Anti-SLC34A2 antibody or antigen binding fragment thereof and application thereof

Abstract

The invention relates to an anti-SLC34A2 antibody or an antigen binding fragment thereof and application thereof, the anti-SLC34A2 antibody or the antigen binding fragment thereof lays a good foundation for immunotherapy such as ADC (Analog to Digital Converter) and CAR-T (Chimeric Antigen Receptor-T) of a NaPi2b target by virtue of high-level affinity of the anti-SLC34A2 antibody or the antigen binding fragment thereof, breaks through the bottleneck of the prior art by virtue of multi-dimensional optimization, and provides a more efficient and safer solution for precise therapy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an antibody or antigen-binding fragment thereof against SLC34A2 and application thereof. BACKGROUND

[0002] Ovarian cancer is one of the diseases with the highest mortality rate among gynecological malignancies, and its treatment has long relied on platinum-based chemotherapy and PARP inhibitors. However, the prognosis of patients with platinum resistance is still not ideal, and new targeted therapies are urgently needed. In recent years, sodium-dependent phosphate transporter NaPi2b (SLC34A2) has become a research hotspot due to its high expression in various solid tumors such as ovarian cancer and lung cancer, and its limited distribution in normal tissues. However, its clinical translation is still limited by three major challenges: the spatiotemporal heterogeneity of tumor antigen expression, the lack of a standardized biomarker detection system, and the insufficient adaptability of existing antibodies to complex tumor microenvironments. In the prior art, Lifastuzumab Vedotin (LIFA) developed by Genentech is the first NaPi2b-targeted ADC to enter clinical trials, which achieves targeted killing through a monomethyl auristatin E (MMAE) payload and a protease-sensitive linker. Phase II clinical trial data showed that LIFA had an objective response rate (ORR) of 36% in platinum-resistant ovarian cancer patients with high NaPi2b expression, but the overall efficacy did not reach a statistically significant difference. Upliftamab Rilsodotin (UpRi) developed by Mersana Therapeutics has a higher drug-to-antibody ratio through innovative design, showing better safety characteristics in I / II phase trials. The ORR of patients with a NaPi2b tumor proportion score (TPS) of 75% or higher was 34%, but its detection system still cannot effectively identify subgroups with membrane expression loss. In addition, the MX35 antibody in early research achieved intraperitoneal targeted therapy through radioisotope coupling, but failed to achieve clinical translation due to antigen distribution heterogeneity and radiation dose control difficulties. The above technologies collectively expose the shortcomings of existing solutions: the antibody design is limited to a single epitope, which is difficult to adapt to the complex expression patterns of NaPi2b in tumors (such as membrane surface, cytoplasm, or mixed localization), and the biomarker detection lacks multi-dimensional integration, resulting in a high false negative rate. SUMMARY

[0003] To solve the above problems in the prior art, the application provides a technical solution to solve the above problems. Specifically, In a first aspect, the present application provides an anti-SLC34A2 antibody or an antigen-binding fragment thereof, comprising CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region; and / or, CDR-L1, CDR-L2 and CDR-L3 of a light chain variable region, wherein, the amino acid sequence of CDR-H1 comprises NX1LIE (SEQ ID NO: 34), the amino acid sequence of any one of SEQ ID NOs: 1-2, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 1-2; the amino acid sequence of CDR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 5-6, IINPGSGGX2NYX3X4KFKX5 (SEQ ID NO: 35), or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 5-6, IINPGSGGX2NYX3X4KFKX5 (SEQ ID NO: 35); the amino acid sequence of CDR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 10-11, GETX6VX7AX8SDX9DMDX 10 (SEQ ID NO: 36), or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 10-11, GETX6VX7AX8SDX9DMDX 10 (SEQ ID NO: 36); the amino acid sequence of CDR-L1 comprises the amino acid sequence of any one of SEQ ID NOs: 15-17, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 15-17; the amino acid sequence of CDR-L2 comprises the amino acid sequence of any one of SEQ ID NOs: 18-20, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 18-20; the amino acid sequence of CDR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 21-22, QQYX 11 X 12 X 13 PLT (SEQ ID NO: 37), the amino acid sequence of any one of SEQ ID NOs: 23, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 21-22, QQYX 11 X 12 X 13an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NO: 37, SEQ ID NO: 23.

[0004] wherein, X1-X 13 is any natural amino acid residue, for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), valine (V).

[0005] In one embodiment of the application, X1in SEQ ID NO: 34 represents Y or F; X2in SEQ ID NO: 35 represents N or T, X3X4represents NV, NE or SE, X5represents D or G; X6in SEQ ID NO: 36 represents T or S, X7represents V or I, X8represents T or K, X9represents Y or C, X 10 represents Y or N; X 11 represents Y or N; X 12 represents Y or N; X 13 represents L or T.

[0006] Preferably, the CDR-H1 comprises an amino acid sequence represented by any one of SEQ ID NO: 1-4 or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NO: 1-4.

[0007] Preferably, the CDR-H2 comprises an amino acid sequence represented by any one of SEQ ID NO: 5-9 or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NO: 5-9.

[0008] Preferably, the CDR-H3 comprises an amino acid sequence represented by any one of SEQ ID NO: 10-14 or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NO: 10-14.

[0009] Preferably, the CDR-L3 comprises an amino acid sequence represented by any one of SEQ ID NO: 21-23 or an amino acid sequence having at least 80% identity to the amino acid sequence represented by any one of SEQ ID NO: 21-23.

[0010] In one embodiment of the present application, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 comprise any one of the following groups: A) SEQ ID NO: 1, 5, 10; B) SEQ ID NO: 2, 6, 11; C) SEQ ID NO: 3, 7, 12; D) SEQ ID NO: 4, 8, 13; E) SEQ ID NO: 3, 9, 14.

[0011] In one embodiment of the present application, the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 comprise any one of the following groups: A) SEQ ID NO: 15, 18, 21; B) SEQ ID NO: 16, 19, 22; C) SEQ ID NO: 17, 20, 23.

[0012] Preferably, the amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are arranged in the order from N-terminal to C-terminal. In the present application, the division of amino acids of antibody CDR region adopts Kabat numbering system.

[0013] Preferably, the heavy chain of the anti-SLC34A2 antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences shown in SEQ ID NO: 24-28 or an amino acid sequence having at least 80% identity to any one of the amino acid sequences shown in SEQ ID NO: 24-28.

[0014] Preferably, the light chain of the anti-SLC34A2 antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences shown in SEQ ID NO: 29-33 or an amino acid sequence having at least 80% identity to any one of the amino acid sequences shown in SEQ ID NO: 29-33.

[0015] Preferably, the antibody or antigen-binding fragment thereof of the present application is a whole antibody, a bi- or multi-specific antibody, a bi- or multi-specific single-chain antibody, a single-chain antibody (scFv), a nanobody, a dAb fragment, a Fab, a Fab', a F(ab')2, a linear antibody or a Fv antibody.

[0016] Preferably, the antibody or antigen-binding fragment thereof binds to SLC34A2 protein.

[0017] In a second aspect of the present application, there is provided a method for producing an anti-SLC34A2 antibody or antigen-binding fragment thereof as described above, the method comprising immunizing a non-human animal with an SLC34A2 antigen and screening for an antibody or antigen-binding fragment thereof that specifically binds to SLC34A2.

[0018] Preferably, the non-human animal is a non-human mammal. Further preferably, the non-human mammal is a rodent.

[0019] In one embodiment of the present application, the non-human animal is a rat or a mouse.

[0020] In a third aspect of the present application, there is provided a biological material comprising: 1) a nucleic acid sequence encoding an anti-SLC34A2 antibody or antigen-binding fragment thereof as described above or obtained by the method as described above; 2) a vector comprising the nucleic acid of 1); and 3) a cell comprising the nucleic acid of 1) and / or the vector of 2). Preferably, the nucleic acid comprises DNA and / or mRNA.

[0021] In some embodiments, the nucleic acid is DNA, which encodes an antibody or antigen-binding fragment as described above.

[0022] Preferably, the vector is capable of being expressed in a prokaryotic or eukaryotic cell.

[0023] For example, the expression vector can be introduced into a cell by transient transfection or stable transfection.

[0024] Preferably, the cell can be a eukaryotic cell or a prokaryotic cell.

[0025] The eukaryotic cell includes animal and plant cells, such as T cells, yeast cells, HEK293 cells, CHO cells, and the like.

[0026] The prokaryotic cell is, for example, E. coli and the like.

[0027] In a fourth aspect of the present application, there is provided a method for producing an anti-SLC34A2 antibody or antigen-binding fragment thereof as described above, the method comprising culturing a cell as described above to express the anti-SLC34A2 antibody or antigen-binding fragment thereof.

[0028] In a fifth aspect of the present application, there is provided a product for treating and / or preventing a disease, the product comprising any one of:

[0029] ​A) the anti-SLC34A2 antibody or antigen-binding fragment thereof described above or the anti-SLC34A2 antibody or antigen-binding fragment thereof obtained by the preparation method described above; or B) the biomaterial described above.

[0030] Preferably, the product can be a kit or a drug or a chip or an antibody-drug conjugate, etc.

[0031] Preferably, the disease is a disease associated with SLC34A2, and the disease includes but is not limited to ovarian cancer, non-small cell lung cancer, breast cancer, thyroid cancer, endometrial cancer, or pulmonary alveolar microlithiasis.

[0032] In a sixth aspect of the present application, the A) to B) described above are used in the preparation of a product for treating and / or preventing a disease associated with SLC34A2.

[0033] The disease includes but is not limited to ovarian cancer, non-small cell lung cancer, breast cancer, thyroid cancer, endometrial cancer, or pulmonary alveolar microlithiasis.

[0034] The product can be a drug.

[0035] The product can be a kit or a chip.

[0036] In a seventh aspect of the present application, a detection method of SLC34A2 is provided, and the detection method comprises combining a sample to be detected with the anti-SLC34A2 antibody or antigen-binding fragment thereof described above or the anti-SLC34A2 antibody or antigen-binding fragment thereof obtained by the preparation method described above, and then detecting the content of a complex formed by SLC34A2 or a variant thereof and the anti-SLC34A2 antibody or antigen-binding fragment thereof.

[0037] The detection method is for detecting the presence or content of SLC34A2. The presence means the presence or absence, and the content can be the expression amount or the protein concentration, etc.

[0038] Preferably, the method comprises diagnosis or treatment of a disease.

[0039] Preferably, the method does not involve diagnosis or treatment of a disease.

[0040] In an eighth aspect of the present application, a method for treating and / or preventing a disease is provided, and the method comprises applying the product for treating and / or preventing a disease described above to an individual.

[0041] The disease includes but is not limited to ovarian cancer, non-small cell lung cancer, breast cancer, thyroid cancer, endometrial cancer, or pulmonary alveolar microlithiasis.

[0042] The "medicament" of the present application can be used for treating a human or a non-human animal, such as a non-human mammal. The medicament can comprise a pharmaceutically acceptable carrier, adjuvant or salt as commonly used in the art.

[0043] The medicament can be administered by any suitable route, such as a gastrointestinal route (e.g., oral) or a non-gastrointestinal route (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.).

[0044] The medicament can be in any suitable dosage form, such as a gastrointestinal dosage form or a non-gastrointestinal dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nose drops, sublingual tablets, suppositories, aerosols, effervescent tablets, dripping pills, gels, etc. The various dosage forms of the medicament can be prepared according to conventional methods in the pharmaceutical field.

[0045] The "pharmaceutically acceptable" of the present application means neither significantly stimulating the organism nor inhibiting the biological activity and characteristics of the active substance of the administered product.

[0046] The "… method" of the present application can be for the diagnosis, treatment and / or prevention of a disease, or for the diagnosis, treatment and / or prevention of a non-disease.

[0047] The "antigen-binding fragment" of the present application is a part of an antibody that retains the specific binding activity of the antibody, i.e., any part of an antibody can specifically bind to an epitope on the target molecule of the antibody. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd and variants of these fragments. For example, a heavy chain and / or a light chain of an antibody, a heavy chain variable region and / or a light chain variable region of an antibody, or a single or more than two CDRs from a heavy chain or a light chain of an antibody. Among them, Nanobody or single-domain antibody refers to the variable domain of heavy chain (VHH) of an antibody, which has independent antigen binding activity.

[0048] Single-chain antibody is an antibody formed by connecting the variable domain of the heavy chain and the variable domain of the light chain through a linker.

[0049] Fab, i.e., a monovalent fragment consisting of VL, VH, CL and CH1 domains.

[0050] Fab', i.e., a Fab fragment having one or more cysteine residues at the C-terminus of the CH1 domain.

[0051] F(ab')2, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.

[0052] Fd, an Fd fragment consisting of the VH and CH1 domains.

[0053] Fv, a fragment consisting of the VL and VH domains of a single arm of an antibody.

[0054] dAb fragment, a fragment consisting of a VH domain.

[0055] The "linear antibodies" described herein comprise one or more pairs of tandem antibody fragments, which can be Fd segments (VH-CH1), single-chain Fv (scFv), Fab fragments, or single-domain antibodies (VHH), connected together via linkers.

[0056] wherein VH represents a heavy chain variable region, VL represents a light chain variable region, CH represents a heavy chain constant region, and CL represents a light chain constant region.

[0057] The "comprising" or "including" described herein is open-ended, when used in describing compositions of matter, such as proteins or nucleic acids, the proteins or nucleic acids can be composed of the recited sequences, or can have additional amino acids or nucleotides at one or both ends of the proteins or nucleic acids, but still have the same or similar activity as the original sequences.

[0058] The "homology" or "identity" as used herein refers to the degree of sequence relatedness between a test sequence and a reference sequence as determined by the alignment of sequences and comparison of sequence elements. The skilled artisan can adjust the sequence to meet the actual work needs, so that the use of the sequence has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology / identity compared with the sequence obtained by the prior art.

[0059] The "individual" as used herein can be a human or a non-human mammal, which can be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, and the like. Preferably, the non-human mammal includes but is not limited to a pig, a cow, a sheep, a horse, a donkey, a fox, a raccoon dog, a mink, a camel, a dog, a cat, a rabbit, a mouse (e.g., a rat, a mouse, a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel), or a monkey, and the like.

[0060] The "treatment" as used herein means to slow down, interrupt, stop, control, reduce, or reverse the progression or severity of a sign, a symptom, a disorder, a disease, or a disease-related sign, symptom, disorder, or the like, after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, disorders, or the like.

[0061] The "prevention" as used herein means a way to prevent or delay the occurrence of a disease or a disorder or a symptom in the body.

[0062] The "diagnosis" as used herein means to ascertain whether a patient has a disease or a disorder in the past, at the time of diagnosis, or in the future, or to ascertain the progression or possible future progression of a disease.

[0063] The beneficial effects of the present application: The present patent designs five new antibodies targeting NaPi2b, which are directed against different epitopes of NaPi2b to adapt to the complex expression pattern of NaPi2b in tumors, have better affinity than the two positive antibodies reported previously, and lay a good foundation for immunotherapy such as ADC and CAR-T targeting this target, break through the existing technical bottleneck through multidimensional optimization, and provide more efficient and safe solutions for precision treatment. BRIEF DESCRIPTION OF DRAWINGS

[0064] Figure 1 SDS-PAGE detection of SLC34A2-MP-F13-mFc recombinant protein preparation.

[0065] Figure 2 SDS-PAGE detection of SLC34A2-MP-F17-hFc recombinant protein preparation.

[0066] Figure 3 SDS-PAGE detection of 5 recombinant antibodies expression.

[0067] Figure 4 Antibody EC 50 Overlay plot results.

[0068] Figure 5 IHC experiment staining results. DETAILED DESCRIPTION

[0069] In order to enable the personnel in the technical field to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor should belong to the scope of protection of the present application.

[0070] The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0071] Some of the materials are shown in the following table.

[0072] Table 1 Sources of main reagents Example 1 Screening and preparation of antibodies targeting SLC34A2 1. Preparation of SLC34A2-MP-F13-mFc and SLC34A2-MP-F17-hFc recombinant proteins 1.1 Preparation of SLC34A2-MP-F13-mFc recombinant protein 1.1.1 Expression process a. Transfect HEK293F cells growing in exponential phase with a viability of greater than 98%; dilute the cell density to 2 x 10 6 cells / mL; b. Place the flask in a 5% CO2, constant temperature shaker at 37°C, 120 rpm constant temperature shaking for 10 min before transfection; c. Prepare two 15 mL sterile centrifuge tubes, add 5 mL plasmid dilution buffer and 100 μg sterile plasmid DNA to one of the tubes, and mix gently; add 5 mL transfection reagent dilution buffer and 0.5 mL transfection reagent to the other centrifuge tube, and mix gently; d. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and mix gently; e. Prepare the plasmid-carrier complex at room temperature for 10 min; f. Take the cells from the constant temperature shaker, add the prepared plasmid-carrier complex while shaking, and return to the CO2 constant temperature shaker for shaking culture; g. Add 2 mL of feed after 24 h of transfection to increase the expression of the product; h. On the fourth day after transfection, collect the cell culture medium, centrifuge at 1000 g at 4°C for 15 min, and take the supernatant for purification.

[0073] 1.1.2 Protein A column purification a. Wash 5 column volumes with water, then wash 5 column volumes with 20 mM PB + 150 mL NaCl (pH 7.0), and blow the column material with a gun head, and make sure that the column material does not clump; b. Filter the sample liquid with a 0.45 μm filter, and add 0.15 M NaCl to the protein sample liquid; c. Sample, and let the protein drop drop by drop, and sample at least 2 times; d. Wash the impurities with 20 mM PB + 150 mL NaCl (pH 7.0). One drop should be colorless when detected with G250; e. Elute with 0.1 M glycine (pH 3.5), control the flow rate, and neutralize with 2 M Tris-HCl (pH 8.0) while connecting, and the pH paper should be light green. Stop elution when one drop is colorless when detected with G250; f. Note: After the protein is eluted, continue to elute the column with 3.5% acetic acid for 20 column volumes. Slowly elute 5 column volumes (like the elution of the target protein), and quickly elute 15 column volumes. Meanwhile, blow the column material with a gun head, and make sure that the column material does not clump; g. Put the eluted protein into dialysis bag, 4°C, 5L lxPBS dialysis, after 2h dialysis, change 5L lxPBS to continue dialysis; h. The next day, ultrafiltration, SDS-PAGE and quality control concentration and purity.

[0074] SLC34A2-MP-F17-hFc was obtained by a similar method.

[0075] 1.1.3 Experimental results The experimental results are shown in Figure 1 and 2 The protein was arranged for quality control, and the test information is shown in Table 2: Table 2 Protein test results 1.2 The protein was subjected to quality control detection, and it was confirmed that it could be used for subsequent immunization.

[0076] 1.3 Construction of MC38-SLC34A2 stable cells 1.3.1 Cell preparation of MC38-SLC34A2 pool cells a. Virus packaging: Take HEK293T cells in good condition and in logarithmic growth phase for lentivirus packaging. Mix the plasmid and use PEI reagent to transiently transfect HEK239T cells, mix gently, and then put it in the incubator for 72h, then collect the virus supernatant for standby.

[0077] b. Virus infection: inoculate an appropriate amount of HEK239T cells into a 10cm culture dish, and after the cells adhere, add the virus supernatant according to virus supernatant: complete culture medium = 1:1, culture for 8h, and then replace the fresh culture medium for continuous culture.

[0078] c. Drug screening: After 48h of cell culture, inoculate an appropriate amount of cells into a 6-well cell culture plate, and use the corresponding antibiotic in the scheme to perform gradient drug screening on the infected cells, and flow cytometry detection after one week of drug screening.

[0079] 1.3.2 FACS detection of MC38-SLC34A2 pool cells 1.3.2.1 FACS detection process a. Cell treatment: collect MC38-SLC34A2 pool cells, centrifuge, discard supernatant, resuspend with PBS to wash off the culture medium, add 10% normal non-immune goat serum (diluted with PBS) for resuspension, and block the cells at 4°C for 45min (incubate on a silent mixer); b. Incubate the primary antibody: divide the cells into tubes, about 3x10 5Cells, centrifuged at 1300 rpm for 4 min to remove the blocking solution, and the antibody RebmAb200 (CSB-RA021581MA1HU) and the isotype control hIgG1 were diluted to 20 μg / mL with 5% goat serum. The primary antibody diluent was added, and the cells were incubated at 4°C for 45 min (incubated on a silent mixer, and note that the light was avoided). c. Incubation of secondary antibody: centrifuge to remove the primary antibody diluent, resuspend in PBS, and wash the remaining primary antibody; dilute the goat anti-human secondary antibody-APC with 5% goat serum, and add 50 μL of the corresponding secondary antibody diluent to each tube, and incubate at 4°C for 35 min (incubate on a silent mixer, and note that the light is avoided); after incubation, centrifuge to remove the secondary antibody diluent, resuspend in PBS, and wash the remaining secondary antibody. The cells are detected by flow cytometry.

[0080] The MC38-SLC34A2 pool APC flow cytometry results show that the SLC34A2 antibody has no background on the MC38 cells. The cells that are positive in the MC38-EF detection are selected for further monoclonal and FACS detection.

[0081] 1.3.3 Monoclonalization of MC38-SLC34A2-EF pool cells a. Count the cells and inoculate the monoclonal cells; b. Observe the growth state of the clones during the period, and replace the liquid in time. The drug is used to maintain the monoclonalization period; c. When the clones grow to a sufficient number, select the clones with a good growth state for subsequent flow cytometry detection; d. Select the best single clone for expansion, and the qualified clones are frozen and delivered after mycoplasma detection.

[0082] 1.3.4 FACS detection of monoclonal cells and mycoplasma detection 1.3.5 Select the best cell with the best expression effect in the flow cytometry detection results for further subcloning and FACS detection and mycoplasma detection The cell strain with the best peak chart, positive rate, and MFI results and negative mycoplasma detection results (referred to as "MC38-SLC34A2" below) is selected for further fusion screening and flow cytometry verification.

[0083] 2. Mouse immunization and fusion screening 2.1 Immunization grouping and immunization time arrangement Table 3 Immunization grouping 2.2 Mouse immunization process a. Protein processing: Freund's adjuvant: take the protein diluted with PBS and mixed with Freund's adjuvant at a volume ratio of 1:1, emulsified at 4°C for 3-5 min to ensure the shortest emulsification time, Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for the subsequent boost; b. Protein injection: Freund's adjuvant: transfer the emulsified protein into a 1 mL syringe, and remove the bubbles in the syringe. Foam box ice bag storage (emulsified on the same day, immunized on the same day). Take the mice to be immunized from the cage and place them in a specially designed fixing frame, and perform multiple subcutaneous injections on the back (when immunized for the first time, generally 5-6 points, among which the neck and the back of the limbs near the center are the best). When injecting, remove the mouse hair at the injection site and disinfect the exposed skin with alcohol, lift the skin to form a triangle, and insert the needle at an angle of 15 degrees relative to the skin (insert the needle from the middle of the triangle to prevent piercing the skin and causing the immunogen to leak), the injection depth is 1-2 cm, and be careful not to pierce the muscle; c. Immune cycle: 2 weeks apart each time. A total of 4 times, a total of 8 mice.

[0084] 2.3 Serum titer detection after the third immunization 2.3.1 Titer detection process a. Coating: Dilute the antigen to 2 μg / ml with coating solution CB, 100 μL / well, and add it to the enzyme-labeled plate, and incubate at 4°C overnight.

[0085] b. Blocking: Take out the enzyme-labeled plate and pat the liquid in the well, 5% skimmed milk blocking (dissolved in PBS), 200 μL / well, add to the enzyme-labeled plate, incubate at 37°C for 2 h, and wash the plate 3 times with TBS; c. Sample addition: Dilute the immune serum with PBS at 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000, 100 μL per well, and incubate at 37°C for 1 h. Wash the plate 3 times with TBS.

[0086] d. Add secondary antibody: Dilute the goat anti-mouse secondary antibody-HRP 1:10000 enzyme, 100 μL / well, incubate at 37°C for 40 min, and wash the plate 5 times with TBS; e. Color development: Add TMB substrate, 90 μL / well, and incubate at 37°C for 5-20 min in the dark.

[0087] f. Termination: Add termination solution, 50 μL / well, and read on the enzyme-labeled instrument (wavelength 450 nm). The maximum dilution of the positive reaction is the serum titer of the immunized mouse.

[0088] 2.4 Serum titer detection after the fourth immunization The detection process is the same as above. 2.5.1 FACS detection process a. Cell processing: collect MC38 cells, MC38-SLC34A4 cells, centrifuge, discard the supernatant, wash with PBS to remove the culture medium, resuspend with 10% normal non-immune goat serum (diluted with PBS), block the cells at 4°C for 45 min (incubate on a silent mixer); b. Incubate the first antibody: divide the cells into tubes, about 3x10 5 cells per tube, centrifuge at 1300 rpm for 4 min to remove the blocking solution, dilute the immune serum with 5% goat serum at 1:200, dilute the positive antibody to 20 μg / ml, add the immune serum and the first antibody diluent respectively, incubate at 4°C for 45 min (incubate on a silent mixer); c. Incubate the second antibody: centrifuge to remove the first antibody diluent, resuspend with PBS and centrifuge to remove residual first antibody; dilute the corresponding goat anti-human secondary antibody-APC and donkey anti-mouse secondary antibody-APC with 5% goat serum, add 50 μL of the corresponding secondary antibody diluent per tube, incubate at 4°C for 35 min (incubate on a silent mixer, note to avoid light); after incubation, centrifuge to remove the secondary antibody diluent, resuspend with PBS and wash to remove residual secondary antibody, and detect the cells on a flow cytometer.

[0089] Three and four immune sera were diluted 1 / 200, and the FACS detection results showed that there was a slight background on MC38 cells, and the four immune serum flow sorting was Balb / c>AJ. According to the MFI value of the four immune serum, Balb / c-no (SLC34A2-MP-F13-mFC immunization) was arranged for fusion.

[0090] 2.6 Shock process Select Balb / c mice for shock, shock method: take 100 μg of protein, dilute with PBS to 200 μL, and inject intraperitoneally into Balb / c mice.

[0091] 2.7 Fusion 2.7.1 Preparation of immune spleen cells Take the shocked Balb / c mice, exsanguinate by removing the eyeball, completely decapitate, collect the blood and separate it, and the serum is used as a positive control serum for antibody detection. Sterilely cut open the peritoneum, remove the spleen and grind it into single cells, and then wash it twice with serum-free DMEM for fusion.

[0092] 2.7.2 Cell electrofusion a. Take out 9 ml of fusion pool, soak in an equal volume of 75% ethanol for 10 min, rinse twice with sterile deionized water, and rinse twice with BTX fusion buffer; b. Mix the logarithmically growing myeloma cells (SP2 / 0) and the prepared spleen cells at a ratio of 1:1 for electrofusion; c. After completing the electrofusion procedure, let the cells stand for 2-3 minutes, then collect them in 40 mL of HAT selection medium and incubate them in a 37°C 5% CO2 incubator for 1 hour. d. Supplement with HAT selection medium, plate cells in 96-well plates, and incubate at 37°C in a 5% CO2 incubator; e. Replace the entire medium with HT complete medium once on day 5-6, and incubate at 37℃ in a 5% CO2 incubator for 24-48 hours before detecting the supernatant.

[0093] 2.8 ELISA screening and flow cytometry detection of raw well supernatant SLC34A2-MP-F17-hFc positive screening and hFc negative screening were selected. Stable cells were used for FACS detection. Forty fusion plates were prepared, and 27 Balb / c- cells without subclones were selected.

[0094] Subclonal cell lines were further screened by ELISA and then analyzed by flow cytometry. All subclonal cell supernatants were subjected to flow cytometry, sorted according to Mean-MFI, and then sequenced (9D11D7, 11B8F6, 58E12D9, 62H9E3, 63C2H7).

[0095] 2.9 Sequencing Flowchart for 5 Cell Lines 2.9.1 RNA Extraction a. Take 1 dish (1x10 7 Cells (total of 10^2) were centrifuged, the supernatant was discarded, 1 mL of Trizol was added, vortexed to mix, and incubated on ice for 5 min. After complete lysis, 200 μL of chloroform was added, the mixture was vigorously shaken for 15 s, and then incubated at room temperature for 5 min until layers appeared. b. Centrifugation: 4℃, 12000rpm, 15min; transfer the colorless aqueous phase from the top layer to a new EP tube and add 500μL of isopropanol; c. Remove the supernatant and retain the precipitate at 4℃, 12000rpm, and 10min; d. Add 1 mL of 75% anhydrous ethanol to each tube, gently blow and wash the precipitate; e. At 4℃, 12000rpm, and 5min, aspirate the residual ethanol waste liquid; then centrifuge and aspirate until only precipitate remains at the bottom of the tube (operate on ice). f. Dissolve in 50 μL of RNase-free water.

[0096] 2.9.2 cDNA Synthesis a. RNA denaturation: After RNA extraction, take 15 μL and put it into a PCR tube, denature at 65℃ for 5 min, and place on ice immediately after the experiment is terminated.

[0097] b. Reverse transcription (cDNA synthesis): The reverse transcriptase system was configured according to the following table, mixed, and then subjected to reverse transcription reaction at 37°C for 15 min and enzyme inactivation reaction at 98°C for 5 min.

[0098] Table 4 Reverse transcription system 2.9.3 PCR amplification and recovery The cDNA was used as a template to amplify the light chain and the heavy chain by PCR. The solution was sequentially added to the PCR tube from the most to the least according to the following system (the template cDNA was added last). Table 5 PCR system After adding, mix the solution with a pipette, and then perform PCR amplification according to the following procedure: Table 6 PCR steps After amplification, run 1% DNA gel, analyze the size with a gel imaging analysis system, cut the gel, and then recover the product with a DNA purification and recovery kit for standby.

[0099] 2.9.4 Vector and product digestion The light chain vector / product digestion was mixed according to the following table, and then subjected to enzyme digestion at 37°C overnight. Table 7 Mixing system The heavy chain vector / product digestion was mixed according to the following table, and then subjected to enzyme digestion at 50°C overnight.

[0100] Table 8 Mixing system After enzyme digestion, use a DNA purification and recovery kit for recovery. The PCR product can be recovered without running gel, and the vector needs to be recovered after running DNA gel.

[0101] 2.9.5 Ligation and transformation The heavy chain product and the heavy chain vector, and the light chain product and the light chain vector were subjected to ligation reaction, and the reaction system (10 μL) was as follows: Table 9 Reaction system

[0102] 2.9.6 Bacterial detection and sequencing analysis Single colonies were picked for bacterial detection, and positive single colonies were sent for measurement. Single colonies were inoculated into 300 μL of LB medium containing corresponding antibiotics and cultured for 3-4 h, and then sequenced by Wuhan Jin Kai Rui Biological Sequencing. The effective sequence was recorded and updated by sequencing analysis.

[0103] 2.9.7 Sequencing results The sequencing results of the cell lines are as follows: Table 10 Amino acid sequences of CDR1, CDR2 and CDR3 of antibodies Table 11 Amino acid sequences of heavy and light chains of antibodies Recombinant expression of 9D11D7, 11B8F6, 58E12D9, 62H9E3 and 63C2H7 was selected.

[0104] 2.10 Recombinant antibody expression and purification of 5 cell lines 2.10.1 Recombinant expression process a. Transfect HEK293F cells growing in the exponential phase with a survival rate of more than 98%; dilute the cell density to 2x10 6 cells / mL; b. Place the flask in a 5% CO2 constant temperature shaker, and start transfection after constant temperature shaking at 37°C and 120 rpm for 10 min; c. Prepare two sterile centrifuge tubes of 15 mL, add 5 mL of plasmid dilution buffer and 100 μg of sterile plasmid DNA to one of them, and mix gently; take another centrifuge tube, add 5 mL of transfection reagent dilution buffer and 0.5 mL of transfection reagent, and mix gently; d. Transfer all the liquid in the centrifuge tube containing the transfection reagent to the centrifuge tube containing the plasmid, and mix gently; e. Prepare the plasmid-carrier complex at room temperature for 10 min; f. Take the cells out of the constant temperature shaker, add the prepared plasmid-carrier complex while shaking, and put them back into the CO2 constant temperature shaker for shaking culture; g. Add 2 mL of feed after 24 h of transfection to increase the expression of the product 2.10.2 Protein A column purification The specific steps can refer to 1.1.2.

[0105] 2.10.3 Experimental results As Figure 3 shown in Table 12, the purity and yield of the five recombinant antibodies meet the requirements, and ELISA and FACS detection are arranged.

[0106] Table 12 Expression and purification results 2.11 ELISA detection of 5 recombinant antibodies 2.11.1 Reagent preparation (1) CB buffer: accurately weigh 2.352 g of sodium bicarbonate and 2.332 g of sodium carbonate, dissolve in about 900 mL of primary water, and mix well with a magnetic stirrer. After dilution to 1 L with primary water, filter the prepared CB buffer through a 0.22 μm microporous filter and store separately; (2) Blocking solution, protein diluent and antibody diluent: weigh 4 g of skimmed milk powder, dilute with 100 mL of PBS, and stir to dissolve and mix well. Prepare fresh before blocking or diluting antibodies.

[0107] 2.11.2 Experimental procedure 2.11.2.1 SLC34A2-MP-F13-mFC protein coating According to the concentration of the target protein, dilute SLC34A2-MP-F13-mFC to 5 μg / mL and 2 μg / mL with CB buffer, and add 100 μL / well to the enzyme-labeled plate. Seal the enzyme-labeled plate with a sealing film and incubate at 4°C overnight. When coating, set up a blank control group, a negative control group, a positive control group and an experimental group. The blank control group directly adds CB buffer without protein; the negative control group and the experimental group are coated with SLC34A2-MP-F13-mFC protein; the positive control group is coated with RebmAb200 (CSB-RA021581MA1HU) antibody, 10H1.11.4B (CSB-RA021581MA2HU) antibody and 5 recombinant antibodies.

[0108] 2.11.2.2 Blocking After overnight coating, take the enzyme-labeled plate out of the refrigerator and warm it at room temperature for about 10 min. After warming, shake off the CB buffer, stack 4 layers of absorbent paper, and tap the enzyme-labeled plate on the absorbent paper to remove the remaining coating liquid in the wells. Wash the plate with PBS once, shake dry, and tap the plate on the absorbent paper to remove as much liquid as possible from the wells. After tapping, add about 300 μL of blocking solution (4% skimmed milk powder) to each well, and fill the wells to the top. Incubate the enzyme-labeled plate in a 37°C incubator for 2 h.

[0109] 2.11.2.3 RebmAb200 (CSB-RA021581MA1HU) antibody, 10H1.11.4B (CSB-RA021581MA2HU) antibody and 5 recombinant antibody incubation Before incubation, dilute RebmAb200 (CSB-RA021581MA1HU) antibody, 10H1.11.4B (CSB-RA021581MA2HU) antibody, and 5 recombinant antibodies to a certain concentration as the highest concentration with protein diluent containing 4% skimmed milk powder, and dilute them one by one to 18 concentration groups in a 2-fold gradient. After blocking, shake out the blocking solution in the wells, tap the plate on the absorbent paper, and try to tap out the liquid in the wells. Add the recombinant antibody diluent of each concentration group to the wells, 100 μL / well, then seal with a sealing film, and incubate the enzyme-labeled plate in a 37°C incubator for 1 h.

[0110] 2.11.2.4 Antibody incubation Dilute HRP-labeled goat anti-human IgG with an antibody diluent containing 4% skimmed milk powder at a ratio of 1:10000 and mix well with a suspension instrument. After antibody incubation, shake out the protein diluent in the wells, wash 3 times with PBST, 1 min each time, and then wash 3 times with PBS, 1 min each time. After washing, shake out the liquid in the wells and tap the plate on the absorbent paper to try to tap out the liquid in the wells. Add the antibody to the wells, 100 μL / well, seal with a sealing film, and incubate at 37°C for 1 h.

[0111] 2.11.2.5 Add TMB developing solution After antibody incubation, shake out the antibody solution in the wells, wash 3 times with PBST, and then wash 3 times with PBS, 1 min each time. After washing, shake out the liquid in the wells and tap the plate on the absorbent paper to try to tap out the liquid in the wells. Add TMB developing solution, 100 μL / well, seal with a sealing film. Incubate at 37°C for 10-20 min.

[0112] 2.11.2.6 Stop the reaction Directly add stop solution 50 μL / well to the enzyme-labeled well, immediately put it into the enzyme-labeled instrument, and read at 450 nm wavelength.

[0113] 2.11.2.7 Data arrangement and analysis After reading the absorbance value, export the EXCEL table. Mark the corresponding groups, then copy the data into the Graphpad software, plot, read the EC 50 data, and finally export the curve graph.

[0114] Table 13 ELISA detection results The ELISA detection results show that: SLC34A2-MP-F13-mFC coating detection results show that: 5 antibodies are better than RebmAb200 (CSB-RA021581MA1HU), 58E12D9, 62H9E3, 63C2H7 and 10H1.11.4B (CSB-RA021581MA2HU) have little difference.

[0115] 2.12 FACS detection of 5 recombinant antibodies 2.12.1 Flow detection process a. Cell processing: collect MC38 cells, MC38-SLC34A2 cells, centrifuge, discard supernatant, wash with PBS to remove medium, resuspend with 10% normal non-immune goat serum (diluted with PBS), block cells at 4°C for 45min (incubate on a silent mixer); b. Incubate the first antibody: divide the cells into tubes, about 3x10 5 cells per tube, centrifuge at 1300 rpm for 4 min to remove the blocking solution, dilute the positive antibody to 20 μg / ml with 5% goat serum, incubate the supernatant, add 100 μL of antibody diluent and supernatant, respectively, and incubate at 4°C for 45 min; c. Incubate the second antibody: centrifuge to remove the first antibody diluent, resuspend with PBS and centrifuge to remove the remaining first antibody; dilute goat anti-human secondary antibody-APC and donkey anti-mouse secondary antibody-APC (final concentration 0.005 mg / mL) at a dilution ratio of 1:200 with 5% goat serum, add 50 μL of corresponding secondary antibody diluent to each tube, incubate at 4°C for 35 min (incubate on a silent mixer, note to avoid light); after incubation, centrifuge to remove the secondary antibody diluent, resuspend with PBS and wash to remove the remaining secondary antibody, and detect the cells on a flow cytometer.

[0116] 2.12.2 Flow detection results Table 14 Flow detection results using MC38-SLC34A2 cells Table 15 Flow detection results of control group using MC38 cells 5 recombinant antibodies + 2 positive antibodies EC 50 Superposition chart results are shown in Table 16 and Figure 4 : Table 16 EC of each antibody 50 results FACS detection of recombinant antibodies showed that 9D11D7, 58E12D9, 62H9E3, 63C2H7 and two positive antibodies had little difference, and the antibody affinity was good.

[0117] IHC experiment: The pathological section of ovarian cancer patient (ethics number S2024-615-01) or ovarian cancer cell line NIH-OVA3 was inoculated in 96-well plates at 1 x10 4 -2 x10 4 Inoculate in 96-well plates, and after adhering to stretch to monolayer, discard the culture solution, wash with PBS, fix with 4% paraformaldehyde for 20 min, wash with PBS, treat with 3% hydrogen peroxide for 15 min, wash with PBS for 5 min / time x 3, block with 10% goat serum for 15 min, respectively add humanized Xueshuan antibody and humanized experimental primary antibody 4°C overnight, wash with PBS for 5 min / time x 3, respectively add corresponding HRP-labeled corresponding anti-human IgG secondary antibody, incubate at room temperature for 30 min, wash with PBS for 5 min / time x 3, develop with DAB for 10 min, terminate with distilled water, then dye with DAPI in the dark for 15 min, wash away the unbound dye, and observe and evaluate the staining effect under a bright field and fluorescence microscope. The results are shown in Figure 5 As shown, the five antibodies can be used to observe the expression of SLC34A2.

[0118] 3. Summary This project uses mammalian and E. coli expression systems to express SLC34A2-MP-F13-mFc recombinant protein for immunization, a total of 8 mice are immunized, Babl / c (SLC34A2-MP-F13-mFc) is selected by serum ELISA and FACS detection for fusion. Select SLC34A2-MP-F17-hFc positive screening, hFc negative screening, and use stable cells for FACS detection, 40 plates of fusion are laid out, 27 strains of subcloning are selected, 5 strains of cells are sequenced and arranged for recombinant expression. A total of 5 antibodies are recombinantly expressed, and the antibody affinities of the 5 antibodies are all good.

[0119] Although the present application has been described in detail by preferred embodiments, the present application is not limited thereto. Various equivalent modifications or replacements can be made to the embodiments of the present application by those skilled in the art without departing from the spirit and essence of the present application, and these modifications or replacements shall be within the scope of the present application. Any person skilled in the art within the technical scope disclosed by the present application can easily think of changes or replacements, which shall be covered within the protection scope of the present application.

Claims

1. An anti-SLC34A2 antibody or antigen-binding fragment thereof, characterized in that, The anti-SLC34A2 antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region, and / or CDR-L1, CDR-L2 and CDR-L3 of a light chain variable region, wherein, the amino acid sequence of CDR-H1 comprises NX1LIE (SEQ ID NO: 34), the amino acid sequence of any one of SEQ ID NOs: 1-2, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 1-2; the amino acid sequence of CDR-H2 comprises any one of SEQ ID NOs: 5-6, IINPGSGGX2NYX3X4KFKX5 (SEQ ID NO: 35), or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 5-6, IINPGSGGX2NYX3X4KFKX5 (SEQ ID NO: 35); the amino acid sequence of any one of SEQ ID NOs: 10-11, GETX6VX7AX8SDX9DMDX 10 the amino acid sequence of any one of SEQ ID NOs: 10-11, GETX6VX7AX8SDX9DMDX 10 the amino acid sequence of any one of SEQ ID NOs: 10-11, GETX6VX7AX8SDX9DMDX the amino acid sequence of CDR-L1 comprises any one of SEQ ID NOs: 15-17, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 15-17; the amino acid sequence of CDR-L2 comprises any one of SEQ ID NOs: 18-20, or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 18-20; The amino acid sequence of CDR-L3 contains QQYX 11 X 12 X 13 The amino acid sequence shown in either PLT (SEQ ID NO: 37) or SEQ ID NO: 23, or the sequence associated with QQYX 11 X 12 X 13 The amino acid sequences shown in either PLT (SEQ ID NO: 37) or SEQ ID NO: 23 have at least 80% amino acid sequence identity; X1- X 13 is any natural amino acid residue.

2. The anti-SLC34A2 antibody or antigen-binding fragment thereof of claim 1, wherein, In SEQ ID NO: 34, X1 represents Y or F; in SEQ ID NO: 35, X2 represents N or T, X3 and X4 represent NV, NE, or SE, and X5 represents D or G; in SEQ ID NO: 36, X6 represents T or S, X7 represents V or I, X8 represents T or K, X9 represents Y or C, and X... 10 Represents Y or N; X in SEQ ID NO: 37 11 X 12 Represents YS or SK, X 13 It represents L or T.

3. The anti-SLC34A2 antibody or antigen-binding fragment thereof of claim 1 or 2, wherein, the CDR-H1 comprises the amino acid sequence of any one of SEQ ID NOs: 1-4 or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 1-4; the CDR-H2 comprises the amino acid sequence of any one of SEQ ID NOs: 5-9 or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 5-9; the CDR-H3 comprises the amino acid sequence of any one of SEQ ID NOs: 10-14 or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 10-14; the CDR-L3 comprises the amino acid sequence of any one of SEQ ID NOs: 21-23 or an amino acid sequence having at least 80% identity to the amino acid sequence of any one of SEQ ID NOs: 21-23.

4. The anti-SLC34A2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The anti-SLC34A2 antibody or antigen-binding fragment thereof is a whole antibody, a bi- or multi-specific antibody, a bi- or multi-specific single-chain antibody, a single-chain antibody (scFv), a nanobody, a dAb fragment, a Fab, a Fab', a F(ab')2, a linear antibody, or a Fv antibody.

5. The anti-SLC34A2 antibody or antigen-binding fragment thereof of any one of claims 1-4, which binds to SLC34A2.

6. The anti-SLC34A2 antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein, the heavy chain of the anti-SLC34A2 antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 24-28 or an amino acid sequence having at least 80% identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 24-28, and / or, the light chain of the anti-SLC34A2 antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 29-33 or an amino acid sequence having at least 80% identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 29-33.

7. A method of producing an anti-SLC34A2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that, The method of preparation comprises immunizing a non-human animal with an SLC34A2 antigen and screening for an antibody or antigen-binding fragment thereof that specifically binds to SLC34A2.

8. A biomaterial, characterized by, The biological material comprises: 1) a nucleic acid sequence encoding the anti-SLC34A2 antibody or antigen-binding fragment thereof of any one of claims 1-6 or the anti-SLC34A2 antibody or antigen-binding fragment thereof obtained by the method of preparation of claim 7; 2) a vector comprising the nucleic acid of 1); 3) a cell comprising the nucleic acid of 1) and / or the vector of 2).

9. A product for the treatment and / or prophylaxis of a disease, characterized in that The product for treating and / or preventing a disease comprises any one of: A) the anti-SLC34A2 antibody or antigen-binding fragment thereof of any one of claims 1-6 or the anti-SLC34A2 antibody or antigen-binding fragment thereof obtained by the method of preparation of claim 7; or, B) the biological material of claim 8.

10. Use of the anti-SLC34A2 antibody or antigen-binding fragment thereof of any one of claims 1-6, the anti-SLC34A2 antibody or antigen-binding fragment thereof obtained by the method of preparation of claim 7, or the biological material of claim 8 in the manufacture of a product for treating and / or preventing a disease associated with SLC34A2.

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