Monoclonal antibody specifically targeting human-derived slc19a1 and use thereof

Abstract

The present application relates to the technical field of antibodies, in particular to a monoclonal antibody specifically targeting human SLC19A1 and application thereof.The present application provides a monoclonal antibody specifically targeting human SLC19A1 and application thereof.The present application obtains human SLC19A1 protein by expression and purification through a eukaryotic expression system; then uses the obtained SLC19A1 protein as an immunogen to immunize mice to generate antibodies; finally, a monoclonal antibody specifically recognizing and combining SLC19A1 protein is obtained through flow enrichment and screening, and the epitope recognized and combined by the antibody is identified.It is verified that the monoclonal antibody provided by the present application can specifically recognize and combine the amino acid residues of human SLC19A1 protein, has high affinity, can be used as a molecular tool for detection of SLC19A1 protein, has high sensitivity and specificity, and has application advantages in detection of human SLC19A1 protein.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to a monoclonal antibody that specifically targets human SLC19A1 and its applications. Background Technology

[0002] SLC19A1 (solute carrier family 19 member 1), also known as reduced folate carrier (RFC1), is a highly conserved 12-transmembrane transporter in the solute carrier family. SLC19A1 actively pumps reduced folate (5-MTHF) and various antifolate drugs (such as methotrexate and pemetrexed) from the extracellular space into the cytoplasm, playing a crucial role in maintaining normal one-carbon metabolism for growth and development, as well as in the uptake of immunomodulatory drugs. Furthermore, SLC19A1 can activate the cGAS-STING immune signaling pathway by transporting cyclic dinucleotides (CDNs), playing an important role in pathogen immunity and tumor immunity. Given the key role of SLC19A1 in tumor immunity, inflammatory diseases, and folic acid metabolism, the development of high-affinity and high-specificity SLC19A1 antibodies can not only serve as molecular detection tools to assist in the detection and functional study of SLC19A1 protein expression, but also as potential biological agents to block CDN uptake or synergistically treat antifolate.

[0003] The principle of monoclonal antibody technology lies in the fact that B lymphocytes can produce antibodies but cannot divide indefinitely in vitro; while tumor cells, although they can be passaged indefinitely in vitro, cannot produce antibodies. The hybridoma cells obtained by fusing these two cell types possess the characteristics of both parental cell lines. The process of obtaining monoclonal antibodies includes animal immunization, cell fusion, cell screening, cloning, and characterization. Monoclonal antibodies can recognize specific single antigenic epitopes and exhibit high specificity.

[0004] Functional studies of the SLC19A1 protein and the development of related antibody drugs require the development of highly specific monoclonal antibodies. However, monoclonal antibodies that specifically target human SLC19A1 and bind to specific sites are still relatively scarce. In view of this, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a monoclonal antibody that specifically targets human SLC19A1 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.17, the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO.18, and the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO.19; 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.20, the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO.21, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.22.

[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. 14 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 14; 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. 16 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 16.

[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 SLC19A1 protein or cells expressing human SLC19A1 protein in a sample; (2) Application in detecting the presence or level of human SLC19A1 protein in a sample; (3) Application in detecting the presence or level of cells expressing human SLC19A1 protein in a sample.

[0015] Eighthly, the present invention provides a reagent or pharmaceutical composition for detecting human SLC19A1 protein, wherein the reagent or pharmaceutical composition for detecting human SLC19A1 protein comprises the antibody or its antigen-binding fragment, or comprises the bispecific antibody or multispecific antibody, or comprises the antibody conjugate.

[0016] Beneficial effects: This invention provides a monoclonal antibody specifically targeting human SLC19A1 and its applications. The invention involves expressing and purifying human SLC19A1 protein using a eukaryotic expression system; then using the purified SLC19A1 protein as an immunogen to immunize mice to generate antibodies; finally, flow cytometry enrichment and screening are used to obtain monoclonal antibodies that specifically recognize and bind to the SLC19A1 protein, and the epitopes recognized and bound by the antibodies are identified. Verification shows that the monoclonal antibody provided by this invention can specifically recognize and bind to amino acid residues of the human SLC19A1 protein, exhibiting high affinity. It can be used as a molecular tool for the detection of SLC19A1 protein, demonstrating high sensitivity and specificity, and has application advantages in the detection of human SLC19A1 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 are the affinity assay results for antibody 4 in Example 3. Figure 2 The results are the affinity assay results for antibody 10 in Example 3.

[0019] Figure 3 The results are from the flow cytometry analysis of antibody 4 in Example 3.

[0020] Figure 4 The results are from the flow cytometry analysis of antibody 10 in Example 3.

[0021] Figure 5 The results of Western blot detection of SLC19A1 protein with antibody No. 4 in Example 3 are shown.

[0022] Figure 6 The SLC19A1 protein was detected by Western blot using antibody No. 10 in Example 3. Detailed Implementation

[0023] This invention provides a monoclonal antibody that specifically targets human SLC19A1 and binds to a specific site. It can serve as a highly sensitive detection tool to aid in the study of SLC19A1 function and mechanism, and can also serve as a potential biological agent for subsequent drug development applications.

[0024] This invention first purifies and expresses the human SLC19A1 protein (its amino acid sequence is shown in SEQ ID NO.1) using an insect cell eukaryotic expression system. The purified protein is then used as an immunogen to immunize mice to generate corresponding antibodies. The BCR sequences of positive B cells from these immunized mice are then enriched by flow cytometry and analyzed to obtain potential monoclonal antibody sequences that specifically recognize and bind to SLC19A1. Subsequently, expression vectors for these monoclonal antibody sequences are constructed, and expression and purification are performed using 293F cells. Finally, the purified antibodies are screened by flow cytometry using cells expressing the human SLC19A1 protein and cells expressing a chimeric protein that partially replaces the mouse SLC19A1 protein. The monoclonal antibodies that specifically recognize and bind to SLC19A1 are ultimately selected, and the binding epitopes and functions of the corresponding antibodies are identified.

[0025] Among them, the amino acid sequence (SEQ ID NO.1) of the human SLC19A1 protein is as follows: MVPSSPAVEKQVPVEPGPDPELRSWRHLVCYLCFYGFMAQIRPGESFITPYLLGPDKNFTREQVTNEITPVLSYSYLAVLVPVFLLTDYLRYTPVLLLQGLSFVSVWLLLLLGHSVAHMQLMELFYSVTMAARIAYSSYIFSLVRPARYQRVAGYSRAAVLLGVFTSSVLGQLLVTVGRVSFSTLNYISLAFLTFSVVLALFLKRPKRSLFFNRDDRGRCETSASELERMNPGPGGKLGHALRVACGDSVLARMLRELGDSLRRPQLRLWSLWWVFNSAGYYLVVYYVHILWNEVDPTTNSARVYNGAADAASTLLGAITSFAAGFVKIRWARWSKLLIAGVTATQAGLVFLLAHTRHPSSIWLCYAAFVLFRGSYQFLVPIATFQIASSLSKELCALVFGVNTFFATIVKTIITFIVSDVRGLGLPVRKQFQLYSVYFLILSIIYFLGAMLDGLRHCQRGHHPRQPPAQGLRSAAEEKAAQALSVQDKGLGGLQPAQSPPLSPEDSLGAVGPASLEQRQSDPYLAQAPAPQAAEFLSPVTTPSPCTLCSAQASGPEAADETCPQLAVHPPGVSKLGLQCLPSDGVQNVNQ。

[0026] The amino acid sequence (SEQ ID NO.2) of the murine SLC19A1 protein is as follows: MVPTGQVAEKQAYEEPRQDHELKSWRCLVFYLCFFGFMAQLRPGESFITPFLLERKFTKEQVTNEIIPMLPYSHLAVLVPVFLLTDYLRYKPVLVLQCLSFVCVWLLLLLGTSVVHMQLMEVFYSVTM AARIAYSSYIFSLVHPSRYQRMASYSRAAVLLGVFISSVLGQALVTVGHISTYTLNCVSLGFILFSLVLSLFLKRPKRSLFFNRSTLARGALPCELDQMHPGPDRPETRKLDRMLGTCRDSFLVRMLS ELVENARQPQLRLWCLWWVFNSSGYYLITYYVHVLWRSTDSSLSYNGAVDAASTLLSAITSFSAGFLSIRWTLWSKLVIAGVIAIQASLVFCMFQIRDIWVCYVTFVLFRGAYQFLVPIATFQIASSL SKELCALVFGINTFLATALKTCITLVVSDKRGLGLQVRDQFRIYFIYFLMLLSITCFAWAGLDGLRYCQRGRHQPLAQAQELRSPLETSVQAISLQDGDLRGPQPSAPQLLSEDGMEDDRGDLRVEAKA.

[0027] In a more specific embodiment, the monoclonal antibody No. 4 prepared by the present invention can specifically recognize and bind to amino acid residues 215-225 of the human SLC19A1 protein, and can be used as a molecular tool for Western blot detection of SLC19A1 protein.

[0028] In a more specific embodiment, the monoclonal antibody No. 10 prepared by the present invention can specifically recognize and bind to amino acid residues 235-252 of human SLC19A1 protein, and can be used as a molecular tool to detect SLC19A1 protein in its natural, undenatured state.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Example 1 This example demonstrates the expression and purification of human SLC19A1 protein. The specific steps are as follows: (1) Gene synthesis and vector construction.

[0034] Synthetic human Slc19a1 The gene (GenBank: NM_001352512.2), fused with an 8*His tag at its C-terminus, was constructed into the insect cell expression vector pFastBac. The constructed plasmid vector was transformed into *E. coli* competent TOP10 strain, amplified, and the plasmid was extracted. Sequencing confirmed the sequence was correct, yielding the target plasmid vector pFAST-SLC19A1-His. This plasmid vector was then transformed into *E. coli* DH10Bac strain and plated on blue-white screening plates for selection. After colony growth, white colonies were selected for propagation and extraction of baculovirus (Bacmid).

[0035] (2) Protein expression and purification.

[0036] 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, dilute the mixture to 800 μL Grace medium and add it to pre-coated six-well sf9 cells (5 × 10⁶ cells per well). 5 (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 2000 rpm to obtain P1 virus. Then, P1 virus was added to 10 mL of sf9 cells at a ratio of 5%, and after culturing for another 72 hours, the supernatant was collected by centrifugation to obtain P2 virus. P2 virus was then further inoculated into 50 mL of sf9 cells, and after another 72 hours, the supernatant was collected by centrifugation to obtain P3 virus. P3 virus was inoculated into 1 L of 2×10⁶ 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 40 mL of Solution 1 (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 20 mMimidazole, 0.035% β-ME, 10% glycerol).

[0037] The resuspended cells were sonicated and disrupted. A final concentration of 1% (w / v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace) and 0.1% (w / v) Cholesteryl Hemisuccinate TrisSalt (CHS, Anatrace) was added, followed by centrifugation at 18,000 rpm for 30 minutes at 4°C. The precipitate was discarded, and the supernatant protein sample was collected. The supernatant was added to a pre-equilibrated nickel column. After all the protein was loaded, the column was washed with 5 column volumes of solution 1 supplemented with 0.06% glyco-diosgenin (GDN, Anatrace). The protein was then eluted with solution 2 (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 500 mM imidazole, 10% glycerol, 0.12% GDN). The eluted protein was concentrated to 500 μL by centrifugation in a 30 kDa concentrator. The protein was then further purified using a Superdex 200 gel chromatography column equilibrated with Solution 3 (20 mM Hepes pH 7.5, 150 mM NaCl, 0.01% GDN). After SDS-PAGE gel chromatography confirmed the purified target protein, it was concentrated to a concentration of 10 mg / ml, flash-frozen in liquid nitrogen, and stored at -80°C for later use.

[0038] Similar to the steps above, the SLC19A1 protein fused with the Flag-His tag at the C-terminus (SLC19A1-Flag-His) was further constructed, expressed, and purified for subsequent specific B cell enrichment.

[0039] Example 2 This example describes the preparation of a monoclonal antibody, and the specific steps are as follows: (1) Immunize BALB / c mice.

[0040] The purified SLC19A1 protein sample was diluted to 100 μg / mL with solution 3. CpG1826 (Invitrogen) diluted with PBS to a concentration of 150 μg / mL was mixed 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. 0.2 mL of suspension 2 was injected subcutaneously at multiple sites into 6-week-old BALB / c mice. On days 30 and 60 after the initial immunization, 0.2 mL of suspension 3 was injected twice more in the same manner. On day 90, a booster immunization was performed using SLC19A1 protein diluted to 25 μg / mL with 0.2 mL of unadjuvanted PBS.

[0041] (2) B cell enrichment and single-cell sequencing analysis of B cell receptor (BCR).

[0042] Seven days after booster immunization, mice were sacrificed, and spleen cells were isolated and extracted. After grinding and digestion, a spleen cell suspension was prepared. After co-incubation for 20 minutes with blocking buffer (PBS, 2 mM EDTA, 10% mouse serum, TruStain FcX™-labeled anti-mouse CD16 / 32 antibody (1:100, Biolegend)), the cells were co-incubated with previously purified SLC19A1-Flag-His protein for 30 minutes. Subsequently, the cells were washed once with FACS buffer (PBS, 2 mM EDTA, 2% BSA); after centrifugation, the suspension was prepared using a mixture containing APC-eFluor... TM 780-labeled anti-mouse CD3e antibody (Invitrogen), PerCP-Cy5.5-labeled anti-mouse B220 antibody (BioLegend), PE-Cy7-labeled anti-mouse CD19 antibody (BioLegend), Alexa Fluor TMCells were stained with fluorescently labeled antibodies, including 488-labeled anti-mouse GL7 antibody (Invitrogen), BV421-labeled anti-mouse CD38 antibody (BioLegend), DYKDDDDYTag (PE, BioLegend), and the LIVE / DEAD™ Fixable Violet Dead Cell Stain Kit (Yellow, Invitrogen), for 30 minutes. After washing once with FACS buffer, cells were centrifuged and resuspended. Flow cytometry was then performed to separate CD38 antibodies. - B220 + CD19 + CD38 - GL7 + Flag + B cells. The sorted positive cells were subjected to single-cell BCR sequencing (10x genomic) and analyzed using Cell Ranger Loupe VDJBrowser 4.0.0 software. The light and heavy chain sequences corresponding to antibodies 4 and 10 were selected for antibody cloning and expression.

[0043] Among them, the heavy chain variable region nucleic acid sequence of antibody No. 4 (SEQ ID NO.3) is: CAGATCCTGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTTATGCCTTCACAGTCTATTCAATGCACTGGGTGAAAACAGACTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAG CCAACATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGAAACCTCTGCCAGTACTGCCTATTTGCAGATCAACAATATCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGGGGGTACCGCCCTCCGGACTACTGGGGTCCAGGAACCTCAGTCACCGTCTCCTCAG.

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

[0045] The light chain variable region nucleic acid sequence (SEQ ID NO.5) of antibody 4 is as follows: AAATTATGATGACACAGTCGCCATCTCTGGCTGTGTCTGCAGGAGAAGGTCACTTTGAACTGTAAGTCCAGTCAAAGTGTTTTATACAGTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGG CATCCACTCGGGAATCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGAAGTGTACAAGCTGAAGACCTGGCAGTTTTATTACTGTCTTAACTACCTCTCCTCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAGAC.

[0046] The light chain variable region protein sequence (SEQ ID NO.6) of antibody 4 is as follows: NIMMTQSPSSLAVSAGEKVTLNCKSSQSVLYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTIRSVQAEDLAVYYCLNYLSSWTFGGGTKLEIR.

[0047] The amino acid sequences of each CDR in the variable region of antibody 4 are as follows: Heavy chain CDR1 protein sequence (SEQ ID NO.7): GYAFTVY.

[0048] Heavy chain CDR2 protein sequence (SEQ ID NO.8): NTETGE.

[0049] Heavy chain CDR3 protein sequence (SEQ ID NO.9): CARGYRPPDYW.

[0050] Light chain CDR1 protein sequence (SEQ ID NO.10): KSSQSVLYSSNQKNYLA.

[0051] Light chain CDR2 protein sequence (SEQ ID NO.11): WASTRES.

[0052] Light chain CDR3 protein sequence (SEQ ID NO.12): CLNYLSSWTF.

[0053] The heavy chain variable region nucleic acid sequence (SEQ ID NO.13) of antibody 10 is as follows: CAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTTGGGCTACACACTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATAGGAGATATTCATCAAGGAGGTAGTGGTACTGCCTACA ATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACATCTGAGGACTCTGCTGTCTATTACTGTACAAGAGAGGGACTTAACTGGGACTGGTACTTCGATATCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAG.

[0054] The heavy chain variable region protein sequence (SEQ ID NO.14) of antibody 10 is as follows: QVQLQQSGAELVRPGASVKLSCKALGYTLTDYEMHWVKQTPVHGLEWIGDIHQGGSGTAYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCTREGLNWDWYFDIWGAGTTVTVSS.

[0055] The light chain variable region nucleic acid sequence (SEQ ID NO.15) of antibody 10 is as follows: GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTACTGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTT CCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTCCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC.

[0056] The light chain variable region protein sequence (SEQ ID NO.16) of antibody 10 is as follows: DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSTGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPPTFGGGTKLEIK.

[0057] The amino acid sequences of each CDR in the variable region of antibody 10 are as follows: Heavy chain CDR1 protein sequence (SEQ ID NO.17): GYTLTDY.

[0058] Heavy chain CDR2 protein sequence (SEQ ID NO.18): HQGGSG.

[0059] Heavy chain CDR3 protein sequence (SEQ ID NO.19): CTREGLNWDWYFDIW.

[0060] Light chain CDR1 protein sequence (SEQ ID NO.20): RSSQSIVHSTGNTYLE.

[0061] Light chain CDR2 protein sequence (SEQ ID NO.21): KVSNRFS.

[0062] Light chain CDR3 protein sequence (SEQ ID NO.22): CFQGSHVPPTF.

[0063] (3) Expression and purification of target antibody The target antibody sequence obtained in the previous step was analyzed to obtain the corresponding light and heavy chain sequences. After eukaryotic codon optimization, the light chain variable region sequence was constructed into a pTT3 plasmid containing the mouse light chain κ chain constant region, and the heavy chain variable region sequence was constructed into a pTT3 plasmid containing the mouse heavy chain lgG2a constant region. After the constructed vector was verified by sequencing, it was transformed into E. coli competent cells for amplification, and plasmid extraction was performed using an endotoxin-free plasmid extraction kit.

[0064] The extracted light and heavy chain expression plasmids were co-transfected into 293F cells using PEI. The cells were cultured in a shaker at 37°C and 5% CO2 for 5 days, and the supernatant was collected. The collected cell supernatant was filtered and loaded onto a PBS-equilibrated Protein A column. After washing with 5 column volumes of precipitate, the cells were eluted with 0.1 M Glycine (pH 2.7). The eluted sample was concentrated and the solution was replaced with PBS.

[0065] 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.

[0066] First, SLC19A1 protein was diluted to six concentration gradients (0.8 μM, 0.4 μM, 0.2 μM, 0.1 μM, 0.05 μM, 0.025 μM) using Solution 3 (20 mM Hepes pH 7.5, 150 mM NaCl, 0.01% GDN). The antibody to be tested was then diluted to a concentration of 25 μg / ml. After setting up the program on the biomembrane interferometer (ForteBio Octet Red96), detection was performed using Octet ProG Biosensors. The final data were processed to obtain the affinity (K). D ), of which antibody 4 has a high affinity (K D The value was 42.42 ± 3.47 nM. Figure 1 The affinity of antibody 10 was 75.22 ± 4.28 nM. Figure 2 ).

[0067] (2) Flow cytometry identification of antibody-binding epitopes.

[0068] To identify the binding epitopes of the above monoclonal antibodies, a series of truncated and chimeric variants of human SLC19A1 were designed (T1 (amino acids 215-252 replaced with amino acids 213-252 of the mouse protein); T2 (amino acids 215-225 replaced with amino acids 213-222 of the mouse protein); T3 (amino acids 235-252 replaced with amino acids 232-252 of the mouse protein)). These were then 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 in PBS containing 2% PFA for 20 minutes. The cell pellet was then collected by centrifugation at 1500 rpm and resuspended in perforation buffer. After another centrifugation at 1500 rpm, the pellet was collected, and the antibody to be tested, diluted 1:1000 with perforation buffer, was added to the sample and incubated for 30 minutes. After centrifugation to discard the supernatant, the samples were resuspended in PBS and washed to remove excess unbound antibodies. Next, the samples were resuspended in a 1:1000 dilution of goat anti-mouse IgG secondary antibody diluted with membrane rupture buffer and incubated for 30 minutes. Centrifugation was then performed again, and excess secondary antibody was washed away with PBS. Finally, the samples resuspended in PBS were subjected to flow cytometry screening. Flow cytometry screening first used the pTT3 empty vector as a control to screen for a batch of monoclonal antibodies that could significantly bind to human SLC19A1. Then, cells expressing the SLC19A1 chimeric protein T1, which replaced the mouse protein sequence, were further screened to obtain antibodies 4 and 10 that specifically recognize and bind to amino acid residues 215-252 of the human SLC19A1 protein. Figure 3 , Figure 4 Furthermore, flow cytometry results of cells expressing SLC19A1 chimeric T2 and T3 showed that antibody 4 specifically binds to amino acid residues 215-225. Figure 3 ), while antibody 10 specifically binds to amino acid residues 235-252 ( Figure 4 ).

[0069] (3) Western blot identification of antibody-binding epitopes.

[0070] Furthermore, the detection function of the monoclonal antibodies was further analyzed by Western blot. The previously purified human SLC19A1 protein was subjected to SDS-PAGE and then transferred to an NC membrane. After blocking with blocking buffer for 1 hour, antibodies 4 and 10 were diluted 1000-fold with antibody dilution buffer as primary antibodies 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 thorough washing with PBST, the membrane was developed. The results showed that antibody 4 had a significant positive band (…). Figure 5 ), while antibody number 10 did not ( Figure 6This indicates that antibody 4 likely recognizes a linear epitope of SLC19A1 and can be used for Western blot detection of the SLC19A1 protein. Antibody 10, on the other hand, likely recognizes a spatial epitope of SLC19A1 and can be used for detection of the native, non-denaturing conformation of the SLC19A1 protein.

[0071] 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, 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.17, the amino acid sequences of the heavy chain CDR2 are shown in SEQ ID NO.18, and the amino acid sequences of the heavy chain CDR3 are shown in SEQ ID NO.19; the amino acid sequences of the light chain CDR1 of the antibody or its antigen-binding fragment are shown in SEQ ID NO.20, the amino acid sequences of the light chain CDR2 are shown in SEQ ID NO.21, and the amino acid sequences of the light chain CDR3 are shown in SEQ ID NO.

22.

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 as shown in SEQ ID NO. 14 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO. 14; 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. 16 or has at least 80% similarity to the amino acid sequence shown in SEQ ID NO.

16.

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 antibody, Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, and single-chain antibody.

4. A bispecific antibody or multispecific antibody comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

5. 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.

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

7. An antibody conjugate, characterized in that, It is obtained by conjugating the antibody or its antigen-binding fragment according to any one of claims 1 to 3 or the bispecific antibody or multispecific antibody according to claim 4 with a label or protein; 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.

8. A 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.

9. Any one of the following applications of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the bispecific antibody or multispecific antibody according to claim 4, or the nucleic acid molecule according to claim 5, or the biomaterial according to claim 6, or the antibody conjugate according to claim 7: (1) Use in the preparation of products for detecting the presence or level of human SLC19A1 protein or cells expressing human SLC19A1 protein in a sample; (2) Application in detecting the presence or level of human SLC19A1 protein in a sample; (3) Application in detecting the presence or level of cells expressing human SLC19A1 protein in a sample.

10. A reagent or pharmaceutical composition for detecting human SLC19A1 protein, characterized in that, The reagent or pharmaceutical composition for detecting human SLC19A1 protein comprises the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3, or comprises the bispecific antibody or multispecific antibody as described in claim 4, or comprises the antibody conjugate as described in claim 7.

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