate solution at pH 9.6 and adjust the concentration to 25 ng / ml. Add 100 μl of the prepared coating solution to each well of a 96-well microplate. Place the microplate in a refrigerator at 4°C for 12–16 h.

[0039] 2) Wash the plate 5 times using an ELISA fully automated plate washer, and add 200 μl of 2% BSA to each well and block overnight.

[0040] 3) Wash the ELISA plate 5 times using an automated plate washer. Dilute the collected serum samples 5-fold with sample diluent, add 50 μl of the sample to each well, and incubate at 37°C for 30 min.

[0041] 4) Wash the plate 5 times using an automated ELISA plate washer. Dilute the HRP-labeled antigen at a ratio of 1:10000, 50 μl per well, and incubate at 37°C for 30 min.

[0042] 5) Wash the plate 5 times using an automated ELISA plate washer. Add 100 μl of TMB to each well, and after 10 min, add 50 μl of ELISA stop solution to terminate the reaction.

[0043] 6) Place the microplate into the microplate reader, shake the plate and read the absorbance value at 450 nm, plot the graph and calculate the result.

[0044] The results showed that the OD value of anti-S1PR3 antibody in serum samples from MASLD patients was higher than that in serum samples from OB patients. Figure 1 ).

[0045] Example 2: Isolation of anti-S1PR3 specific B cells Isolation of PBMCs from anti-S1PR3 antibody-positive specimens: 1) Dilute the blood sample to be separated with an equal volume of PBS.

[0046] 2) Add an equal volume of lymphocyte separation solution to the centrifuge tube to the diluted blood sample. Spread the diluted blood sample evenly on the surface of the separation solution, keeping the interface between the two liquid surfaces clear.

[0047] 3) Centrifuge at 800 rpm for 30 minutes at room temperature, using the centrifuge's slow acceleration and deceleration function.

[0048] 4) After centrifugation, carefully aspirate the white film layer into a new centrifuge tube, dilute with PBS, and mix by inverting.

[0049] 5) Centrifuge at room temperature, 250g, for 10 minutes and discard the supernatant. Repeat washing twice.

[0050] 6) Resuspend using PBS buffer and count.

[0051] Specific B cell sorting: 1) Incubate streptomycin carrying a fluorescent tag and recombinant human S1PR3 protein A98~A114 epitope polypeptide carrying a biotin tag at a 1:4 molar ratio at 4°C for 60 min.

[0052] 2) Wash PBMCs with PBS, add 0.1 μl of fixable viabiling dye and 5 μl of FcR blocking solution, and incubate at 4°C for 15 min.

[0053] 3) After incubation, wash twice with PBS, add 0.5 μl of BIO-200, 1 μl of SA-BV421 / S1PR3-biotin, 1 μl of SA-APC / S1PR3-biotin, and 0.2 μl of SA-PE, and incubate at 4℃ for 60 min.

[0054] 4) After incubation, wash twice with PBS, add 5 μl each of anti-human CD19-FITC and anti-human CD45-APC-Cy7, and incubate at 4℃ for 30 min.

[0055] 5) Wash once with PBS after incubation.

[0056] 6) After filtering the above-treated cell suspension through a flow cytometer, single cells are sorted into 96-well plates using a flow cytometer.

[0057] The results showed that after flow cytometry sorting, CD19-FITC was selected from the separated PBMCs. + S1PR3-BV421 + S1PR3-APC + SA-PE - Specific B cells ( Figure 2 ).

[0058] Example 3: Anti-S1PR3 specific single B cell BCR sequencing 1) Add Bio-Single Cell Sequencing Library Preparation Reagent to the 96-well plate containing the single B cells collected and sorted above.

[0059] 2) Add cell lysis buffer to the sorted 96-well cell culture plates. The oligo-dT buffer is used to capture mRNA. The full-length cDNA coding strand was obtained using SMART-seq3xpress technology, and the oligo-dT index was used for subsequent cDNA library enrichment.

[0060] 3) The cDNA sequence of BCR was obtained by amplification using universal PCR primers provided on the Oligo. The BCR product was enriched through two rounds of specific semi-nested PCR.

[0061] 4) Human antibody variable region genome primers were used for PCR amplification and library construction.

[0062] 5) Preliminary analysis of the sequencing library. Statistical analysis of the BCR mapping rate of the samples, and the proportion of alignments to heavy and light chains (IGH, IGκ, IGλ). Individual cells are separated using the index and assembled using BCRV(D)J to obtain complete BCR sequencing results.

[0063] 6) By comparing with the IMGT antibody genome database, it was confirmed that mismatched, redundant and duplicate antibody gene sequences were removed.

[0064] 7) Use Clustal Omega software to perform phylogenetic tree analysis and screen and confirm candidate antibody gene sequences.

[0065] Example 4: Expression and purification of genetically engineered antibodies Preparation of antibody expression plasmids: The variable region nucleic acid sequence of the candidate anti-S1PR3 antibody (2E3) was synthesized from the whole genome.

[0066] Double enzyme digestion of the full-molecule anti-S1PR3 antibody expression plasmid: pFUSE-CHIg-hG1, pFUSE-CLIg-h λ The double enzyme digestion reaction system of the template vector is shown in Table 1: Table 1: Double enzyme digestion reaction system

[0067] The reaction conditions were: enzyme digestion overnight at 37 ℃.

[0068] 1) 1% agarose gel electrophoresis, followed by UV-cut gel recovery.

[0069] 2) Purify the target DNA fragment using a gel extraction kit and elute with deionized water.

[0070] 3) The heavy chain variable region nucleic acid of antibody 2E3 was recombined into the expression plasmid pFUSE-CHIg-hG1 containing the human IgG1 heavy chain constant region nucleic acid gene using in-fusion PCR; the light chain variable region nucleic acid of antibody 2E3 was recombined into the expression plasmid pFUSE-CLIg-hG1 containing the human light chain constant region nucleic acid. λ middle.

[0071] 4) Take 1µl of the reaction solution to transform competent bacteria, spread it on ampicillin-resistant LB agar plates, and incubate overnight at 37°C.

[0072] 5) Select positive clones the next day and send them for sequencing. Preserve the bacterial strain of clones with correct sequencing results, expand the culture, and extract plasmids.

[0073] Antibody expression in eukaryotic cell expression systems: 1) Process 293Freestyle cells, centrifuge, resuspend in 293F Hi-exp medium, stain with trypan blue, determine cell viability and count cells, and resuspend an appropriate amount of cells in a cell culture flask to achieve a cell density of 3 × 10⁻⁶ cells / year. 6 cells / ml. Cell viability ≥97%.

[0074] 2) Take 400µl of PEI solution and mix it with 2100µl of 293F Hi-exp medium. After mixing, let it stand for 5 minutes to obtain the transfection reagent dilution solution.

[0075] 3) Simultaneously prepare a plasmid dilution solution. Take 40µg of heavy chain plasmid and 60µg of light chain plasmid and mix them with 293F Hi-exp medium to make a total volume of 2.5ml.

[0076] 4) Slowly add the previously mixed transfection reagent dilution to the plasmid dilution, mix well, and incubate at 37°C for 30 min to allow the plasmid-transfection reagent complex to fully react and form.

[0077] 5) Slowly add the incubated plasmid-transfection reagent complex to the cultured cells to be transfected while shaking the flask. Incubate in a shaker incubator at 37°C, 8% CO2, and 125 rpm.

[0078] 6) 22 h after transfection, add 500 µl of 5% 293F Hi-exp feed and 450 µl of glucose concentrate to the cell shake flask while shaking it, and then put it back into the shaker incubator to continue culturing.

[0079] 7) Three days after transfection, add 670 µl of glucose concentrate to the cell shake flask and continue to culture in the shaker incubator.

[0080] 8) Collect the cell culture supernatant 5-7 days after transfection.

[0081] Affinity chromatography purification of antibodies: 1) Filter the collected cell culture supernatant through a 0.22 μm filter membrane.

[0082] 2) Add affinity chromatography equilibration solution at a volume ratio of 1:1 and pre-cool in a refrigerator at 4°C.

[0083] 3) Use the AKTA purifier100 instrument and select Protein A purification column to purify antibody proteins and collect the eluent.

[0084] 4) Use an ultrafiltration tube with a 30kDa cutoff and centrifuge at 4000 rpm for 20 min to obtain antibody ultrafiltrate.

[0085] 5) Measure the antibody concentration, label, adjust the volume, and then aliquot and freeze.

[0086] Figure 3 The image shows a protein UV detection pattern obtained using AKTA purifier100 affinity chromatography. In the early stage, the antibody adsorbs onto the Protein A column, and in the later stage, a sharp elution peak is visible after the antibody is eluted. Figure 4 The assays were performed using SDS-PAGE and Coomassie Brilliant Blue staining. Clear bands were observed at 55 kDa (antibody heavy chain) and 27 kDa (antibody light chain) in the supernatant and antibody elution lanes of 293 Freestyle cell culture cells co-transfected with the antibody expression plasmid. These bands are consistent with the theoretical values ​​for the antibody heavy and light chains, indicating that the antibody protein was specifically purified and enriched.

[0087] Example 5: Antibody Binding Ability Detection ELISA assay of binding of 2E3 antibody to specific peptides (A98~A114) of recombinant S1PR3 protein.

[0088] 1) Dilute the S1PR3 specific peptide with carbonate solution at pH 9.6 and plate it; 100 µl per well, concentration 5 ng / ml, incubate overnight at 4°C.

[0089] 2) Wash the plate 5 times using an automated ELISA plate washer, add 200µl of 2% BSA solution to each well to block, and incubate at 37°C for 2 hours.

[0090] 3) Wash the plate 5 times using an automated ELISA plate washer, serially dilute the anti-S1PR3 antibody (2E3) to an initial concentration of 4 µg / µl, 100 µl per well, set up 3 replicates, and incubate at 37°C for 2 h.

[0091] 4) Wash the plate 5 times using an automated ELISA plate washer, dilute the HRP-labeled goat anti-human secondary antibody at a ratio of 1:10000, 100µl per well, and incubate at 37℃ for 1h.

[0092] 5) Wash the plate 5 times using an ELISA fully automated plate washer, adding 100µl TMB to each well.

[0093] 6) After 5 minutes, add 50 µl of ELISA stop solution to terminate the reaction, and immediately place it under a microplate reader at a wavelength of 450 nm to measure the absorbance.

[0094] See results Figure 5 This indicates that the anti-S1PR3 antibody (2E3) can specifically bind to the S1PR3 protein-specific peptide, and the binding force of the antibody is positively correlated with the quality of the coated antigen.

[0095] Example 6: Antibody Binding Specificity Detection Western blot analysis of the binding ability of 2E3 antibody to S1PR3 antigen. 1) Collect HepG2 cells and HEK293T cells, add 100µl of cell lysis buffer to extract proteins.

[0096] 2) Add 5x SDS loading buffer and boil at 100℃ for 10 minutes.

[0097] 3) Prepare 10% SDS-PAGE and load 20µl per well. The total protein of HepG2 cells and the total protein of HEK293T cells are in one group. Load the two groups with protein markers in between and run the gel at 120V constant voltage for 45min.

[0098] 4) 200mA constant current membrane transfer for 90min.

[0099] 5) Seal with 5% skim milk for 60 minutes.

[0100] 6) Cut the PVDF membrane from the middle marker and incubate it with commercial S1PR3 antibody (1:1000 purchased from Proteintech cat NO#55204-AP) and 2E3 (1µg / ml) antibody at 4°C overnight.

[0101] 7) Wash 3 times with TBST, 10 min each time, add goat anti-rabbit-HRP secondary antibody and goat anti-human-HRP secondary antibody respectively, and incubate at room temperature for 1 h.

[0102] 8) Wash 5 times with TBST, 10 minutes each time.

[0103] 9) Exposure.

[0104] See results Figure 62E3 can bind to the S1PR3 protein in HepG2 cells, showing a band size that is the same as that of the commercial S1PR3 antibody indicator protein. No S1PR3 band was shown in the negative control HEK293T cells.

[0105] Example 7: Antibody Affinity Detection The instrument used in this experiment was the BLItz molecular interaction analyzer from Fortebio.

[0106] 1) Place the Protein A sensor corresponding to BLItz into a PBST solution (containing 0.2% BSA) and activate it for more than 10 minutes.

[0107] 2) Dilute the S1PR3 protein to 100 nM, 200 nM, 400 nM, 800 nM, and 1600 nM according to its molecular weight, and prepare a blank control of 0 nM.

[0108] 3) Dilute the antibody to 50µg / ml.

[0109] 4) Place the activated sensor into the instrument and run the baseline.

[0110] 5) After the baseline stabilizes, add 4µl of anti-S1PR3 antibody (2E3) for curing.

[0111] 6) Place the sensor in the PBST and run the baseline.

[0112] 7) Add different concentrations of S1PR3-specific peptides to the loading wells to bind with the sensor. A new sensor needs to be replaced each time.

[0113] 8) Place the sensor in a glycine solution at pH 1.7 for 5 seconds, then in PBST for 5 seconds, repeating this process 3 times for a total of 3 times to perform the regeneration operation.

[0114] 9) Analyze the binding and dissociation curves to obtain the affinity constant.

[0115] See results Figure 7 The affinity constant KD value for the interaction between the anti-S1PR3 antibody (2E3) and the S1PR3-specific peptide was 8.528 × 10⁻⁶. -8 M.

[0116] Example 8: Effect of antibody on Huh7 cell viability Detecting the effect of S1PR3 antibody 2E3 on Huh7 cell proliferation 1) One day before treatment, lay 3x10 mm PVC sheets in each well of a 96-well plate. 3 One Huh7 cell.

[0117] 2) After serially diluting the 2E3 antibody to a maximum concentration of 100µg / ml, mix it with Huh7 cell culture medium and replace the old culture medium.

[0118] 3) After 48 hours, dilute CCK-8 to a concentration of 10µL / 100µL and replace the old culture medium.

[0119] 4) OD450nm was measured 4 hours later.

[0120] Test results as follows Figure 8 2E3 concentrations of 4 µg / ml and 20 µg / ml significantly promoted the proliferation of Huh7 cells.

[0121] Example 9: Effect of antibody on lipid accumulation in Huh7 cells 1) Take Huh7 cells in the logarithmic growth phase, at 5 x 10⁻⁶ cells / year. 4 Seeds were placed per well into a 24-well plate and cultured for 12 hours to allow adhesion.

[0122] 2) Dilute the 2E3 antibody to a concentration of 4 µg / ml with fresh culture medium, and replace the old culture medium with 0.5 mM OA and 0.25 mM PA inducer, and continue culturing for 48 h.

[0123] 3) Fix with 4% paraformaldehyde for 30 min, then rinse twice with PBS.

[0124] 4) Add 1 ml of Oil Red O staining solution to each well and stain at room temperature for 30 min.

[0125] 5) Observe the number and size of red lipid droplets in the cells under a microscope and take pictures.

[0126] 6) Add 0.2 ml of lysis buffer to each well and determine the triglyceride content using the Nanjing Jiancheng triglyceride test kit. Detect the OD value at 500 nm using a microplate reader. Simultaneously, determine the protein content using the Beyotime BCA kit.

[0127] Test results as follows Figure 9 Compared to the control group, the 2E3 antibody reduced intracellular lipid accumulation in Huh7 cells by 28.91%.

[0128] Example 10. Detection of body weight in high-fat diet-induced MASLD mice using 2E3 antibody. 1) Six-week-old SPF-grade male C57BL / 6J mice were induced to be obese by a 60% high-fat diet for 6 weeks and then randomly divided into four groups: control group (HFD-PBS), low-dose group (HFD-5), medium-dose group (HFD-10), and high-dose group (HFD-20).

[0129] 2) On the basis of a high-fat diet, mice in each group were given antibody treatment at 5 mg / kg, 10 mg / kg and 20 mg / kg respectively via tail vein injection twice a week according to their grouping. The control group was treated with the same volume of PBS.

[0130] 3) Weigh the mice twice a week at a fixed time until the end of the 56-day experiment.

[0131] 4) Plot the curve of mouse weight change.

[0132] Test results as follows Figure 10 Compared to the control group, the mice treated with the medium and high doses showed a stable body weight trend, while the mice in the control group showed a continuous increase in body weight. At the end of the experiment, the body weight of mice in both the medium and high dose treatment groups was significantly lower than that of mice in the control group.

[0133] Example 11. Effect of 2E3 antibody on lipid accumulation in the liver of high-fat diet-induced MASLD mice 1) After the administration was completed, the mice were sacrificed and the liver tissue was removed and fixed with 4% paraformaldehyde for 24 h.

[0134] 2) OCT embedding, preparation of frozen sections, section thickness 6-10μm.

[0135] 3) Remove the frozen sections from the -20℃ freezer and allow them to warm to room temperature for 5-10 minutes. Then, immerse the sections in 60% isopropanol for 20 seconds.

[0136] 4) Immerse the sections in Oil Red O staining solution for 10 min, then differentiate them in 60% isopropanol to remove excess dye, and rinse with distilled water.

[0137] 5) Counterstain the sections in hematoxylin for 2 minutes, rinse with distilled water, and mount with glycerin-gelatin mounting medium.

[0138] 6) Take 50mg of liver homogenate and use a fully automated biochemical analyzer to determine the TG and TC content in the liver.

[0139] Test results as follows Figure 11 The liver TG content of mice treated with medium and high doses was significantly lower than that of mice in the control group.

[0140] Example 12. Effect of serological detection of anti-S1PR3 specific antibody on high-fat diet-induced MASLD mice 1) After fasting for 12 hours, mice were anesthetized with isoflurane, and 0.6-1.0 ml of blood was collected by removing the eyeballs and injected into a dry, sterile 1.5 ml centrifuge tube.

[0141] 2) Let it stand at room temperature for 30-60 minutes to allow the blood to coagulate fully, and the blood clot to shrink and precipitate serum.

[0142] 3) Centrifuge at 3000 rpm for 20 min at 4℃, and gently aspirate the upper layer of pale yellow serum with a pipette.

[0143] 4) Use a fully automated biochemical analyzer to determine the levels of TG, TC, ALT and AST in serum.

[0144] Test results as follows Figure 12 The serum TG levels in mice treated with different doses were significantly lower than those in the control group, while the ALT and AST levels were also lower, indicating that the anti-S1PR3 specific antibody can improve liver function and lipid metabolism.

[0145] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. A fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment, characterized in that, It contains VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3, VLCDR1 with amino acid sequences as shown in SEQ ID NO: 4, VLCDR2 with GNS and VLCDR3 with amino acid sequences as shown in SEQ ID NO:

5.

2. The fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment according to claim 1, characterized in that, It contains a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 6 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

7.

3. The fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment according to claim 1, characterized in that, It contains a heavy chain with an amino acid sequence as shown in SEQ ID NO: 10 and a light chain with an amino acid sequence as shown in SEQ ID NO:

11.

4. The fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment can specifically bind to amino acid residues 98-114 in the extracellular region of the human sphingosine-1-phosphate receptor 3 protein, as shown in SEQ ID NO:

14.

5. The fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody is a monoclonal antibody; the antigen-binding fragment of the antibody is selected from Fab, Fab', F(ab')2, Fv or scFv.

6. A nucleic acid encoding the fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment as described in any one of claims 1-5.

7. The nucleic acid according to claim 6, characterized in that, It also contains nucleic acid sequences encoding the heavy chain variable region as shown in SEQ ID NO: 8 and nucleic acid sequences encoding the light chain variable region as shown in SEQ ID NO:

9.

8. The nucleic acid according to claim 6, characterized in that, It also contains nucleic acid sequences encoding the heavy chain as shown in SEQ ID NO: 12 and nucleic acid sequences encoding the light chain as shown in SEQ ID NO:

13.

9. An expression vector comprising the nucleic acid of claim 6.

10. A transgenic cell line comprising the nucleic acid of claim 6 or the expression vector of claim 9.

11. A pharmaceutical composition, characterized in that, It comprises the fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment as described in any one of claims 1-5, and a pharmaceutically acceptable carrier or excipient.

12. A detection reagent or kit for detecting human sphingosine-1-phosphate receptor 3, characterized in that, The detection reagent or kit contains the fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment as described in any one of claims 1-5.

13. The use of the fully human anti-human sphingosine-1-phosphate receptor 3 antibody or its antigen-binding fragment as described in any one of claims 1-5 in the preparation of a medicament for the prevention or treatment of metabolic dysfunction-associated fatty liver disease (MASLD).

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