SLC15A4-activating antibodies and uses thereof

Abstract

The present application belongs to the technical field of antibodies, and particularly relates to SLC15A4-activating antibodies and application thereof. Specifically, the present disclosure provides SLC15A4 antibodies or antigen-binding fragments thereof, which can promote the binding of SLC15A4 to TASL. The present disclosure also provides biomaterials related to the SLC15A4 antibodies or antigen-binding fragments thereof, conjugates, chimeric antigen receptors, modified immune cells or compositions containing the SLC15A4 antibodies or antigen-binding fragments thereof, and applications thereof.

Description

Technical Field

[0001] This invention belongs to the field of antibody technology, specifically relating to SLC15A4 activating antibody and its applications. Background Technology

[0002] SLC15A4, also known as PHT1, is a member of the SLC15 family of solute carriers and is mainly expressed in antigen-presenting cells, such as plasmacytic dendritic cells (pDCs) and B cells. SLC15A4 is an endosomally resident proton-coupled amino acid transporter that primarily mediates the transport of L-histidine and oligopeptides.

[0003] Studies have shown that SLC15A4 can influence NOD1 / 2-dependent innate immune responses through ligands of nucleotide-binding oligomerization domain 1 (NOD1) and nucleotide-binding oligomerization domain 2 (NOD2). - / - NOD1 / 2-mediated cytokine production was significantly reduced in mice. Furthermore, SLC15A4 is a key component involved in the activation of the Toll-like receptor (TLR) 7 / 8 / 9 pathway. These studies support the use of specific activation of SLC15A4 as a potential therapeutic strategy for diseases such as malignancies.

[0004] Recent studies have found that SLC15A4 forms a complex with TASL (TLR adaptor interacting with SLC15A4 on the lysosome), encoded by the X chromosome open reading frame 21 (CXorf21), a gene associated with systemic lupus erythematosus (SLE). This complex recruits TASL to the lysosome, where it activates the transcription factor IRF5 via the conserved pLxIS motif at the C-terminus of TASL, thereby activating the expression of inflammatory factors and the production of type I interferon. A recently reported small-molecule inhibitor of SLC15A4 can inhibit TASL recruitment or promote SLC15A4 degradation by locking SLC15A4 in a conformation that opens towards the lysosomal lumen, thus suppressing downstream TLR signaling activation. However, no targeted drugs capable of activating SLC15A4 have yet been reported. Summary of the Invention

[0005] To address one of the aforementioned technical problems in the prior art, this disclosure provides an SLC15A4 antibody or its antigen-binding fragment, which can effectively bind to SLC15A4, promote the formation of a complex between SLC15A4 and TASL, and promote the activation of downstream TLR (TLR7 / 8 / 9) signaling pathways, thereby promoting the production of type I interferons and inflammatory factors downstream of the pathways. This can be used for the prevention and treatment of SLC15A4-related diseases (e.g., tumors). Furthermore, this activating antibody can lock the inward (cytoplasmic) opening conformation of SLC15A4, and can be developed as a molecular biology tool for tracking specific SLC15A4 conformations, as well as as a tool to assist in the screening of SLC15A4-activating small molecules.

[0006] According to one aspect of this disclosure, an SLC15A4 antibody or an antigen-binding fragment thereof is provided, said antibody or antigen-binding fragment thereof promoting the formation of a complex between SLC15A4 and TASL.

[0007] In some embodiments, the antibody or its antigen-binding fragment can stabilize SLC15A4 in an inward (cytoplasmic) opening conformation.

[0008] In some embodiments, the antibody or its antigen-binding fragment binds to a portion of the NTD domain corresponding to human SLC15A4, and / or all or part of the CTD domain.

[0009] In some embodiments, the antibody or its antigen-binding fragment binds to a region corresponding to amino acids 63-72 and / or 348-532 of human SLC15A4.

[0010] In some embodiments, the antibody or its antigen-binding fragment may bind to regions corresponding to amino acids 63-72, 348-366, 429-453 and / or 511-532 of human SLC15A4; further, it may bind to regions corresponding to amino acids 429-453 and / or 511-532 of human SLC15A4.

[0011] In some embodiments, the antibody or its antigen-binding fragment may bind to one or more amino acid residues corresponding to A65, E353, T359, T434, I435, V442, V443, D448, D523, F524, N526, and N528 of human SLC15A4; further, it may bind to one or more amino acid residues corresponding to T434, I435, V442, V443, D448, D523, N526, and N528.

[0012] In some embodiments, the amino acid sequence of the human SLC15A4 comprises:

[0013] a1)SEQ ID NO: 65; or

[0014] a2) An amino acid sequence of SEQ ID NO: 65 that has undergone substitution, deletion, and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO: 65; or

[0015] a3) has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or 85% homology with SEQ ID NO: 65 and has the same function as the protein shown in SEQ ID NO: 65.

[0016] SLC15A4 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

[0017] Heavy chain variable region comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 10; and

[0018] The light chain variable region contains CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region as shown in SEQ ID NO: 24.

[0019] In some embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 can be defined by any definition scheme conventionally used by those skilled in the art. In some embodiments, CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined by the definition schemes of Kabat, Chothia, IMGT, Martin, Contact, AbM, or combinations thereof, or determined by Cell Ranger Loupe VDJ Browser software (preferably Cell Ranger Loupe VDJ Browser 4.0.0 software).

[0020] SLC15A4 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

[0021] Heavy chain variable regions, comprising CDR-H1, CDR-H2, and CDR-H3; and

[0022] The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3;

[0023] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are as shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 27, respectively; or

[0024] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 26, and SEQ ID NO: 27, respectively. The amino acid sequence of CDR-L2 is: STS; or

[0025] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are as shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 27, respectively; or

[0026] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are as shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively.

[0027] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 33, respectively.

[0028] In some implementations...

[0029] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 27, respectively. The CDRs are defined using the Kabat scheme; or

[0030] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 26, and SEQ ID NO: 27, respectively. The amino acid sequence of CDR-L2 is STS. The CDR is defined according to the IMGT scheme.

[0031] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 27, respectively, and the CDRs are defined according to the Chothia scheme; or

[0032] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively. The CDR is defined using the Contact definition scheme.

[0033] The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 33, respectively. The CDRs are determined using Cell Ranger Loupe VDJ Browser software (preferably Cell Ranger Loupe VDJ Browser 4.0.0 software).

[0034] In some embodiments, the amino acid sequence of the heavy chain variable region comprises:

[0035] b1)SEQ ID NO:10; or

[0036] b2) An amino acid sequence of SEQ ID NO:10 that has undergone substitution, deletion, and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO:10; or

[0037] b3) An amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, or 75% homology to SEQ ID NO:10 and having the same function as the protein shown in SEQ ID NO:10; and / or

[0038] The amino acid sequence of the light chain variable region includes:

[0039] c1)SEQ ID NO:24; or

[0040] c2) An amino acid sequence of SEQ ID NO:24 that has undergone substitution, deletion, and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO:24; or

[0041] c3) has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO:24 and has the same function as the protein shown in SEQ ID NO:24.

[0042] In some embodiments, the antibody may be, but is not limited to, IgA, IgD, IgE, IgG, or IgM.

[0043] In some embodiments, the antibody may be of the IgG type, such as IgG1, IgG2, IgG3 or IgG4.

[0044] In some embodiments, the antigen-binding fragment may be one known in the art. In specific embodiments, the antigen-binding fragment may include scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, dsFv.

[0045] In some embodiments, the antibody may be a biantibody, a bispecific antibody, or a multispecific antibody.

[0046] In some embodiments, the antibody or its antigen-binding fragment may further include a heavy chain constant region and / or a light chain constant region.

[0047] In some embodiments, the heavy chain constant region may be selected from the IgG1, IgG2, IgG3 or IgG4 heavy chain constant regions.

[0048] In some embodiments, the heavy chain constant region may be the IgG2a heavy chain constant region.

[0049] In some implementations, the light chain constant region may be a κ light chain constant region or a λ light chain constant region.

[0050] In some embodiments, the antibody or antigen-binding fragment includes, but is not limited to, murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0051] The antibody or antigen-binding fragment thereof disclosed herein can promote the formation of a complex between SLC15A4 and TASL. In some embodiments, the antibody or antigen-binding fragment thereof disclosed herein binds partially to the NTD domain of SLC15A4 and / or all or part to the C-terminal domain (CTD). In specific embodiments, the antibody or antigen-binding fragment thereof disclosed herein binds to loops 1-2 (63-72), 7-8 (348-366), 9-10 (429-453), and / or loops 11-12 (511-532) of SLC15A4. In specific embodiments, the antibody or antigen-binding fragment thereof disclosed herein can form hydrogen bonds, hydrophobic interactions, or salt bridges with any one or more sites among A65, E353, T359, T434, I435, V442, V443, D448, D523, F524, N526, and N528.

[0052] The antibody or antigen-binding fragment disclosed herein can bind to SLC15A4, stabilizing SLC15A4 in an inward (cytoplasmic) opening conformation, thereby promoting the formation of a complex between SLC15A4 and TASL.

[0053] In some embodiments, the antibodies or antigen-binding fragments of the present disclosure can promote the expression of type I interferon and inflammatory cytokines, promote the activation of the TLR7 / 8 / 9 signaling pathway, and / or promote the expression of type I interferon and inflammatory cytokines in the THP1 cell line.

[0054] According to another aspect of this disclosure, biological materials related to the SLC15A4 antibody or its antigen-binding fragment described above are provided, said biological materials comprising any one of n1)-n9):

[0055] n1) A nucleic acid molecule encoding the SLC15A4 antibody or its antigen-binding fragment disclosed herein;

[0056] n2) contains an expression cassette containing the nucleic acid molecule described in n1);

[0057] n3) A carrier containing the nucleic acid molecule described in n1);

[0058] n4) A carrier containing the expression box described in n2);

[0059] n5) A cell containing the nucleic acid molecules described in n1);

[0060] n6) Cells containing the expression cassette described in n2);

[0061] n7) Cells containing the carrier described in n3);

[0062] n8) contains cells containing the carrier described in n4);

[0063] n9) Cells containing the SLC15A4 antibody or its antigen-binding fragment as disclosed herein.

[0064] In some embodiments, any of the cells described in n5)-n9) does not contain reproductive material.

[0065] In some embodiments, any of the vectors n3)-n4) includes a prokaryotic expression vector and a eukaryotic expression vector.

[0066] In some embodiments, the eukaryotic expression vector includes yeast expression vectors, mammalian expression vectors, insect expression vectors, etc.

[0067] In some embodiments, any one of the cells (n5)-n9) is selected from prokaryotic cells and eukaryotic cells.

[0068] In some embodiments, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces.

[0069] In some embodiments, the eukaryotic cells include yeast cells, mammalian cells, insect cells, etc.

[0070] In some embodiments, the mammal is selected from humans, monkeys, mice, rats, hamsters, goats, sheep, cattle, pigs, dogs, and cats.

[0071] In some embodiments, the mammalian cells include CHO cells, 293 cells, 293T cells, Vero cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells.

[0072] In some embodiments, the nucleic acid molecule encoding the SLC15A4 antibody or antigen-binding fragment thereof disclosed herein comprises a nucleic acid molecule encoding the heavy chain variable region of the SLC15A4 antibody or antigen-binding fragment thereof disclosed herein and a nucleic acid molecule encoding the light chain variable region of the SLC15A4 antibody or antigen-binding fragment thereof disclosed herein.

[0073] The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the SLC15A4 antibody or its antigen-binding fragment disclosed herein comprises:

[0074] d1)SEQ ID NO: 23; or

[0075] d2) A nucleotide sequence of SEQ ID NO: 23 that has undergone substitution, deletion, and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 23; or

[0076] d3) A nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, or 75% homology with SEQ ID NO: 23 and having the same function as the nucleic acid molecule shown in SEQ ID NO: 23;

[0077] The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the SLC15A4 antibody or its antigen-binding fragment disclosed herein comprises:

[0078] e1)SEQ ID NO: 25; or

[0079] e2) A nucleotide sequence of SEQ ID NO: 25 that has undergone substitution, deletion, and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 25; or

[0080] e3) has at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO: 25, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 25.

[0081] According to another aspect of this disclosure, a conjugate is provided comprising: the SLC15A4 antibody or its antigen-binding fragment described above, and a conjugation portion; said conjugation portion is selected from at least one of detectable markers, drugs, toxins, cytokines, antibodies, antibody Fc fragments, antibody scFv fragments, radionuclides, enzymes, gold nanoparticles / nanorobars, magnetic nanoparticles, and viral capsid proteins.

[0082] In some embodiments, the detectable marker is a fluorescent or luminescent marker.

[0083] In some embodiments, the detectable marker is selected from any one of acridine ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase, and alkaline phosphatase.

[0084] In some embodiments, the radionuclide is at least one of diagnostic isotopes and therapeutic isotopes.

[0085] In some embodiments, the diagnostic isotope is selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, and Re-188.

[0086] In some embodiments, the therapeutic isotope is selected from at least one of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, and Yb-177.

[0087] In some embodiments, the drug is a cytotoxic drug.

[0088] In some embodiments, the cytotoxic drug is selected from at least one of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, caustic agents, aminopicrines, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, and antimitotic agents.

[0089] In some embodiments, the cytotoxic drug is selected from at least one of auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines, or benzodiazepine-containing drugs (e.g., at least one of pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines, vincristine, and vinca.

[0090] According to another aspect of this disclosure, an antibody-drug conjugate is provided, comprising: the SLC15A4 antibody or its antigen-binding fragment described above; and a drug covalently linked to the antibody or its antigen-binding fragment.

[0091] In some embodiments, the antibodies or antigen-binding fragments described herein may be covalently linked to therapeutic agents, such as cytotoxins, drugs (e.g., immunosuppressants), or radiotoxins, to obtain the antibody-drug conjugates.

[0092] In some embodiments, the antibodies or antigen-binding fragments described in this disclosure may also be conjugated with radioisotopes to produce cytotoxic radiopharmaceuticals, also known as radioimmunoconjugates.

[0093] According to another aspect of this disclosure, a chimeric antigen receptor is provided, comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises the aforementioned SLC15A4 antibody or its antigen-binding fragment.

[0094] According to another aspect of this disclosure, a modified immune cell is provided that includes the chimeric antigen receptor described above.

[0095] According to another aspect of this disclosure, a method for preparing the SLC15A4 antibody or its antigen-binding fragment described above is provided, which is obtained by culturing cells in the biological material described above.

[0096] According to another aspect of this disclosure, the use of the SLC15A4 antibody or its antigen-binding fragment, biological material, or conjugate described above in this disclosure in the preparation of a product having any one of the functions e1)-e2):

[0097] e1) Detect the presence or content of SLC15A4 in the sample;

[0098] e2) Diagnosis or prognostic assessment of SLC15A4-related diseases.

[0099] In some embodiments, the product comprises at least one of reagents, detection plates, chips, test strips, and kits.

[0100] In some implementations, the SLC15A4-related diseases include autoimmune diseases.

[0101] In some embodiments, the autoimmune disease includes at least one of arthritis, systemic lupus erythematosus, ankylosing spondylitis, psoriasis, neurological disorders, vitiligo, asthma, inflammatory bowel disease, peritonitis, lung injury, pneumonia, nephritis, neuroinflammatory diseases, and hepatitis.

[0102] In some embodiments, the neurological disease includes at least one of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, multiple sclerosis, encephalitis, brain tumor, traumatic brain injury, and immune encephalopathy.

[0103] In some embodiments, the encephalitis includes at least one of fungal encephalitis, bacterial encephalitis, viral encephalitis, and anti-N-methyl-D-aspartate receptor encephalitis.

[0104] According to another aspect of this disclosure, a product is provided that comprises the SLC15A4 antibody described above or its antigen-binding fragment, or conjugate thereof.

[0105] In some embodiments, the product has at least one of the functions e1)-e2):

[0106] e1) Detect the presence or content of SLC15A4 in the sample;

[0107] e2) Diagnosis or prognostic assessment of SLC15A4-related diseases.

[0108] In some embodiments, the product comprises at least one of reagents, detection plates, chips, test strips, and kits.

[0109] In some embodiments, the SLC15A4-related diseases include autoimmune diseases (preferably the autoimmune diseases described above in this disclosure).

[0110] According to another aspect of this disclosure, the use of the SLC15A4 antibody or its antigen-binding fragment, biological material, or conjugate described above in any of f1)-f3) is provided:

[0111] f1) Tracking the specific conformation of SLC15A4;

[0112] f2) Assisted screening of SLC15A4-activated small molecules;

[0113] f3) Prepare a product, which is used in f1) and / or f2).

[0114] According to another aspect of this disclosure, the use of the SLC15A4 antibody or its antigen-binding fragment, biological material, conjugate, chimeric antigen receptor, or modified immune cell described herein in the preparation of a medicament is provided.

[0115] In some embodiments, the drug is used to treat and / or prevent tumors.

[0116] In some embodiments, the tumor includes at least one of a solid tumor and a hematoma.

[0117] In some embodiments, the solid tumors include liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal carcinoma, lung cancer, gastric cancer, adrenocortical carcinoma, adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, atypical teratoma, rhabdomyosarcoma, basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt lymphoma, carcinoid tumor, cardiac tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, germinal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial endodermal tumor, gonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip cancer, oral cancer, and medrogenic carcinoma. At least one of the following: mesothelioma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, nasal and sinus carcinoma, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumor, islet cell tumor, papilloma, paraganglioma, sinus and nasal cavity carcinoma, parathyroid carcinoma, penile cancer, pharyngeal cancer, pituitary adenoma, pleural pulmonary blastoma, primary peritoneal carcinoma, retinoblastoma, salivary gland tumor, sarcoma, Cézare syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, and single myeloma; further, breast cancer, gastric cancer, ovarian cancer, colon cancer, lung cancer, bladder cancer, prostate cancer, pancreatic cancer, or liver cancer.

[0118] In some embodiments, the hematologic malignancy is selected from at least one of B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphoblastic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell tumor, Waldenstrom's macroglobulinemia, and preleukemia.

[0119] According to another aspect of the present invention, a pharmaceutical composition is provided comprising the SLC15A4 antibody or its antigen-binding fragment disclosed herein, biological material, conjugate, chimeric antigen receptor, or modified immune cell.

[0120] In some embodiments, the pharmaceutical composition is used to treat and / or prevent tumors (preferably the tumors described above in this disclosure).

[0121] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0122] In some embodiments, the pharmaceutically acceptable carrier may be a carrier conventional in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents, etc.

[0123] In some embodiments, the pharmaceutical composition also contains other active ingredients for preventing and treating tumors.

[0124] In some embodiments, the other active antitumor ingredients may be selected from antibiotic derivatives, such as daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycin, procainamide (photomycin), mitomycin, and actinomycin; platinum complexes, such as cisplatin, oxalool and carboplatin, procarbazine, hydroxyurea, mitotane, and aminoglutamine; taxanes, such as paclitaxel and docetaxel; camptothecin, including synthetic topotecan-like compounds; antimetabolites, such as chlorouracil, 5-FU, chlorouracil, capecitabine, and cytarabine, interferon α-2b, glutamate, procainamide, mercaptopurine, and 6-thioguanine; folic acid analogs, such as norpterin, Methotrexate, pteropterin, and trimethotropic acid; purine analogs, such as fludarabine, 6-mercaptopurine, thiamine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmoflurane, cytarabine, dideoxyuridine, doceflurane, exetabine, and fluorouridine; antiproliferative / antimitotic agents, such as natural products like vinca alkaloids (vincrine, vincristine), nocodazole, epopycin, vinorelbine, and epipodophyllotoxin (etoposide, teniposide), nitrogen mustard, cyclophosphamide, and analogs (melphalan, chlorambucil, hexamethylmelamine, and thiotepa), alkylnitrosoureas (carmustine), and analogs, streptozotocin, and triazine (dacarbazine); Alkylating agents, such as thiotepa and cyclophosphamide; DNA damaging agents, such as actinomycin, acridine, busulfan, carboplatin, chlorambucil, isophosphamide, melphalan, chloroethylamine, mitomycin, mitoxantrone, nitrosourea, procarbazine, tesolete, teniposide, etoposide, and triethylene thiophosphamide; anti-estrogens, such as tamoxifen, raloxifene, droloxifen, 4-hydroxytamoxifen, traxifene, keoxifene, LY117018, onnaprisone, and toremifene; anti-androgens, such as flutamide, nilumet, bicalutamide, leuprolide, and goserelin; aromatase inhibitors, such as aminoglutathione, megestrol acetate, exemestane, formetanan, and famethoxazole. Trazol, vorazole, letrozole, and anastrozole; hormones such as medroxyprogesterone, estradiol phosphate sodium, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianilide, and testrolide; corticosteroids such as cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone; nitrogen mustard derivatives such as melfarin, chlorambucil, methylchloroethylamine (nitrogen mustard), and thiotepa; antiplatelet drugs; antimigration agents; antisecretory agents (breveldin); antiadrenergics such as aminoglutamine, mitotane, and triclocysteine; growth factor inhibitors, fibroblast growth factor inhibitors, angiotensin receptor blockers, and nitric oxide donors;Cell cycle inhibitors; topoisomerase inhibitors (doxorubicin, daunorubicin, daunorubicin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan, and irinotecan); growth factor signal transduction kinase inhibitors; dysfunction inducers; toxins, such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin, and caspase activators; fibrinolytic agents, such as tissue plasminogen activator, streptokinase, urokinase, etc. Aspirin, dipyridamole, ticlopidine, and clopidogrel; cyclic melamine and melamine, including alpha-vinyl melamine, ethyltrimethylamine, ethyltrimethylphosphoramide, ethyltrimethylphosphoramide, and methyltrimethylamine; lichenin; carlistatin; anti-angiogenic agents such as retinoids and their derivatives, 2-methoxyestradiol, suramin, and squalamine; tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, and cartilage derivatives. Inhibitors, platelet factor 4, protamine sulfate (clupeine), sulfated chitin derivatives (prepared from mitten crab shells), sulfated polysaccharide-peptide-polysaccharide complex (sp-pg), astrococcalin, and matrix metabolism regulators, including proline analogs (l-azetidine-2-carboxylic acid (LACA)), cis-hydroxyproline, d,I-3,4-dehydroproline, thioproline, α,O'-bipyridine, β-aminopropionitrile fumarate, 4-propyl-5- (4-pyridyl)-2(3h)-oxazolone, mitoxantrone, heparin, interferon, chymotrypsin inhibitors, β-cyclodextrin, tetradecyl sulfate, ependycin, fumonisin, sodium gold thiomalate, d-penicillamine, β-1-anticollagenase serum, α-2-antifibrinolytic enzyme, bismuth subcitrate, lobenzyl ether disodium, n-2-carboxyphenyl-4-chlorophthalate disodium or "CCA", thalidomide, angiotensin-converting steroids, carboxyaminoimidazoles and metalloproteinase inhibitors such as BB-94; immunotherapeutic agents, etc.

[0125] In some embodiments, the tumor immunotherapy agent comprises at least one of the following: monoclonal antibody, immune checkpoint inhibitor, immune cell, oncolytic virus, and tumor vaccine.

[0126] In some embodiments, the target of the monoclonal antibody is selected from one of CD20, HER2, VEGF / VEGFR, EGFR, CD19, FGL1, CD47, CD3, CD30, CD33, CD38, CD52, αVβ3, α5β1, FAP, Tenascin, CEA, EPCAM, PSMA, GAN-GD2, GAN-GD3, GM2, and IGF-IR.

[0127] In some embodiments, the immune checkpoint inhibitor is an inhibitor that acts on negative co-stimulatory (co-inhibitory) molecules of T cells and / or their respective ligands.

[0128] In some embodiments, the negative co-stimulatory (co-inhibitory) molecules acting on T cells and / or their respective ligands are selected from one or more of CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2, B7-H3, B7-H4, B7-H6, A2aR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, CD20, CD96, CD73, CD160, STING, CEA, CD47, PVRIG, LAIR1, 2B4, KIR, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR, and DcR3.

[0129] In some embodiments, the inhibitors acting on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands comprise any one of (b1) to (b3):

[0130] (b1) Antibodies that specifically bind (neutralize) negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands to T cells;

[0131] (b2) Specific binding (neutralization) of ligand proteins or peptides that act on negative costimulatory (co-inhibitory) molecules and / or their respective ligands on T cells;

[0132] (b3) Non-protein compounds that specifically bind (neutralize) to negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands on T cells.

[0133] In some embodiments, the inhibitor of the ligand of the T-cell negative co-stimulatory (co-inhibitory) molecule is selected from: CTLA-4 antibodies (e.g., ipilimumab, ticilimumab; CP-675,206), AAGEN-1884, ATOR-1015, MGD019 (PD-1 / CTLA-4 bispecific antibody)), PD-1 antibodies (e.g., nivolumab, pembrolizumab, tremilimumab). mumab), tislelizumab (BGB-A317), spartalizumab, MEDI0680, PDR001, FAZ053, MGA012 (retifanlimab), sintilimab, toripalimab, cemiplimab, MGD019 (PD-1 / CTLA-4 bispecific antibody), MGD013 (tebotelimab,PD-1 / LAG-3 bispecific antibodies), PD-L1 antibodies (e.g., atezolizumab, camrelizumab, durvalumab, avelumab, LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105), PD-L2 antibodies, B7-1 antibodies, B7-2 antibodies, B7-H3 antibodies (e.g., enoblituzumab) (mab), MGD009, MGC018), B7-H4 antibody, B7-H6 antibody, A2aR antibody (CPI-444, PBF509), IDO antibody (e.g., GDC0919 (navoximod), epacadostat, indoximid, BMS986205), TIM-3 antibody (e.g., TSR022 (TIM-3 monoclonal antibody), MBG453 (TIM-3 monoclonal antibody)), BTLA antibody, VISTA antibody, TIGIT antibody (e.g., BMS-986207, AB) 154, COM902 (CGEN-15137), OMP-313M32), LAG-3 antibodies (e.g., BMS 986016, MK-4280 (28G-10), REGN3767, GSK2831781, IMP731 (H5L7BW), BAP050, IMP-701 (LAG-5250), IMP321, TSR-033, LAG525, BI 754111, FS-118, MGD013 (tebotelimab, PD-1 / LAG-3 bispecific antibody)), CD40 antibodies (e.g., BMS3h-56, lucatumumab (HCD122 and CHIR-12.12), CHIR-5.9 or dacetuzumab (huS2C6, PRO) 64553, RG3636, SGN 14, SGN-40), CD20 antibodies (e.g., rituximab (RITUXAN; IDEC-102; IDEC-C2B8), ABP 798, ofatumumab or obinutuzumab), CD96 antibodies, CD73 antibodies (e.g., MEDI9447 (oleclumab)), CD160 antibodies (e.g., BY55), STING antibodies, CEA antibodies (e.g., cergutuzumab amunaleukin (RG7813),RO-6895882 or RG7802 (RO6958688)), CD47 antibodies (e.g., HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231 or Effi-DEM), PVRIG antibodies (e.g., COM701 (CGEN-15029)), LAIR1 antibodies, 2B4 antibodies, KIR antibodies (e.g., lirilumab (1-7F9, B)). MS-986015, IPH2101), IPH4102), CEACAM1 antibody (e.g., CM-24 (MK-6018)), GARP antibody (e.g., ARGX-115), PS antibody, CSF1R antibody (e.g., pexidartinib, LY3022855, FPA008, BLZ945), CD94 / NKG2A antibody, TDO antibody, TNFR antibody, and DcR3 antibody are selected from one or more of these.

[0134] In some embodiments, the immune cells comprise at least one of: chimeric antigen receptor T cells (CAR-T), chimeric antigen receptor NK cells (CAR-NK), T cell receptor chimeric T cells (TCR-T), tumor-infiltrating immune cells (TILs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, and natural killer (NK) cells.

[0135] In some embodiments, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, Coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, poliovirus, influenza virus, mumps virus, and parvovirus; and further comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus.

[0136] In some embodiments, the alpha virus comprises at least one of M1 virus and Getta virus.

[0137] In some embodiments, the tumor vaccine comprises at least one of dendritic cell (DC) vaccines, nucleic acid vaccines, and peptide vaccines.

[0138] In some embodiments, the route of administration of the pharmaceutical composition may be parenteral, injection, oral, or topical. The pharmaceutical composition may be formulated into a form suitable for administration, such as a solid, semi-solid, or liquid form, and may be an aqueous solution, non-aqueous solution, or suspension, or in the form of powder, tablet, capsule, granules, injection, or infusion.

[0139] According to another aspect of this disclosure, a method for preventing and / or treating a disease in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of this disclosure described above.

[0140] In some embodiments, the disease includes a tumor (preferably the tumor described above in this disclosure).

[0141] In some embodiments, the subject may include mammals, such as humans or non-human mammals. In some embodiments, the non-human mammals may include, but are not limited to, non-human primates (e.g., monkeys, orangutans), mice, rats, hamsters, gerbils, cats, dogs, guinea pigs, rabbits, horses, sheep, cattle, pigs, etc.

[0142] According to another aspect of the present invention, a method is provided, comprising the steps of employing the product described above;

[0143] The method is used for any one of e1)-e2):

[0144] e1) Detect the presence or content of SLC15A4 in the sample;

[0145] e2) Diagnosis or prognostic assessment of SLC15A4-related diseases.

[0146] In some embodiments, the SLC15A4-related diseases include autoimmune diseases (preferably the autoimmune diseases described above in this disclosure). Attached Figure Description

[0147] Figure 1 The SLC15A4 monoclonal antibody screening strategy is shown: [The strategy is described in the original text.] Figure 1 A shows the antibody immunization and screening strategy; Figure 1 B displays TASL's build strategy; Figure 1 C shows the chromatography and SDS-PAGE results of TASL protein; Figure 1 D shows the chromatographic and SDS-PAGE results of SLC15A4 (32-558) and TASL complex proteins used for immunoassay.

[0148] Figure 2 The SLC15A4 monoclonal antibody 235 (Fab235) binds to the SLC15A4 on the membrane surface and mediates endocytosis; among which, Figure 2 A shows SLC15A4 bound to the membrane surface and membrane-localized SLC15A4 mutants; Figure 2 B-2D imaging reveals that 235 binds to SLC15A4 on the surface of the THP1 membrane and mediates endocytosis.

[0149] Figure 3The SCL15A4 monoclonal antibody Fab 235 fragment (Fab235) was shown to promote TLR7 / 8 pathway activation: [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] Figure 3 A shows that different concentrations of Fab235 promote the expression of TNFα activated by the TLR7 / 8 pathway; Figure 3 B shows that different concentrations of Fab235 promote the expression of IL6, which is activated by the TLR7 / 8 pathway; Figure 3 C shows that different concentrations of Fab235 promote the expression of IFNB1, which is activated by the TLR7 / 8 pathway; Figure 3 D shows that Fab235 promotes the expression of IL6, which is activated by TLR7 / 8 signaling, in THP1 induced by different concentrations of R848. Figure 3 E showed that Fab235 promoted the expression of TNFα, which was activated by TLR7 / 8 signaling, in THP1 induced by different concentrations of R848.

[0150] Figure 4 The SLC15A4 monoclonal antibody Fab 235 fragment (Fab235) was shown to promote R848-induced THP1 activation and differentiation: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Figure 4 A shows that Fab235 promotes R848-induced THP1 activation and adhesion; Figure 4 B shows a flow cytometry plot of Fab235 promoting R848-induced upregulation of CD14 and HLA-DR expression on the surface of THP1 membrane; Figure 4 Figure C shows the statistical results of Fab235 promoting R848-induced upregulation of CD14 and HLA-DR expression on the surface of THP1 membrane.

[0151] Figure 5 The SLC15A4 monoclonal antibody 235 (Fab235) shows that it binds to SLC15A4-dependent LL7-8, LL8-9, and LL11-12: where, Figure 5 A shows the construction strategy for the SLC15A4 mutant; Figure 5 B shows the ability of 235 to bind to the SLC15A4 mutant.

[0152] Figure 6 The SLC15A4 monoclonal antibody 235 binds to SLC15A4, stabilizing SLC15A4 and maintaining its cytoplasmic-side opening conformation. Figure 6 A, Figure 6 B displays 235 combined with SLC15A4; Figure 6 C-6G shows the interaction site between 235 and SLC15A4; Figure 6 L shows that 235 cannot bind effectively after mutation at the key site of SLC15A4; Figure 6 I-6J shows the area of ​​the action surface of 235 and SLC15A4 CTD and NTD in the inward and outward opening conformations, respectively.

[0153] Figure 7 The nucleotide and amino acid sequences of the variable region of the heavy chain of SLC15A4 monoclonal antibody 235 are shown.

[0154] Figure 8 The nucleotide and amino acid sequences of the variable region of the light chain of SLC15A4 monoclonal antibody 235 are shown.

[0155] Figure 9 The amino acid sequence of the variable region of the heavy chain of SLC15A4 monoclonal antibody 235 and its germline-derived antibody is shown.

[0156] Figure 10 The amino acid sequence of the variable region of the light chain of SLC15A4 monoclonal antibody 235 and its germline-derived antibody is shown. Detailed Implementation

[0157] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0158] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0159] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0160] The terms “SLC15A4” and “SLC15A4 antigen” are used interchangeably to include variants, isotypes, species homologs of human SLC15A4, and analogs that share at least one common epitope with SLC15A4. Therefore, the antibodies of the present invention may cross-react with SLC15A4 from species other than humans in certain circumstances, or with other proteins structurally related to human SLC15A4 (e.g., human SLC15A4 homologs). In other circumstances, the antibodies may be completely specific to mouse SLC15A4 and do not exhibit species or other types of cross-reactivity.

[0161] The term "antibody" in this disclosure encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific or trispecific antibodies), single-chain molecules, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0162] The term "monoclonal antibody" in this disclosure refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that, apart from possibly trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, typically present in small quantities), the individual antibodies comprised in the group are identical and / or bind to the same epitopes. Unlike polyclonal antibody formulations, which typically comprise different antibodies targeting different antigenic determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen.

[0163] The terms “antibody or antigen-binding fragment thereof” and “antibody” are used interchangeably herein to refer to antibodies that are substantially similar in structure to natural antibodies. “Natural antibody” refers to a naturally occurring immunoglobulin molecule. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. Each heavy chain has a variable region (VH) (also called a variable heavy chain domain, heavy chain variable domain, or heavy chain variable region) and three constant domains (CH1, CH2, and CH3) (also called heavy chain constant regions) from the N-terminus to the C-terminus. Each light chain has a variable region (VL) (also called a variable light chain domain, light chain variable domain, or light chain variable region) and a light chain constant domain (CL) (also called light chain constant regions) from the N-terminus to the C-terminus. The heavy chain of an antibody can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), and can be further subdivided into subtypes such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chain of an antibody, based on the amino acid sequence of its constant domain, can be one of two types: k-light chains and λ-light chains.

[0164] Within the light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0165] The term "variable region" or "variable domain" in this disclosure refers to a domain in the heavy or light chain of an antibody that participates in antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen binding specificity.

[0166] The term "variable" in this disclosure refers to the fact that certain segments of the variable domain are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the variable domains of the light and heavy chains. The more highly conserved portions of the variable domain are called frame regions (FRs). The variable domains of the native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, linked by three HVRs that form loops and, in some cases, form part of a β-sheet structure. The HVRs in each chain are held together tightly by the FRs and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, MD (1991)). Constant domains do not directly participate in antibody-antigen binding but have other effector functions, such as participating in antibody-dependent cytotoxicity.

[0167] The term "hypervariant region" or "HVR" in this disclosure refers to a region in the variable domain region of an antibody that is highly variable in sequence and / or forms a structurally defined loop ("hypervariant loop"). Typically, a natural tetrachain antibody contains six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). HVRs typically contain amino acid residues from the hypervariant loop and / or from the "complementarity-determining region (CDR)," the amino acid residues from the CDR having the highest sequence variability and / or being involved in antigen recognition.

[0168] As used herein, the term "complementarity-determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, such as the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, the Martin numbering system, the Contact numbering system, the Honegger numbering system, the Gelfand numbering system, or combinations thereof. For a given antibody, those skilled in the art will readily identify the CDR as defined by each numbering system. The correspondence between different numbering systems is well known to those skilled in the art, and some commonly used software can be used to define HCDR1-3 and LCDR1-3. For example, the Kabat antibody numbering system, an immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1991). The Chothia antibody numbering system, an immunoglobulin numbering system proposed by Chothia et al., is a classic rule for identifying CDR region boundaries based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883; Al-Lazikani et al., 1997, JMB 273:927-948). The IMGT antibody numbering system is based on the international ImMunoGeneTics information (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. The Martin numbering system is a new numbering scheme recommended by Martin et al., focusing on the structural alignment of different frame regions of unconventional lengths; it can be completed using the Chothia numbering system corrected by ABnum software. The Contact numbering scheme defines the CDR region based on existing antibody complex crystal structure data. The Honegger (also known as the AHo numbering scheme) is based on structural alignment of 3D structures covering observed length variations of immunoglobulin variable regions, allowing the definition of structurally conserved Cα positions, thus deriving appropriate FR regions and CDR lengths.

[0169] "Frame" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) as follows: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0170] An antibody's "class" refers to the type of constant domain or constant region possessed by its heavy chain. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant domains of the heavy chain corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ.

[0171] "Humanized antibodies" comprise amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, humanized antibodies comprise at least one, typically two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to the HVRs of the non-human antibody, and all or substantially all FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally comprise at least a portion of the antibody constant region derived from a human antibody. Antibodies in a "humanized form," such as non-human antibodies, refer to antibodies that have undergone humanization.

[0172] "Humanized antibodies" have an amino acid sequence that corresponds to that of antibodies produced by humans or human cells, or derived from non-human antibodies using sequences encoded by human antibody libraries or other human antibodies. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0173] As used herein, the term "substitution" or "replacement" of amino acids can refer to the substitution of a conserved amino acid residue, wherein the amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, if an amino acid in a polypeptide is replaced by another amino acid from the same side chain family, such substitution is considered conserved. In another respect, a string of amino acids can be conservatively replaced by a structurally similar string that differs in the order and / or composition of its side chain family members.

[0174] The terms “polynucleotide,” “nucleic acid,” or “nucleotide sequence” in this disclosure refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term “nucleic acid molecule” refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0175] An "antibody fragment" or "antigen-binding fragment" contains a portion of a complete antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv; bisomatic antibodies, trisomatic antibodies, tetrasomatic antibodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0176] After obtaining the DNA fragments encoding the VH and / or VL of the antibody, these DNA fragments can be further manipulated using recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding the VH and / or VL is operatively linked to another DNA fragment encoding a different protein, such as the antibody constant region or a flexible linker. As used herein, the term "operatively linked" means that two DNA fragments are joined together such that the amino acid sequences encoded by both fragments remain within the reading frame.

[0177] By operatively linking DNA encoding the VH region to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3), isolated DNA encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are well known in the art (see, for example, Kabat, BA et al. (1991), Sequences of Proteins of Immunologicl Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments including these regions can be obtained by PCR amplification. The heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but IgG1 or IgG2a constant regions are most preferred. For Fab fragment heavy chain genes, the DNA encoding the VH region can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0178] By operatively linking the DNA encoding VL to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are well known in the art (see, for example, Kabat, BA et al. (1991), Sequences of Proteins of Immunologic Interest, 5th ed., Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments including these regions can be obtained by PCR amplification. The light chain constant region can be either a κ or λ constant region, but a κ constant region is preferred.

[0179] To generate the scFv gene, the DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible linker, such as the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as continuous single-stranded proteins with their VH and VL regions linked by the flexible linker.

[0180] The terms "antigen-binding domain" or "antigen-binding site" used in this disclosure refer to the portion of an antigen-binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen-binding domain" refers to a portion of an antibody containing a region that specifically binds to and is complementary to a portion or all of the antigen. In cases where the antigen molecule is large, the antigen-binding molecule may bind only a specific portion of the antigen, referred to as an epitope. The antigen-binding domain may be provided by, for example, one or more variable domains (also called variable regions). Preferably, the antigen-binding domain comprises a variable region (VL) of the antibody light chain and a variable region (VH) of the antibody heavy chain. In one aspect, the antigen-binding domain is capable of binding its antigen and blocking or partially blocking the function of said antigen.

[0181] The term "antigenic determinant" in this disclosure is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a continuous amino acid sequence or a conformation composed of different regions of non-continuous amino acids) to which an antigen-binding moiety binds, thereby forming an antigen-binding moiety-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum, and / or in the extracellular matrix (ECM). Unless otherwise stated, proteins used as antigens in this invention can be any naturally occurring form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). Antigens can also be human proteins, or antigens can be "full-length," unprocessed proteins, and any form of protein produced by intracellular processing, or naturally occurring protein variants, such as splice variants or allelic variants.

[0182] "Specific binding" refers to the selective binding to an antigen, distinguishable from unwanted or nonspecific binding. The ability of an antigen-binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) and conventional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of the antigen-binding molecule to unrelated proteins is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen. In some embodiments, the dissociation constant (Kd) of the antigen-binding molecule is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10). -7 M or lower, such as 10 -7 M to 10 -13 M, for example, 10 -9 M to 10 -13 M).

[0183] "Affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its bound ligand (e.g., an antigen). Binding affinity is typically expressed as a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constants (Koff and Kon, respectively). Therefore, equivalent affinity can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured using conventional methods known in the art, such as surface plasmon resonance (SPR).

[0184] The term "isolated" nucleic acid molecule or polynucleotide in this disclosure refers to a nucleic acid molecule, DNA, or RNA, that has been separated from its natural environment. In this invention, the recombinant polynucleotide encoding a polypeptide contained in the vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules typically found in cells containing the polynucleotide molecule, but which are located extrachromosomally or at chromosomal locations different from their natural chromosomal locations. Isolated RNA molecules include in vivo or in vitro RNA transcripts of this invention, in positive and negative strand forms, and in double strand forms. The isolated polynucleotides or nucleic acids of this invention further include synthetically generated molecules of this type. Additionally, the polynucleotide or nucleic acid may be or may include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0185] The terms "vector" or "expression vector" and "expression construct" used herein are used interchangeably to describe a DNA molecule to which a specific gene, operatively linked, is introduced into a target cell and directed for expression. The vector comprises a vector as a self-replicating nucleic acid structure and a vector incorporated into the genome of the host cell into which it has been introduced. The expression vector of the present invention comprises an expression cassette. The expression vector can be transcribed into a large amount of stable mRNA. Once the expression vector is in the target cell, a ribonucleic acid molecule or protein encoded by the gene is generated by cellular transcription and / or translation mechanisms. The term "expression cassette" in this disclosure refers to a recombinant or synthetically produced polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in the target cell. Recombinant expression cassettes can be introduced into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, in addition to other sequences, the recombinant expression cassette portion of the expression vector includes the nucleic acid sequence to be transcribed and a promoter.

[0186] The term "antibody-drug conjugate" or "ADC" refers to a binding protein (such as an antibody or its antibody- or antigen-binding fragment) chemically linked to one or more chemical drugs. In a preferred embodiment, an ADC comprises a binding protein, a drug, and a connector linking the binding protein to the drug. These conjugates are also referred to as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are called "immunotoxins." Cytotoxins or cytotoxic agents include any agent that is harmful to cells (e.g., killing). Examples include paclitaxel, cytochalasin B, bacitracin D, ethidium bromide, emetine, mitomycin, epipodophyllotoxin glucoside, epipodophyllotoxin thiophene glycoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraquinone, mitoxantrone, scintillans, actinomycin D, L-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and their analogues or homologues. Therapeutic agents also include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), causative agents (e.g., nitrogen mustard, thioepa, phenylalanine mustard, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozomycin, mitomycin C, and cis-dichlorodiamineplatin(II)(D)). DP (cisplatin), amiodarone derivatives (e.g., daunorubicin (formerly known as doxorubicin) and doxorubicin), antibiotics (e.g., actinomycin D, bleomycin, scintillan, and atrazodine (AMC)), and antimitotic agents (e.g., vincristine and vinblastine). Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies or antigen-binding fragments thereof disclosed herein include buprofen, scutellarin, metansin, auristatin, and their derivatives.

[0187] Cytotoxins can be conjugated to antibodies or antigen-binding fragments thereof disclosed herein using adapter techniques available in the art. Examples of adapter types already used for conjugating cytotoxins to antibodies or antigen-binding fragments thereof include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing adapters. Alternatively, adapters may be selected that are readily cleaved at low pH or readily cleaved by proteases, such as proteases preferentially expressed in tumor tissues, like cathepsins (e.g., cathepsins B, C, D).

[0188] As used herein, the term "radioisotope" refers to an antibody or its antigen-binding fragment that can be conjugated for diagnostic or therapeutic purposes, including but not limited to iodine-131, indium-111, yttrium-90, and lutetium-177. Methods for preparing radioimmunoconjugates are known in the art.

[0189] The term "chimeric antigen receptor" or "CAR" refers to a receptor that possesses desired antigen specificity and signal transduction domains to propagate intracellular signals upon antigen binding. For example, T lymphocytes recognize specific antigens via the interaction of T cell receptors (TCRs) with short peptides presented by class I or II major histocompatibility complex (MHC) molecules. For initial activation and clonal expansion, naïve T cells depend on antigen-presenting cells (APCs) that provide additional co-stimulatory signals. In some embodiments, monocytes and macrophages can be engineered to express, for example, chimeric antigen receptors (CARs). Modified cells can be recruited to the tumor microenvironment, where they act as potent immune effectors by infiltrating the tumor and killing target cancer cells. CARs may include antigen-binding domains, transmembrane domains, and intracellular domains. The antigen-binding domain binds to the antigen on the target cell. Examples of cell surface markers that can be used as antigens binding to the antigen-binding domain of a CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (e.g., tumor antigens).

[0190] The term "modified immune cell" refers to immune cells that have been genetically modified to express CAR. In some embodiments, the immune cells include macrophages, monocytes, dendritic cells, T cells (e.g., induced pluripotent stem cell (iPSC) derived T cells), stem cells, regulatory T cells (Treg), natural killer (NK) cells (e.g., induced pluripotent stem cell (iPSC) derived NK cells), γδ cells, natural killer T (NKT) cells, B cells, or cells derived therefrom.

[0191] An "effective amount" of a drug is the amount necessary to produce physiological changes in the cells or tissues to which it is administered. An "effective amount" includes the amount sufficient to improve or prevent the symptoms or signs of a medically diagnosed disease. An effective amount also means the amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of any side effects. An effective amount can be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0192] The "therapeutic effective amount" of a drug (such as a pharmaceutical composition) refers to the amount necessary to effectively achieve the desired therapeutic or preventive effect in terms of dosage, dosing intervals, and time. For example, a therapeutically effective amount of a drug eliminates, mitigates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0193] The terms "individual" or "subject" refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, an individual or subject is a human.

[0194] The term "pharmaceutical composition" refers to a mixture containing an antibody or antibody- or antigen-binding fragment thereof of the present disclosure, biological material, conjugate, chimeric antigen receptor, or modified immune cell, and other chemical components, such as physiological / pharmaceutical-grade carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0195] The term "pharmaceuticalally acceptable excipient" refers to a component in a pharmaceutical composition that, apart from the active ingredient, is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0196] Examples of suitable aqueous or non-aqueous carriers that can be used in the pharmaceutical compositions of this disclosure include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable flowability can be maintained, for example, by applying a coating material such as lecithin, in the case of a dispersion, by maintaining the desired particle size, and by applying a surfactant.

[0197] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. The presence of microorganisms can be prevented by the sterilization procedures described above or by including various antibacterial and antifungal agents such as parabens, chlorobutanol, and phenolic sorbic acid. Isotonic agents, such as sugars and sodium chloride, may also be required in the composition. Additionally, prolonged absorption of injectable drugs can be achieved by including delayed absorption agents, such as aluminum monostearate and gelatin.

[0198] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the provisional preparation of sterile injections or dispersions. The use of these media and reagents for the application of pharmaceutically active substances is well known in the art. Additional active compounds may also be incorporated into the composition.

[0199] Therapeutic compositions must generally be sterile and stable under the conditions of preparation and storage. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersant containing, for example, water, ethanol, polyols (e.g., glycerol, polyethylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For example, by using a coating, such as lecithin, appropriate flowability can be maintained by maintaining the desired particle size in the case of a dispersant, and by using a surfactant. In many cases, the composition preferably contains an isotonic agent, such as sugars, polyols such as mannitol, sorbitol, or sodium oxide. Prolonged absorption of injectable drugs can be achieved by adding delayed absorption agents, such as monostearate and gelatin, to the composition.

[0200] Sterile injection solutions can be prepared by mixing the active compound in a suitable solvent in the required amount, and adding one or a combination of the components listed above as needed, followed by aseptic microfiltration. Dispersants are typically prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and other desired components listed above. For sterile powders used to prepare sterile injection solutions, preferred preparation methods include vacuum drying and freeze-drying (lyophilization), yielding a powder containing the active ingredient plus any additional desired components from the aforementioned aseptically filtered solution.

[0201] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose form varies depending on the subject being treated and the specific route of administration. The amount of active ingredient that can be combined with a carrier material to prepare a single-dose form is generally the amount of the composition that produces the therapeutic effect. Typically, this amount, in 100% terms, ranges from about 0.01% to about 99% of the active ingredient, preferably from about 0.1% to about 70%, and most preferably from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0202] The term "treatment" refers to the administration of an oral or topical therapeutic agent, such as a composition comprising any antibody of the present disclosure or an antibody thereof or an antigen-binding fragment thereof, or a nucleic acid molecule encoding an antibody thereof or an antibody thereof or an antigen-binding fragment thereof, to a patient having one or more diseases or symptoms, and the therapeutic agent having a therapeutic effect on these diseases or symptoms. Typically, the therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent.

[0203] The term "parenteral" administration, as used in this article, refers to a mode of administration other than enteral and local administration, typically via injection, including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. The antibodies or antigen-binding fragments thereof, or pharmaceutical compositions described herein, can be administered intravascularly, subcutaneously, intraperitoneally, intramuscularly, by inhalation, intranasally, via airway instillation, or via intrapleural instillation. The antibodies or antigen-binding fragments thereof, or pharmaceutical compositions described herein, can also be administered in aerosol or spray form, such as nasally; or intrathecally, intramedullaryly, or intraventricularly; or transdermally, percutaneously, locally, enterically, intravaginally, sublingually, or rectally. The antibodies or antigen-binding fragments thereof, or pharmaceutical compositions described herein, can be formulated into various dosage forms as needed, and the physician can determine the beneficial dosage for the patient based on factors such as patient type, age, weight, general disease condition, and route of administration.

[0204] The "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any location where the same nucleotide or amino acid is present in both the target and reference sequences. Vacancies are not nucleotides or amino acids and are not counted in the target sequence. Similarly, vacancies in the reference sequence are not counted because nucleotides or amino acids from the target sequence are included, but those from the reference sequence are excluded.

[0205] The percentage of sequence identity can be calculated as follows: determine the number of positions in both sequences where the same amino acid residue or nucleic acid base appears (the number of matching positions), divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of the percentage of sequence identity between two sequences can be accomplished using software that is readily available online and downloadable. Suitable software programs are available from various sources for protein and nucleotide sequence alignment. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparing two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0206] Those skilled in the art will understand that the reference herein to having “at least 85% sequence identity” compared to a sequence is intended to include all sequences with more than 85% sequence identity, such as including at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity.

[0207] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0208] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0209] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the invention. The reagents and / or kits used in the following embodiments are commercially available or can be synthesized by known methods.

[0210] It should be noted that, unless specific conditions are specified in the examples, experimental conditions should be performed according to standard conditions, manufacturer recommendations, or publicly reported experimental conditions. Reagents or instruments whose manufacturers are not specified are all commercially available, standard products. For reagents whose manufacturers are specified, similar products from other manufacturers are substitutes.

[0211] Example

[0212] Example 1. Production of mouse anti-SLC15A4 antibody

[0213] 1. Preparation of immunogen and tag protein

[0214] The immunogen and antigen-specific B cell-enriched tag proteins used in this disclosure were constructed.

[0215] (1) Primers were designed based on Genbank accession number XM_011537895.2 (SLC15A4 mRNA), and the primer sequences are shown in Table 1.

[0216] Table 1

[0217]

[0218]

[0219] (2) Construction of expression vectors for immunogen and antigen-specific B cell-enriched tag proteins

[0220] First, let's take pFastBac TM Using Dual (Invitrogen) as a template, reverse PCR was performed according to the PrimeSTAR HSDNA Polymerase (TaKaRa) instructions, using primers HF1 and HR1 respectively, to obtain the PCR product pFastBac. TM Dual.TEV.his full-length clip.

[0221] The PCR conditions were as follows: pre-denaturation, 98℃, 2 minutes; denaturation, 98℃, 30 seconds; annealing, 58℃, 30 seconds; extension, 72℃, 2 minutes 30 seconds; a total of 20 cycles were performed, with an additional 10 minutes of denaturation time at the end.

[0222] The PCR products were digested with DpnI enzyme (Thermo Fisher Scientific) for 1-4 hours, and the digested products were transformed into E. coli TOP10 strains to construct the vector pFastBac. TM Dual.TEV.his.

[0223] Then, using synthesized and codon-optimized full-length human SLC15A4 DNA (SEQ ID NO:66) as a template, human SLC15A4 (32-558) fragments were amplified with primers 15F2 and 15R, and human SLC15A4 (32-558) fragments with N-terminal flag tags were amplified with primers 15F3 and 15R. The PCR products of the amplified human SLC15A4 (32-558) fragments and the SLC15A4 (32-558) fragments with N-terminal flag tags were recovered. The PCR products and pFastBac enzymes were digested with BamHI-HF and HindIII-HF. TM The Dual.TEV.his plasmid was recovered and ligated to obtain expression vectors for human SLC15A4(32-558) and human SLC15A4(32-558) with a flag tag attached to the N-terminus. The recombinant plasmids were transformed into DH10-Bac competent cells and plated onto plates containing kanamycin, tetracycline, gentamicin, x-gal, and IPTG. After static incubation at 37°C for 3 days, white single clones were picked and transferred to liquid culture medium. Baculovirus (Bacmid) was extracted the following day and named Bacmid-SLC15A4(32-558) and Bacmid-flag-SLC15A4(32-558), respectively.

[0224] A mouse TASL protein optimized for insect cell codons was synthesized (based on mouse TASL with accession number 71398, by deleting the random sequence of amino acids 35-203 and replacing amino acids 1-18 at the N-terminus with the human TASL sequence (accession number 80231)). [Biotechnology, schematic diagram shown] Figure 1 (as shown in B), the C-terminus carries a Strep tag. The above nucleic acid molecules and pF astBac were digested using BamHI-HF and HindIII-HF enzymes. TMThe plasmid from Dual was recovered and ligated to obtain the expression vector for the chimeric TASL. The recombinant plasmid was transformed into DH10-Ba c competent cells and plated onto plates containing kanamycin, tetracycline, gentamicin, x-gal, and IPTG. After static incubation at 37°C for 3 days, white single clones were picked and transferred to liquid culture medium. After expansion culture, baculovirus (Bacmid) was extracted the next day and named Bacmid-TASL.

[0225] (3) Immunogen and tag protein expression

[0226] The Sf9 cell concentration was diluted to 0.5 × 10⁻⁶. 6 After spreading the cells / mL evenly onto a 6-well plate, 2 mL per well, incubate at 27°C for 30 minutes to allow complete adhesion. Take a sterile EP tube and dilute 10 μL of Bacmid (5 μL each of Bacmid-SLC15A4(32-558) and Bacmid-TASL, or 5 μL each of Bacmid-flag-SLC15A4(32-558) and Bacmid-TASL, or 10 μL of Bacmid-TASL) with 90 μL of Grace medium (Thermo Fisher Scientific). Take another sterile EP tube and dilute 6 μL of transfection reagent cellfectin (Thermo Fisher Scientific) with 100 μL of Grace medium. Mix the two solutions thoroughly and incubate at room temperature for 30 minutes. Add 800 μL of Grace medium to the above mixture; aspirate the medium from the 6-well plate and add 2 mL of Grace medium to wash the cells; add the transfection solution dropwise into the cells and incubate at 27°C for 5 hours; aspirate the transfection solution and add 2 mL of ESF921 insect cell culture medium (Expression Systems) containing 10% fetal bovine serum; after incubation at 27°C for 72 hours, collect the supernatant, which is the P1 virus. Since the virulence of the P1 virus is relatively weak, further amplification of the viral particles is needed for subsequent large-scale expression. Add 10 mL of 2×10⁻⁶ g / mL of the transfection solution to the cells. 6 Add 5% P1 virus to Sf9 insect cells per mL, incubate at 27°C with shaking for 72 hours, and collect the supernatant to obtain P2 virus. Continue this process until P4 virus is finally used as the expression virus.

[0227] (4) Protein expression and purification

[0228] Following the instructions for the Bac-to-Bac baculovirus expression system (Invitrogen), human SLC15A4 (32-558) and the TASL complex were expressed in Sf9 insect cells. The N-terminus of each complex was tagged with a flag, along with TASL. Sf9 insect cells were expressed at a density of 2-2.5 × 10⁻⁶ cells / year. 6 Cells were infected with 2% (v / v) P4 baculovirus at a concentration of cells / mL and cultured with shaking at 27°C for 72 hours before centrifugation to collect the cells. The cells were then resuspended in lysis buffer supplemented with 1 mM PMSF (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM imidazole, 10% glycerol) and homogenized by sonication on ice.

[0229] The protein was incubated at 4°C with gentle agitation for 2 hours using a final concentration of 1% (w / v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace) and 0.1% (w / v) Cholesteryl Hemisuccinate Tris Salt (CHS, Anatrace). After centrifugation at 18,000 rpm for 30 minutes at 4°C to remove insoluble fractions, the supernatant was incubated sequentially with equilibrated nickel column stock and Strep-Tactin, followed by gentle agitation for 1 hour. The resin was then washed with lysis buffer supplemented with 0.06% glyco-diosgenin (GDN, Anatrace). The target protein was then eluted with elution buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 500 mM imidazole, 10% glycerol, 0.12% GDN) and concentrated to 750 μL using a 50 kDa stop-volume concentrate. The protein was then further purified by gel filtration chromatography (SEC) on a Superdex 200, and the peak of the target protein was collected. After SDS-PAGE verification, the target proteins were pooled and concentrated to 5-10 mg / mL and stored at -80°C for later use.

[0230] The SLC15A4 (32-558) and TASL complex protein samples, and the SDS-PAGE results of the TASL samples after Ni affinity chromatography and size exclusion chromatography are as follows: Figure 1 C Figure 1 As shown in D: it has good uniformity and high purity.

[0231] according to Figure 1 An exemplary procedure is shown for immunizing mice, screening antibodies, and expressing and purifying them.

[0232] 2. Immunization of Balb / C mice

[0233] The human SLC15A4 (32-558) and TASL complex protein obtained in step 1 were used as immunogens. 20 μg of the complex protein was mixed with 200 μg of MnJβ adjuvant (Qimeng Biotechnology) to form a 0.2 mL suspension, which was then administered to 6-week-old Balb / c mice via subcutaneous injection at multiple sites. Immunization was repeated monthly for a total of 3 times. A final booster immunization was performed using 20 μg of unadjuvanted SLC15A4 (32-558) and TASL complex protein, and single-cell BCR sequencing analysis was performed 7 days later.

[0234] 3. Single-cell BCR sequencing analysis

[0235] The immunized mice were sacrificed, and spleen cells were harvested and blocked. Specifically, the isolated mouse cells were incubated with blocking buffer (PBS + 2mM EDTA + 10% mouse serum + anti-CD16 / 32 antibody (1:100, Biolegend)) for 20 min. The blocked cells were then incubated with the flag-tagged human SLC15A4 (32-558) and TASL complex obtained in step 1 for 30 min. The cells were washed once with FACS buffer (PBS + 2mM EDTA + 2% BSA), centrifuged, stained with secondary antibody, and CD3+ sorted using BD ArialIII. - B220 + CD19 + CD38 - GL7 + Flag + Single-cell BCR sequencing analysis (10x genomic) was performed on approximately 10,000 B cells. Sequencing data were analyzed using Cell Ranger Loupe VDJ Browser 4.0.0 software to select target sequences.

[0236] 4. Expression of the target antibody

[0237] The light and heavy chains of the target antibody selected in the previous step were subjected to codon optimization analysis, and the optimized sequences of the variable regions of the light and heavy chains were ligated into the pTT3 (Addgene) vector containing the constant region of the mouse light chain κ chain and the constant region of the heavy chain IgG2a, respectively. The mouse antibody constant region sequence was derived from the in vitro hybridoma cell JL2 (Xuyuan Zhang et al. The binding of a monoclonal antibody to the apical region of SCARB2 blocks EV71 infection. PROTEIN & CELL, 2017.8, 8(8):590~600.). The above antibody expression vectors were synthesized at Sangon Biotech and BGI Genomics.

[0238] The synthesized antibody expression vector was extracted using an endotoxin-free plasmid mini-extraction kit (purchased from Tiangen Biotech). The antibody light and heavy chains were then mixed in a 1:1 ratio with PEI transfection reagent and allowed to stand. The mixture was then added to 293F cells (purchased from ATCC) and cultured at 37°C for 5 days in a 5% CO2 shaker. The supernatant was then collected by centrifugation.

[0239] Protein A packing material (Captiva™ PriMAB, cat: CA-PRI-1000) was packed into the column, and the antibody was adsorbed through the affinity chromatography column. The bound antibody was eluted from the affinity chromatography column with 0.1M glycine (pH 2.7) elution buffer. The antibody in the collection tube was used to determine the protein concentration. The liquids in the tubes containing protein were combined, the antibody was diluted with PBS, added to a 30KD ultrafiltration centrifuge tube, and concentrated by centrifugation at 3000 rpm and 4°C. The solution in which the antibody was dissolved was replaced with PBS.

[0240] Example 2: Identification of functional antibodies against SLC15A4 in mice

[0241] 1. Construction of the detection vector

[0242] An antibody detection protein was constructed.

[0243] Primers were designed based on Genbank accession number XM_011537895.2 (SLC15A4 mRNA), and the primer sequences are shown in Table 2.

[0244] Table 2

[0245]

[0246] Using full-length human SLC15A4 DNA (SEQ ID NO:66) as a template, the full-length human SLC15A4 fragment was amplified using 15-F and 15-R. Using the pIRES2-EGFP-puro vector (Addgene) as a template, the IRES-GFP fragment was amplified using IGF and IGR. Then, fusion PCR was performed using 15-F and IGR. The fusion PCR product fragment was ligated into the pTT3 vector (Addgene) to obtain the plasmid pTT3-hSLC15A4-IRES-GFP. In this plasmid, the EGFP gene is located downstream of the SLC15A4 sequence; when SLC15A4 is expressed, the EGFP signal can be observed. Subsequent SLC15A4 mutant expression vectors were constructed using pTT3-hSLC15A4-IRES-GFP as a template.

[0247] 2. SLC15A4 antibody screening

[0248] Using lipofectamine transfection reagent (Thermo Fisher), the pTT3-hSLC15A4-IRES-GFP plasmid was transfected into 293T cells (purchased from ATCC) to obtain cells expressing human SLC15A4, 293T-hSLC15A4-IRES-GFP. The 293T-hSLC15A4-IRES-GFP cells, digested with PBS containing 2 mM EDTA, were fixed with 2% paraformaldehyde for 20 min. The cell pellet was then resuspended in PBS buffer for cell permeabilization (eBioscience). The permeabilized cells were then added to 96-well U-bottom culture plates and incubated for 10 days. 5 Cells / 100 μL / well. Centrifuge at 2200 rpm for 3 minutes, discard the supernatant, add 100 μL / well of the antibody expression supernatant obtained in Example 1, resuspend, and incubate at 4°C for 30 minutes. Wash cells twice with FACS buffer (PBS + 2 mMEDTA + 2% BSA). Then incubate with 100 μL / well of PE-labeled goat anti-mouse IgG antibody (BioLegend, Poly4053, 405307) (1:500 dilution) at 4°C in the dark for 30 minutes, and wash cells twice with FACS buffer. After centrifugation and resuspending, analyze cell GFP and PE signals using flow cytometry (Thermo Analyzer). Wells with double positive results for GFP and PE are antibody-positive wells binding to hSLC15A4. Flow cytometry screening identified well 235 as a monoclonal antibody binding to SLC15A4.

[0249] 3. Small-scale antibody preparation

[0250] The light and heavy chains of the SLC15A4 monoclonal antibody 235 expression vector were mixed in a 1:1 ratio with the transfection reagent PEI and allowed to stand. Then, the mixture was added to 293F cells (purchased from ATCC) and cultured at 37°C in a 5% CO2 shaker for 5 days. The supernatant was then collected by centrifugation.

[0251] Protein A packing material (Captiva™ PriMAB, cat: CA-PRI-1000) was packed into the column, and the antibody was adsorbed through the affinity chromatography column. The bound antibody was eluted from the affinity chromatography column with 0.1M glycine (pH 2.7) elution buffer. The antibody in the collection tube was used to determine the protein concentration. The liquids in the tubes containing protein were combined, the antibody was diluted with PBS, added to a 30KD ultrafiltration centrifuge tube, and concentrated by centrifugation at 3000 rpm and 4°C. The solution in which the antibody was dissolved was replaced with PBS.

[0252] The amino acid sequence of the heavy chain variable region of SLC15A4 monoclonal antibody 235 is: DVQLVESGGGLVQPGGSRKLSCAASGFT FSRFGMHWVRQAPEKGLEWVAYISSGSSNIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCA RSTTIIRAFFDYWGQGTTLTVSS (SEQ ID NO:10), and its nucleotide sequence is shown in SEQ ID NO:23. Figure 7 Table 3 shows the CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain, defined by different CDR schemes.

[0253] Table 3

[0254]

[0255] The amino acid sequence of the light chain variable region of SLC15A4 monoclonal antibody 235 is: QIVLTQSPAIMSASLGERVTMTCTASSGV SSSYLHWYQQKPGSSPRLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPWAF GGGTKLEIK (SEQ ID NO:24), and its nucleotide sequence is shown in SEQ ID NO:25. Figure 8 Table 4 shows the CDR-L1, CDR-L2, and CDR-L3 in the variable region of the light chain, defined by different CDR schemes.

[0256] Table 4

[0257]

[0258] Analysis of the nucleic acid fragment of SLC15A4 monoclonal antibody 235 using the antibody sequence analysis tool igBlast tool (http: / / www.ncbi.nlm.nih.gov / igblast / ) revealed that the V, D, and J genes encoding the variable region of the heavy chain of SLC15A4 monoclonal antibody 235 correspond to the mouse IGHV5-17*02, IGHD2-12*01, and IGHJ2*01 genes, respectively. The comparison results between the amino acid sequence of the heavy chain variable region of SLC15A4 monoclonal antibody 235 and the amino acid sequence of the mouse VDJ region are as follows: Figure 9As shown. The V and J genes encoding the light chain variable region of SLC15A4 monoclonal antibody 235 correspond to the mouse IGKV4-74*01 and IGKJ1*01 genes, respectively. The amino acid sequence of the light chain variable region of SLC15A4 monoclonal antibody 235 is compared with the amino acid sequence of the mouse VJ region as shown. Figure 10 As shown.

[0259] 4. SLC15A4 monoclonal antibody 235 promotes R848-induced THP1 activation and adhesion.

[0260] Preparation of SLC15A4 monoclonal antibody 235Fab fragment: using the Pierce Fab preparation kit (Thermo Scientific) TM The SLC15A4 monoclonal antibody 235Fab fragment was prepared and concentrated to ~10 mg / mL, aliquoted, and cryopreserved. The SLC15A4 monoclonal antibody 235Fab fragment used for functional experiments was processed using Toxin Eraser. TM Endotoxin Removal Kit (GenScript) removes endotoxins.

[0261] Slc15a4 - / - Construction of THP1 cells: The gRNA sequence of SLC15A4, forward(F): caccgCTAACGACAGGATCG CTCCC (SEQ ID NO:34), reverse(R): aaacGGGAGCGATCCTGTCGTTAGc (SEQ ID NO:35), was annealed and then ligated into the lentiCRISPR v2 vector (Addgene) purified by BsmBI-V2 (NEB) via homologous recombination. The vector was then packaged into a lentivirus for infection with THP1 cells using standard lentiviral packaging methods. After selection with puromycin, the infected THP1 cells were cultured as single clones. Sequencing confirmed the frameshift mutation as Slc15a4. - / - THP1 cells.

[0262] SLC15A4 monoclonal antibody 235Fab fragment promotes R848-induced THP1 activation and adhesion: Human monocyte cell line THP1, upon activation, exhibits morphological changes, transforming from a round monocyte morphology to an adherent state or further differentiating into a macrophage morphology. This process is accompanied by increased expression levels of CD14 and HLA-DR. To further demonstrate the promoting effect of SLC15A4 monoclonal antibody 235 on R848-induced THP1 activation and adhesion, THP1 or Slc15A4- / -THP1 cells were cultured at 4 × 10⁻⁶ cells / cells. 5200 μL of the antibody was seeded into 24-well plates, followed by 200 μL of a 40 μg / mL diluted SLC15A4 monoclonal antibody 235Fab fragment, and incubated overnight. The next day, 100 μL of R848 (InvivoGen) was added to the cell culture wells to bring the final concentration to 5 μg / mL, or 100 μL of culture medium was added as a control. Cells were cultured for another 24 hours, and the culture plates were photographed under white light using a Nikon Eclipse Ti2 microscope, or the cells were cultured for another 4 days to detect the levels of HLA-DR and CD14 expression on the cell membrane surface. Figure 4 As shown in Figure A, the 235Fab fragment of the SLC15A4 monoclonal antibody effectively promoted the adhesion of R848-induced THP1 cells. Figure 4 B and Figure 4 As shown in C, the 235Fab fragment of the SLC15A4 monoclonal antibody can effectively promote the expression levels of HLA-DR and CD14 induced by R848.

[0263] 5.235 promotes the activation of the TLR7 / 8 signaling pathway in THP1.

[0264] THP1 is divided into 5x10 5 / 200μL was seeded into a 24-well plate, followed by 200μL of SLC15A4 monoclonal antibody 235Fab fragment at different dilutions (specifically as follows) Figure 3 (As shown in A and B) After incubation overnight, 100 μL of LR848 (InvivoGen) was added to the cell culture wells the next day to bring the final concentration to 5 μg / mL, and the cells were cultured for another 24 hours. Cells and culture supernatant were then collected by centrifugation for cytokine detection.

[0265] Total RNA was extracted from cells using TRIzol (Invitrogen). RNA was then treated with RQ1 RNase-Free DNase (Promega, M6101) to eliminate the influence of genomic DNA. Subsequently, cDNA was synthesized using Oligo(dT) and MMLV reverse transcriptase (Promega, M1705) according to the manufacturer's instructions. Using the cDNA as a template, real-time PCR quantification was performed on a PowerUp SYBR GreenMaster Mix (Applied Biosystems, A25742) instrument on a QuantStudio Q7 (Applied Biosystems) instrument. The primer sequences used are shown in Table 5 below (Actinβ was used as an internal control).

[0266] Table 5

[0267] name Sequence (5'-3') SEQ ID NO: ACTB-F ACCGAGCGCGGCTACAG 36 ACTB-R CTTAATGTCACGCACGATTTCC 37 IFNB1-F GTCTCCTCCAAATTGCTCTC 38 IFNB1-R ACAGGAGCTTCTGACACTGA 39

[0268] The collected cell supernatant was used for ELISA to detect the expression of TNFα and IL6. The ELISA experiment was performed according to the kit instructions. The kits used were as follows: ELISA kits for human TNF (no. 3512-1H-20, MABTECH) and ELISA kits for human IL-6 (no. 3460-1H-20, MABTECH).

[0269] The results are as follows Figure 3 As shown in AC, the SLC15A4 monoclonal antibody 235Fab fragment can effectively promote the expression of IFNB1, IL6 and TNFα induced by R848-induced TLR7 / 8 signal activation in THP1, and the degree of promotion is positively correlated with the concentration of the SLC15A4 monoclonal antibody 235Fab fragment.

[0270] THP1 is divided into 5x10 5 200 μL of the antibody was seeded into 24-well plates, followed by 200 μL of 20 μg / mL SLC15A4 monoclonal antibody 235Fab fragment and incubated overnight. The next day, 100 μL of R848 (InvivoGen) was added to the cell culture wells to achieve final concentrations of 2.5, 5, and 10 μg / mL, respectively, and the cells were cultured for another 24 hours. The cell culture supernatant was then collected by centrifugation for ELISA detection of TNFα and IL6 expression. The ELISA experiments were performed according to the kit instructions, using the following kits: ELISA kits for human TNF (no. 3512-1H-20, MABTECH) and ELISA kits for human IL-6 (no. 3460-1H-20, MABTECH).

[0271] The results are as follows Figure 3 DE shows that the SLC15A4 monoclonal antibody 235Fab fragment can effectively promote the expression of IL6 and TNFα induced by TLR7 / 8 signal activation in THP1 at different concentrations of R848.

[0272] In summary, as Figure 3 As shown in AE, the SLC15A4 monoclonal antibody can promote the activation of the TLR7 / 8 signaling pathway in THP1 to produce cytokines.

[0273] 6. Binding to SLC15A4 antibody on cell membrane surface

[0274] Using Lipofectamine transfection reagent (ThermoFisher), 293T cells (purchased from ATCC) were transfected with plasmids pTT3-hSLC15A4-IRES-GFP, pTT3-hSLC15A4(32-558)-IRES-GFP, and pTT3-hSLC15A4(L14A / L15A)-IRES-GFP. Twenty-four hours after transfection, cells expressing human SLC15A4, GFP-FL (SLC15A4-WT, primarily lysosomal localization), and its membrane-localized mutants GFP-LL / AA (SLC15A4-L14A / L15A, membrane localization) and GFP-32-558 (SLC15A4(32-558), membrane localization) were obtained. These cells were treated with PBS containing 2 mM EDTA. The cell suspension was then added to 96-well U-bottom culture plates at a density of 10⁵ cells / 100 μL / well. After centrifugation at 2200 rpm for 3 minutes, the supernatant was discarded, and 1-2 μg / mL Fab 235 was added. The cells were incubated at 4°C for 30 minutes, washed twice with FACS buffer (PBS + 2 mM EDTA + 2% BSA), centrifuged, and stained with PE goat anti-Myc-tag antibody (Biolegend). The cells were then washed once with FACS buffer. After centrifugation and resuspending, the GFP and PE signals of the cells were analyzed using flow cytometry (BD LSR Fortessa). Wells showing double positive results for GFP and PE were antibody-positive wells that bound the hSLC15A4 membrane surface.

[0275] Figure 2 A shows that the Fab235 antibody can effectively bind to SLC15A4 expressed on the cell membrane surface. Notably, wild-type SLC15A4, primarily located on the endolysosomal membrane, can also be detected by the Fab235 antibody as being expressed on the cell membrane, indicating that this molecule may be cyclically expressed between the endolysosomal membrane and the cell membrane.

[0276] 7.235 antibody-mediated SLC15A4 endocytosis

[0277] To verify whether the 235 antibody can bind to SLC15A4 on the cell membrane surface and mediate endocytosis to exert an inhibitory function, the human monocyte cell line THP1 expressing SLC15A4 was first co-incubated with Fc receptor binding inhibitor antibody (Invitrogen) for 20 minutes, and then co-incubated with 1 μg / mL AF647-labeled 235 antibody for different times. The results were analyzed by flow cytometry and confocal microscopy.

[0278] Figure 2B-2D showed that at 37°C, the ability of AF647-labeled 235 antibody to bind to THP1 cells was significantly enhanced. Confocal microscopy images showed that there was also AF647 signal inside the cells, indicating that AF647-labeled 235 antibody can bind to a small amount of SLC15A4 expressed on the surface of THP1 cells and mediate endocytosis.

[0279] Example 3. Detection of the binding domain of SLC15A4 monoclonal antibody to human SLC15A4

[0280] 1. Construction of expression vectors for replacing σLL1-2 (63-72), σLL3-4 (125-154), σLL5-6 (216-222), σLL7-8 (348-366), σLL9-10 (429-453), and σLL11-12 (511-532) in the extracellular loop region of human SLC15A4.

[0281] (1) Primer design

[0282] Primers were designed based on the sequences of human SLC15A4 and its plasmid. The primer sequences are shown in Table 6.

[0283] Table 6

[0284]

[0285] (2) Plasmid construction

[0286] Using pTT3-hSLC15A4-IRES-GFP from Example 2 as a template, according to PrimeSTAR... According to the HSDNA Polymerase (TaKaRa) instructions, reverse PCR was performed using primers EL1-2-4GS-F and EL1-2-4GS-R to obtain PCR products 1-2; reverse PCR was performed using primers EL3-4-5GS-F and EL3-4-5GS-R to obtain PCR products 3-4; reverse PCR was performed using primers EL5-6-3GS-F and EL5-6-3GS-R to obtain PCR products 5-6; reverse PCR was performed using primers EL7-8-5GS-F and EL7-8-5GS-R to obtain PCR products 7-8; reverse PCR was performed using primers EL9-10-5GS-F and EL9-10-5GS-R to obtain PCR products 9-10; and reverse PCR was performed using primers EL11-12-4GS-F and EL11-12-4GS-R to obtain PCR products 11-12.

[0287] PCR conditions were as follows: pre-denaturation, 98℃, 2 min; denaturation, 98℃, 30 sec; annealing, 58℃, 30 sec; extension, 72℃, 2 min 30 sec; a total of 20 cycles were performed, with an additional 10 min of denaturation time at the end.

[0288] The PCR products were digested with DpnI enzyme (Thermo) for 1-4 hours, and then transformed into Escherichia coli TOP10 strain. Expression vectors were constructed to replace the extracellular loop regions of human SLC15A4 with vectors σLL1-2 (63-72), σLL3-4 (125-154), σLL5-6 (216-222), σLL7-8 (348-366), σLL9-10 (429-453), and σLL11-12 (511-532).

[0289] 2. Obtaining HEK293T cells by replacing the extracellular loop regions of human SLC15A4 cells with GS cells containing σLL1-2 (63-72), σLL3-4 (125-154), σLL5-6 (216-222), σLL7-8 (348-366), σLL9-10 (429-453), and σLL11-12 (511-532).

[0290] Wild-type human SLC15A4 (pTT3-hSLC15A4-IRES-GFP) and GS-replaced human SLC15A4 extracellular loop region expression plasmids σLL1-2 (63-72), σLL3-4 (125-154), σLL5-6 (216-222), σLL7-8 (348-366), σLL9-10 (429-453), and σLL11-12 (511-532) were transfected into HEK293T cell lines using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were treated with PBS containing 2 mM EDTA, and single-cell suspensions were collected to obtain HEK293T cells expressing human SLC15A4 (FL), HEK293T cells expressing human SLC15A4 TM1-TM2 replaced with GS (σLL1-2), and HEK293T cells expressing human SLC15A4 TM1-TM2 replaced with GS (σLL1-2). TM3-TM4 were replaced with GS HEK293T cells (σLL3-4), TM5-TM6 expressing human SLC15A4 were replaced with GS HEK293T cells (σLL5-6), TM7-TM8 expressing human SLC15A4 were replaced with GS HEK293T cells (σLL7-8), TM9-TM10 expressing human SLC15A4 were replaced with GS HEK293T cells (σLL9-10), and TM11-TM12 expressing human SLC15A4 were replaced with GS HEK293T cells (σLL11-12). The cells were fixed with 2% paraformaldehyde for 20 min, and the cell pellet was then resuspended in PBS buffer for antibody binding assays.

[0291] 3. Detection of binding of SLC15A4 monoclonal antibody 235 to various mutants

[0292] Following the method described in Example 2, the binding of SLC15A4 monoclonal antibody 235 to HEK293T cells expressing human SLC15A4 (FL), HEK293T cells expressing human SLC15A4 TM1-TM2 replaced with GS (σLL1-2), HEK293T cells expressing human SLC15A4 TM3-TM4 replaced with GS (σLL3-4), HEK293T cells expressing human SLC15A4 TM5-TM6 replaced with GS (σLL5-6), HEK293T cells expressing human SLC15A4 TM7-TM8 replaced with GS (σLL7-8), HEK293T cells expressing human SLC15A4 TM9-TM10 replaced with GS (σLL9-10), and HEK293T cells expressing human SLC15A4 TM11-TM12 replaced with GS (σLL11-12) mutants were detected.

[0293] The results are as follows Figure 5 As shown: SLC15A4 monoclonal antibody 235 (Fab235) does not bind to HEK293T cells expressing human SLC15A4 TM7-TM8 replaced with GS (σLL7-8) and HEK293T cells expressing human SLC15A4 TM9-TM10 replaced with GS (σLL9-10). The binding of human SLC15A4 TM11-TM12 replaced with GS (σLL11-12) reduces the level by about half. The binding of human SLC15A4 TM1-TM2 replaced with GS (σLL1-2) reduces the level by about 30%. The binding of human SLC15A4 TM3-TM4 replaced with GS (σLL3-4) and human SLC15A4 TM5-TM6 replaced with GS (σLL5-6) is basically unaffected. It is speculated that the SLC15A4 monoclonal antibody 235 binds to the LL1-2 and C-terminal domains (CTD) of the N-terminal domain (NTD) of SLC15A4.

[0294] Example 4. SLC15A4 monoclonal antibody binds to human SLC15A4, stably forming an inward (cytoplasmic) opening conformation.

[0295] 1. Assembly and purification of Nanodisc

[0296] The purified SLC15A4-TASL complex protein, SLC15A4 monoclonal antibody 235Fab fragment, scaffold protein MSP1D1 (amino acid sequence shown in SEQ ID NO:52), and phospholipid POPG (Avanti Polar Lipids, 268550-95-4) were mixed at a molar ratio of 1:1.1:2.2:120 and then gently stirred and incubated on ice for 1 hour. Bio-beads were added to a final concentration of 100 mg / mL and the mixture was continuously rotated overnight at 4°C. The next day, the bio-beads were removed, and the protein was purified using a Superdex 200 buffer pre-equilibrated with a nanodisc buffer. The peak protein was collected, and after SDS-PAGE analysis, it was concentrated to 15 mg / mL using a 50 kDa cutoff concentration tube for cryo-electron microscopy sample preparation.

[0297] 2. Cryo-electron microscopy sample preparation and data collection

[0298] Turn on the Vitrobot Mark IV (Thermo Fisher Scientific) power supply, replace the filter paper, and set the temperature to 8°C and the humidity to 100%. Place the copper cup, cold bridge, sample box, and other tools into the sample preparation foam box and pre-cool with liquid nitrogen for 20 minutes. Slowly introduce ethane gas into the copper cup. Due to the low temperature, the ethane will slowly liquefy. When the liquefied ethane almost fills the copper cup, quickly close the ethane valve. Using tweezers, pick up the grid and place it face up in the Salarus glow discharge instrument. After evacuating the vacuum, perform glow discharge on the grid using oxygen and argon. After 30 seconds, stop the glow discharge, release the vacuum, and remove the grid. When the liquid ethane in the copper cup becomes a solid-liquid mixture due to the low temperature, use tweezers to pick up the grid and fix it onto the Vitrobot Mark IV. Pipette 3 μL of protein sample from the Vitrobot onto the grid and prepare the sample under wait time of 5 s, blot force of 6-2, and blot time of 3-5 s. Quickly transfer the grid from the ethane to a cryostat pre-cooled in liquid nitrogen, and then store the cryostat in a liquid nitrogen tank.

[0299] Samples were initially screened using Talos 120 and Talos 200 electron microscopes. Extensive data collection was then performed on samples that performed well in the initial screening using a Talos Arctica 200kV FEG (Thermo Fisher Scientific). Data collection conditions were: voltage 200kV; pixel size... Electron Dosage The underfocus range is 0.8-1.2μm.

[0300] 3. Cryo-electron microscopy sample preparation

[0301] Taking a batch of data from SLC15A4-TASL-235 as an example, motion correction of electron microscope images was performed using the MotionCor2 program. Micrographs unsuitable for further data processing were manually deleted, and the remaining images were then processed using cryoSPARC v3, with CTF (CT Festimation) evaluation performed using CTFFIND or GCTF scripts. Thousands of particles were selected for two-dimensional classification using a blob picker, generating two-dimensional averages for further template selection. A total of 1,202,084 particles were selected using the template picker. After two rounds of two-dimensional classification, 407,127 particles with clear details were obtained. Next, 50,000 particles from the two-dimensional classification were reconstructed in three dimensions, resulting in a resolution of [resolution missing]. The structure. Subsequent CTF refinement and Bayesian polishing will increase the resolution to [missing information]. After processing the other two batches of data in a similar manner, 173,320 and 146,232 particles were obtained respectively. The REFINE3D dataset, after merging all particles, yielded a resolution of [resolution missing]. The structure was evaluated using ResMap and Golden Standard Fourier Shell Correlation, and all parameters were found to be normal.

[0302] 4. Construction of the SLC15A4 point mutation vector

[0303] Primers were designed based on the sequences of human SLC15A4 and its plasmid. The primer sequences are shown in Table 7.

[0304] Table 7

[0305]

[0306] (2) Plasmid construction

[0307] Using pTT3-hSLC15A4-IRES-GFP from Example 2 as a template, reverse PCR was performed according to the PrimeSTAR HSDNA Polymerase (TaKa Ra) instructions, using primers 442S-443S-435S-F and 442S-443S-435S-R to obtain the PCR product 442S-443S-435S; reverse PCR was performed using primers 523A-526A-528A-F and 523A-526A-528A-R to obtain the PCR product 523A-526A-528A; and reverse PCR was performed using primers 524S-F and 5... Reverse PCR was performed using primers 24S-R to obtain PCR product 524S; reverse PCR was performed using primers A65S-F and A65S-R to obtain PCR product A65S; reverse PCR was performed using primers 353-359-F and 353-359-R to obtain PCR product 353-359; reverse PCR was performed using primers 434-448-F and 434-448-R to obtain PCR product 434-448.

[0308] The PCR products were digested with DpnI enzyme (Thermo) for 1-4 hours, and then transformed into Escherichia coli TOP10 strains. Expression vectors corresponding to the point mutations (V442S / V443S / I435S, D523A / N526A / N528A, F524S, A65S, E353A / T359A, T434A / D448A) were constructed.

[0309] (3) Detection of binding of SLC15A4 monoclonal antibody 235 to various point mutations

[0310] Following the method in Example 3, HEK293T cells expressing the human SLC15A4 point mutation were obtained, and the binding level of SLC15A4 monoclonal antibody 235 to the above-mentioned HEK293T cells expressing the human SLC15A4 point mutation was detected.

[0311] 5. Binding surface analysis of SLC15A4 monoclonal antibody 235 with SLC15A4

[0312] Structural superposition centered on CTD showed that the interaction surface of SLC15A4 with 235 binding to the lysosomal cavity side (outward) was significantly reduced compared to SLC15A5 with the cytoplasmic side (inward).

[0313] like Figure 6As shown, in the SLC15A4-TASL-235 complex structure, 235 binds to the lysosomal surface of SLC15A4. The most significant feature is that hydrophobic interactions almost completely dominate the interface, while hydrogen bonding further strengthens the interaction between SLC15A4 and 235. Specifically, residue A65 of LL1-2 in the N-terminal domain of SLC15A4, residues I435, V442, and V443 of LL9-10 in the C-terminal domain of SLC15A4, and residue F524 of LL11-12, bind to Y32 on CDR1 and CDR3 of the 235 light chain. L F91 L and H92 L And F32 on heavy chains CDR1 and CDR3 H I102 H I103 H and A105 H Hydrophobic interactions are formed. Around these hydrophobic interactions, multiple hydrogen bonds and salt bridges are formed. Residues E353 and T359 on LL7-8 of SLC15A4 interact with the side chains of S29L and R104H on 235 via hydrogen bonds and salt bridges, respectively, further increasing their binding. Furthermore, the side chains T434 and D448 on LL9-10 of SLC15A4 form hydrogen bonds with Y59H and R93L on 235, respectively. The side chains D523, N526, and N528 on LL11-12 of SLC15A4 interact with S53H on 235, respectively. H S56 H and N57 H The side chains form hydrogen bonds. Flow cytometry analysis of the constructed mutant further confirmed the key binding sites mentioned above. Simultaneously, the structure showed that the binding interface between 235 and SLC15A4 is approximately [missing information - likely a number]. The surface area is distributed in the NTD and CTD of SLC15A4, of which the NTD accounts for approximately 235. CTD occupies approximately Compared to the inward-open state of SLC15A4, there is no interaction between the NTD and 235 of the outward-open SLC15A4, and the CTD-antibody binding interface is also reduced by nearly half (approximately). This indicates that 235 is more likely to bind to SLC15A4 in an inward-open state, and the extremely strong interaction between 235 and SLC15A4 can stabilize the inward-open state of SLC15A4.

[0314] The technical solutions disclosed herein are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of this invention fall within the protection scope of this invention.

Claims

1. An SLC15A4 antibody or an antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises: Heavy chain variable region comprising amino acid sequences CDR-H1, CDR-H2, and CDR-H3 as shown in SEQ ID NO: 10; and The light chain variable region contains CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region as shown in SEQ ID NO: 24; The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are defined by the definition schemes of Kabat, Chothia, IMGT, Martin, Contact, or AbM.

2. The SLC15A4 antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 27, respectively. The CDRs are defined using the Kabat scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L3 are shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 26, and SEQ ID NO: 27, respectively. The amino acid sequence of CDR-L2 is STS. The CDR is defined according to the IMGT definition scheme. The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 16, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 27, respectively. The CDRs are defined according to the Chothia scheme; or The amino acid sequences of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively. The CDR is defined using the Contact definition scheme.

3. The SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The amino acid sequence of the heavy chain variable region includes: b1) SEQ ID NO:10; or b2) An amino acid sequence of SEQ ID NO:10 that has undergone substitution, deletion, and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO:10; and / or The amino acid sequence of the light chain variable region includes: c1) SEQ ID NO:24; or c2) An amino acid sequence of SEQ ID NO:24 that has undergone substitution, deletion and / or addition of one or more amino acids and has the same function as the protein shown in SEQ ID NO:

24.

4. The SLC15A4 antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody is of type IgA, IgD, IgE, IgG, or IgM; or The amino acid sequence of the heavy chain variable region includes: b3) An amino acid sequence that shares 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, or 75% homology with SEQ ID NO:10 and has the same function as the protein shown in SEQ ID NO:10; or The amino acid sequence of the light chain variable region includes: c3) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO:24 and has the same amino acid sequence as the protein shown in SEQ ID NO:

24.

5. The SLC15A4 antibody or its antigen-binding fragment according to claim 4, characterized in that, The antibody is of the IgG type.

6. The SLC15A4 antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody is of type IgG1, IgG2, IgG3 or IgG4.

7. The SLC15A4 antibody or its antigen-binding fragment according to claim 3, characterized in that, The antigen-binding fragments include scFv, Fab, Fab', (Fab')2, Fv fragments, dsFv; and / or The antibody or antigen-binding fragment includes murine antibodies, chimeric antibodies, and humanized antibodies.

8. The SLC15A4 antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

9. A biological material relating to the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8, said biological material comprising any one of n1)-n9): n1) A nucleic acid molecule encoding the SLC15A4 antibody or its antigen-binding fragment as described in any one of claims 1-8; n2) An expression cassette containing the nucleic acid molecule described in n1); n3) A carrier containing the nucleic acid molecule described in n1); n4) A carrier containing the expression box described in n2); n5) A cell containing the nucleic acid molecules described in n1); n6) Cells containing the expression cassette described in n2); n7) Cells containing the carrier described in n3); n8) Cells containing the carrier described in n4); n9) Cells comprising the SLC15A4 antibody or its antigen-binding fragment as described in any one of claims 1-8; None of the cells described in n5)-n9) contain reproductive material.

10. The biomaterial according to claim 9, characterized in that, Any of the vectors described in n3)-n4) includes prokaryotic expression vectors and eukaryotic expression vectors.

11. The biomaterial according to claim 10, characterized in that, The eukaryotic expression vectors include yeast expression vectors, mammalian expression vectors, and insect expression vectors.

12. The biomaterial according to claim 9, characterized in that, The nucleic acid molecule encoding the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8 comprises a nucleic acid molecule encoding the heavy chain variable region of the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8 and a nucleic acid molecule encoding the light chain variable region of the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8.

13. The biomaterial according to claim 12, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8 comprises: d1)SEQ ID NO: 23; or d2) A nucleotide sequence of SEQ ID NO: 23 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO: 23; The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8 comprises: e1)SEQ ID NO: 25; or e2) A nucleotide sequence of SEQ ID NO: 25 that has undergone substitution, deletion and / or addition of one or more nucleotides and has the same function as the nucleic acid molecule shown in SEQ ID NO:

25.

14. The biomaterial according to claim 13, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8 comprises: d3) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, or 75% homology with SEQ ID NO: 23, and has the same function as the nucleic acid molecule shown in SEQ ID NO: 23; or The nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8 comprises: e3) has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology with SEQ ID NO: 25 and has the same function as the nucleic acid molecule shown in SEQ ID NO:

25.

15. A conjugate comprising: the SLC15A4 antibody or its antigen-binding fragment as described in any one of claims 1-8, and a conjugation portion; wherein the conjugation portion is selected from at least one of a detectable marker, a radionuclide, gold nanoparticles / nanorobars, and magnetic nanoparticles.

16. The coupling according to claim 15, characterized in that, The detectable marker is a fluorescent or luminescent marker.

17. The coupling according to claim 15, characterized in that, The detectable marker is selected from any one of acridine ester, acridine sulfonamide, luminol, isoluminol, horseradish peroxidase, and alkaline phosphatase; and / or The radionuclide mentioned is a diagnostic isotope.

18. The coupling according to claim 17, characterized in that, The diagnostic isotopes are selected from at least one of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, and Re-188.

19. A chimeric antigen receptor comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the extracellular antigen-binding domain comprises the SLC15A4 antibody or an antigen-binding fragment thereof as described in any one of claims 1-8.

20. A modified immune cell comprising the chimeric antigen receptor of claim 19.

21. The use of the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8, the biomaterial according to any one of claims 9-14, or the conjugate according to any one of claims 15-18 in the preparation of a product, wherein the product has the following function: e1) detecting the presence or content of SLC15A4 in a sample; The product is at least one of reagents, test plates, chips, test strips, and kits.

22. A product comprising the SLC15A4 antibody or its antigen-binding fragment as described in any one of claims 1-8, or the conjugate as described in any one of claims 15-18; The product is at least one of reagents, test plates, chips, test strips, and kits.

23. The product according to claim 22, characterized in that, The product has the following functions: e1) Detect the presence or content of SLC15A4 in the sample.

24. The use of the SLC15A4 antibody or its antigen-binding fragment according to any one of claims 1-8, the biomaterial according to any one of claims 9-14, or the conjugate according to any one of claims 15-18 in any one of f2)-f3): f1) Tracking the specific conformation of SLC15A4; f2) Assisted screening of SLC15A4-activated small molecules; f3) Prepare a product, which is used in f1) and / or f2).

Images