Anti-CD163 monoclonal antibodies, antibody combinations and related applications

Abstract

This invention provides a monoclonal antibody against CD163, antibody combinations, and related applications. Utilizing single-B cell sequencing technology, this invention identifies 19 anti-CD163 antibodies belonging to multiple different antigen-binding epitope groups, offering greater antibody diversity and providing more possibilities for selecting paired antibodies. It is applicable to various scenarios such as ELISA, flow cytometry, proteoblotting, and immunohistochemistry.

Description

Technical Field

[0001] This invention relates to the field of antibodies, and more specifically, to a monoclonal antibody against CD163, antibody combinations, and related applications. Background Technology

[0002] CD163, or scavenger receptor cysteine-rich type 1 protein M130, belongs to the scavenger receptor family and is expressed only on monocytes and macrophages, such as Kupffer cells in the liver and red pulp macrophages in the spleen. It can serve as a specific marker on the surface of M2 macrophages. The CD163 receptor consists of a large extracellular domain, a type I transmembrane domain, and a small intracellular domain. The extracellular domain consists of nine scavenger receptor cysteine-rich (SRCR) domains. CD163 is also the receptor for macrophage endocytosis of the hemoglobin-haptoglobin complex, with the SRCR3 domain proven to be a key domain interacting with the hemoglobin-haptoglobin complex. The CD163-mediated endocytosis during the acute reaction phase may help protect tissues from free hemoglobin-mediated oxidative damage; this process is regulated through calcium-dependent and pH-dependent mechanisms.

[0003] CD163 protein also exists in the blood in a soluble form (sCD163). sCD163 is released by protease cleavage, and its level can be induced by lipopolysaccharide or the Fc fragment of immunoglobulin IgG. sCD163 may have anti-inflammatory effects and is considered an effective biomarker for monitoring macrophage activation. High expression of CD163 plays an important role in suppressing inflammatory responses and clearing components of damaged cells; differences in its expression levels may also affect the proliferation and stemness of glioma cells by mediating casein kinase CK2. The concentration of sCD163 in plasma can serve as a biomarker for inflammatory diseases, such as microbial infections, autoimmune diseases, and cancer. CD163 is also a novel therapeutic target; several drugs targeting CD163 are currently on the market or in clinical development.

[0004] Therefore, the development of CD163 antibodies is of great significance for immunological research and clinical diagnosis, and can support the development of novel therapeutic strategies. However, while anti-CD163 antibodies are important reagents in immunological research and clinical diagnosis, most CD163 antibodies are from mice or derived from hybridoma screening. Hybridoma cell lines are prone to gene drift and mutation, leading to changes in the antibody composition, which may affect production and the reproducibility of results. Furthermore, some hybridoma antibody production involves mouse ascites fluid, making batch-to-batch stability difficult to maintain.

[0005] The current situation indicates that the procurement cycle for imported antibody reagents is long and the prices are high, which is detrimental to supporting the development of domestic scientific and clinical research. Meanwhile, most domestically produced antibodies are polyclonal antibodies or mouse monoclonal antibodies. Polyclonal antibodies are difficult to control batch-to-batch stability, and the production of hybridoma-derived mouse monoclonal antibodies is unstable, with the affinity of most antibodies being uncertain. Therefore, there is an urgent need to provide a new CD163 antibody to increase market selectivity for CD163 antibodies, especially to increase the selectivity of paired antibodies. Summary of the Invention

[0006] The main objective of this invention is to provide an anti-CD163 monoclonal antibody, antibody combination, and related applications to solve the problems of low selectivity and high cost of antibodies in the prior art.

[0007] To achieve the above objectives, according to a first aspect of the present invention, an isolated anti-CD163 monoclonal antibody or its antigen-binding fragment is provided, wherein the monoclonal antibody or its antigen-binding fragment comprises heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3.

[0008] The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from any of the following groups:

[0009] 1) SEQ ID NO: 41: GIDLSIYV, SEQ ID NO: 42: ISSSGNK, SEQ ID NO:

[0010] 43: ARLSGHTVRLDL, SEQ ID NO: 44: QSVYNNNW, SAS and SEQ ID NO: 45:

[0011] LGGYSSTADNT;

[0012] 2) SEQ ID NO: 46: GIDLSIFV, SEQ ID NO: 47: ISYSGNT, SEQ ID NO:

[0013] 48: ARINGDTVRLDL, SEQ ID NO: 49: QSVYNANF, RAS and SEQ ID NO: 50:

[0014] LGGYSSTSDNT;

[0015] 3) SEQ ID NO: 51: GFSLSSYA, SEQ ID NO: 52: ISDGGNT, SEQ ID NO:

[0016] 53: ARAGANSDGDYWDRLDL, SEQ ID NO: 54: QSVYNNNY, DAS and SEQ ID NO: 55: LGGYDDDADTT;

[0017] 4) SEQ ID NO: 56: GIDLSDYV, SEQ ID NO: 57: SSIVGSA, SEQ ID NO:

[0018] 58: ARNPGYSDVI, SEQ ID NO: 59: QSIGSN, IAS and SEQ ID NO: 60:

[0019] QSAYYSTTSSDT;

[0020] 5) SEQ ID NO: 61: GIDLSSYA, SEQ ID NO: 62: ISSSGST, SEQ ID NO:

[0021] 63: ARAGAGGWAYGGYDI, SEQ ID NO: 59: QSIGSN, KAS and SEQ ID NO: 64:

[0022] QSTYYGISYVGA;

[0023] 6) SEQ ID NO: 65: GFSLSTSD, SEQ ID NO: 66: ISGGAIL, SEQ ID NO:

[0024] 67: GRGYDHLDL, SEQ ID NO: 68: ETIGNA, TVF and SEQ ID NO: 69:

[0025] QGGDYDGSYVFA;

[0026] 7) SEQ ID NO: 70: GFSLSSYY, SEQ ID NO: 71: IGPGGNT, SEQ ID NO:

[0027] 72: ARVYSNNDDFDP, SEQ ID NO: 73: QSISSY, RAS and SEQ ID NO: 74:

[0028] HQGYSYSDVDNA;

[0029] 8) SEQ ID NO: 75: GIDLRSNA, SEQ ID NO: 76: ISAGGSV, SEQ ID NO:

[0030] 77: ARAGYGGWSYGGFDP, SEQ ID NO: 59: QSIGSN, KAS and SEQ ID NO: 78:

[0031] QSTYYGVSYVGS;

[0032] 9) SEQ ID NO: 79: GIDLSYYA, SEQ ID NO: 80: LTSGGNT, SEQ ID NO:

[0033] 81: ARDVNQPTAWYFDI, SEQ ID NO: 82: QNIYSN, DAS and SEQ ID NO: 83:

[0034] QGGVYSSDVEIT;

[0035] 10) SEQ ID NO: 70: GFSLSSYY, SEQ ID NO: 84: IGHSGTT, SEQ ID NO:

[0036] 85: ARVYDNSDRLDL, SEQ ID NO: 86: QSISGY, KAS and SEQ ID NO: 87:

[0037] QQGYSYTNVDNT;

[0038] 11) SEQ ID NO: 88: EFSLSSYV, SEQ ID NO: 89: IKPSGNT, SEQ ID NO:

[0039] 90: ARDCYSSDTGYDV, SEQ ID NO: 91: ESIGSR, SAS and SEQ ID NO: 92:

[0040] QCSYDGSGYVGGP;

[0041] 12) SEQ ID NO: 93: GFTLSNNYW, SEQ ID NO: 94: IYGDSSVST, SEQ ID NO: 95: ARDLGGTSLYPDWLDL, SEQ ID NO: 82: QNIYSN, GAS and SEQ ID NO: 96: QGYYYSSGSVDNA;

[0042] 13) SEQ ID NO: 51: GFSLSSYA, SEQ ID NO: 62: ISSSGST, SEQ ID NO:

[0043] 97: ARGVGDNGYLNI, SEQ ID NO: 98: ESISSW, KAS and SEQ ID NO: 99:

[0044] QSVIVGTSFLGG;

[0045] 14) SEQ ID NO: 100: GIDLSSNA, SEQ ID NO: 101: VSGSGHT, SEQ ID NO: 102: ARGAYASDNSYYDI, SEQ ID NO: 103: QNIYRN, TTA and SEQ ID NO:

[0046] 104: QSYYYDLFGTAYD;

[0047] 15) SEQ ID NO: 105: GFSLSSYV, SEQ ID NO: 106: ISASGNG, SEQ ID NO: 107: AREVDYGDYFFNI, SEQ ID NO: 82: QNIYSN, AAS and SEQ ID NO:

[0048] 108: QSAYYSSSADTFA;

[0049] 16) SEQ ID NO: 109: GFSLSSNA, SEQ ID NO: 110: IYGRGDT, SEQ ID NO: 111: ARTYGSNNDYYNI, SEQ ID NO: 73: QSISSY, KAS and SEQ ID NO:

[0050] 112: QQGYSETNVDNP;

[0051] 17) SEQ ID NO: 61: GIDLSSYA, SEQ ID NO: 62: ISSSGST, SEQ ID NO:

[0052] 113: ARCGGSNYFDSCAFNI, SEQ ID NO: 98: ESISSW, GAS and SEQ ID NO:

[0053] 114: QSYYYISSSDSNP;

[0054] 18) SEQ ID NO: 115: GIDLDRYY, SEQ ID NO: 116: INAYGST, SEQ ID NO: 117: ARVGLVGVIHFLDI, SEQ ID NO: 118: EDIYNL, EAS and SEQ ID NO:

[0055] 119: QCAYYGSHYIFT; or

[0056] 19) SEQ ID NO: 61: GIDLSSYA, SEQ ID NO: 120: TNIHDMT, SEQ ID NO:

[0057] 121: ARVFGYAGYGYGTGFYYYGLDL, SEQ ID NO: 122: QSIGYN, KAS and SEQ ID NO: 123: QCTYYGDTYIDA.

[0058] Furthermore, the monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the amino acid sequences of which are selected from any one of the following groups:

[0059] 1) SEQ ID NO: 1 and 21; 2) SEQ ID NO: 2 and 22; 3) SEQ ID NO: 3 and 23;

[0060] 4) SEQ ID NO: 4 and 24; 5) SEQ ID NO: 5 and 25; 6) SEQ ID NO: 6 and 26;

[0061] 7) SEQ ID NO: 7 and 27; 8) SEQ ID NO: 8 and 28; 9) SEQ ID NO: 9 and 29;

[0062] 10) SEQ ID NO: 10 and 30; 11) SEQ ID NO: 11 and 31; 12) SEQ ID NO: 12 and 32;

[0063] 13) SEQ ID NO: 13 and 33; 14) SEQ ID NO: 14 and 34;

[0064] 15) SEQ ID NO: 16 and 36; 16) SEQ ID NO: 17 and 37; 17) SEQ ID NO: 18 and 38;

[0065] 18) SEQ ID NO: 19 and 39; 19) SEQ ID NO: 20 and 40; or

[0066] An amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the sequences in any of groups 1) to 19).

[0067] Furthermore, the monoclonal antibody or its antigen-binding fragment is selected from full-length antibodies, single-chain antibodies, Fab, Fab', Fab'-SH, or F(ab')2.

[0068] Furthermore, the monoclonal antibody or its antigen-binding fragment also includes a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is a constant region of human, mouse, monkey, rabbit or sheep immunoglobulin, preferably a constant region of rabbit IgG; and the light chain constant region is a constant region of rabbit κ type.

[0069] To achieve the above objectives, according to a second aspect of the present invention, an anti-CD163 antibody composition is provided, the antibody composition comprising any one or more of the above-described anti-CD163 monoclonal antibodies or their antigen-binding fragments.

[0070] Furthermore, the antibody composition comprises a combination of two or more antibodies that bind to the same epitope; or a combination of two or more antibodies that bind to different epitopes.

[0071] According to a third aspect of the present invention, a nucleic acid molecule is provided that encodes any of the above-mentioned anti-CD163 monoclonal antibodies or antigen-binding fragments thereof.

[0072] According to a fourth aspect of the present invention, a recombinant expression vector is provided, the recombinant expression vector comprising the above-described nucleic acid molecule.

[0073] According to a fifth aspect of the present invention, a host cell is provided, the host cell being transfected with the above-described recombinant expression vector; preferably, the host cell is selected from prokaryotic cells or eukaryotic cells; preferably, the eukaryotic cell is selected from yeast cells, insect cells or mammalian cells; preferably, the mammalian cell is 293F cells, 293T cells, HEK293 cells, CHO cells or NSO cells.

[0074] According to a sixth aspect of the present invention, a kit is provided comprising any one or more of the above-described anti-CD163 monoclonal antibodies or their antigen-binding fragments, or any one or more of the above-described antibody compositions, nucleic acid molecules, recombinant expression vectors, or host cells.

[0075] Furthermore, the anti-CD163 monoclonal antibody in the kit is a labeled antibody with a detection marker; preferably, the detection marker is selected from any one of the following: enzyme, fluorescent dye, fluorescent protein, isotope, biotin or colloidal gold.

[0076] According to a seventh aspect of the present invention, the use of any of the above-described anti-CD163 monoclonal antibodies or their antigen-binding fragments or any of the above-described anti-CD163 antibody compositions in the preparation of a detection product for detecting CD163 is provided.

[0077] Furthermore, applications include detecting CD163 protein or CD163-expressing cells using any one or more of the following methods: ELISA, proteoblotting, flow cytometry, or immunohistochemistry.

[0078] Furthermore, applications also include combined or conjugated use with other reagents, such as antibodies, proteins, peptides, magnetic beads, chemiluminescent substances, or nucleic acids.

[0079] By applying the technical solution of this invention, multiple anti-CD163 antibodies were discovered for the first time using single-cell B-cell sequencing technology, increasing the selectivity of anti-CD163 antibodies. Furthermore, the antibodies in this application contain multiple groups of antibodies with the same epitope, providing a large number of candidate paired antibodies and thus offering more possibilities for selecting paired antibodies. In addition, the antibodies in this application are all recombinant antibodies. The production method of recombinant antibodies is highly controllable, thus helping to address the problem of poor batch-to-batch stability in existing antibodies.

[0080] In some preferred embodiments, these recombinant antibodies are rabbit-derived antibodies, with a single subtype, precise epitope recognition, and naturally higher affinity and stronger specificity. The multiple rabbit recombinant antibodies recognizing the CD163 protein provided by this invention, through epitope competition assays, identify multiple groups of antibodies with different epitopes within the antibody population, making them applicable to various applications such as ELISA, flow cytometry, Western blotting (WB), and immunohistochemistry (IHC). Attached Figure Description

[0081] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0082] Figure 1A and 1B The diagram shows the protein purification and identification results of the CD163 recombinant protein in Example 1 of the present invention.

[0083] Figure 2 The figure shows the results of rabbit serum titer detection of rabbits immunized with CD163 recombinant protein in Example 2 of the present invention.

[0084] Figure 3 The results of B cell sorting in rabbits immunized with CD163 recombinant protein in Example 3 of the present invention are shown.

[0085] Figure 4 The SDS-PAGE detection results of some representative recombinant antibodies in Example 5 of the present invention are shown.

[0086] Figure 5 The results of the BLI initial screening binding antibody and antibody affinity assay in Example 6 of the present invention are shown.

[0087] Figure 6 The results of the antibody epitope competition experiment grouping in Example 7 of the present invention are shown.

[0088] Figure 7 The flow cytometry results of the antibody binding on 293F cells in Example 8 of the present invention are shown.

[0089] Figure 8 The WB test results of some representative binding antibodies in Example 9 of the present invention are shown.

[0090] Figure 9 The IHC test results of some representative binding antibodies in Example 10 of the present invention are shown. Detailed Implementation

[0091] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0092] Terminology Explanation:

[0093] Recombinant antibodies, also known as genetically engineered antibodies, are monoclonal antibodies produced in vitro using synthetic antibody genes. These synthetic antibody genes are modified and recombined using DNA recombination technology, constructed onto plasmids, and then transferred to suitable host cell lines for antibody expression. Compared to traditional polyclonal and monoclonal antibodies, recombinant antibodies offer several key advantages, including good batch-to-batch consistency, continuous supply, and adaptability to antibody engineering. Recombinant antibodies have significant advantages in scientific research, particularly in addressing reproducibility challenges.

[0094] Antibody: A protein that binds to a specific antigen, broadly referring to all proteins and protein fragments containing a complementarity-determining region (CDR), particularly full-length antibodies. When an antibody is a protein fragment containing a CDR, it can be a substance containing part or all of the antibody's CDR, lacking at least some amino acids present in a full-length antibody but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. In some embodiments, antibodies in this fragment form have the function of specifically recognizing and binding to CD19. In some specific embodiments, such fragments are selected from Fab (composed of the complete light chain and Fd), Fv (composed of VH and VL), ScFv (single-chain antibody with VH and VL linked by a linker peptide), or single-domain antibodies (composed of VH only). Antibodies in this fragment form can be produced by genetic engineering techniques or by enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0095] "Complementarity-determining region" (CDR): This refers to a highly variable region of the heavy and light chains of an immunoglobulin. There are three heavy-chain CDRs and three light-chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to regions containing one or more, or even all, of the major amino acid residues that contribute to the binding affinity of antibodies to the antigens or epitopes they recognize. In other specific embodiments, CDR refers to the highly variable region of the heavy and light chains of an immunoglobulin as defined by IMGT.

[0096] In this application, the complementarity-determining region (CDR) of the heavy chain is represented by HCDR, and the complementarity-determining region of the light chain is represented by LCDR. Commonly used CDR labeling methods in the art include the IMGT numbering system, the Kabat numbering system, the Chothia and Lesk numbering system, and the new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. This application uses the unique IMGT annotation for CDR labeling, but CDR regions labeled by other methods are also within the scope of protection of this application. The International Immunogenetic Database (IMGT) is a database specifically for immunoglobulin (antibody) and T cell receptor genes and their expression. It is the official repository of all immunoglobulin (IG) and T cell receptor (TR) genes and alleles confirmed by the World Health Organization (WHO) / International Union of Immunological Societies (IUIS) Nomenclature Committee. The IMGT annotation standard is a standardized numbering system used in the IMGT database to describe antibodies and variable regions. This numbering system is based on sequence homology and structural characteristics, rather than on the absolute position of the sequence. This means that even in different antibody sequences, positions with the same number may correspond to regions with similar structures and functions. This annotation standard is currently widely adopted.

[0097] As mentioned in the background section, most existing anti-CD163 antibodies are obtained through hybridoma screening. Hybridoma cell lines are prone to gene drift and mutation, leading to changes in the antibody and potentially affecting production and the reproducibility of results. Furthermore, some hybridoma antibody production involves mouse ascites, making batch-to-batch stability difficult to maintain. To improve this situation and meet the needs of immunological research and clinical diagnostic applications, the applicant attempted to utilize existing genetic engineering techniques. This invention selects a soluble recombinant CD163 protein expressed in a eukaryotic system as an immunogen to immunize New Zealand white rabbits. After multiple rounds of immunization, the antiserum titer was detected by enzyme-linked immunosorbent assay (ELISA). Once the serum titer reached 1:20000, a shock immunization was performed, followed by antibody screening using single-B cell sequencing technology. This resulted in a batch of recombinant antibodies with high batch-to-batch stability, relatively high affinity, and high specificity.

[0098] Specifically, the basic steps for screening antibodies according to the present invention are as follows:

[0099] First, peripheral blood was collected to isolate rabbit PBMCs. The cells were incubated with antibodies against T cells and anti-monocytes. Magnetic beads were added, and the cells were negatively selected for B cell separation on a magnetic rack using a magnetic bead separation column. The negatively selected B cells were then incubated with fluorescent secondary antibodies against antigen and anti-rabbit IgG. Finally, antigen-specific IgG was separated using a flow cytometry system. +Single B cells were sorted into 384-well plates. Smart-seq sequencing was then performed on the single B cells in the plates, and the sequencing data were used for BCR analysis of the immune repertoire to obtain 50 pairs of single-cell paired light and heavy chain antibody sequences. The light and heavy chain sequences of the antibodies were then constructed into the recombinant protein expression vector pcDNA3.4(+). The recombinant antibodies were expressed using a eukaryotic protein expression system. The light and heavy chain vector plasmids were co-transfected into HEK 293F cells for expression. Antibody proteins were obtained by affinity chromatography, and the affinity of the antibodies to the antigen was measured using biomembrane interference (BLI) technology, thereby screening out 19 binding antibodies with affinity at the nanomolar or even picomolar level. Finally, the binding antibodies were subjected to epitope competition experiments, flow cytometry (Fc) detection, Western blot (WB) detection, and immunohistochemistry (IHC) detection, ultimately obtaining a high-affinity and high-specificity rabbit recombinant monoclonal antibody group. The amino acid sequences of the complementarity-determining regions (HCDR1, HCDR2, HCDR3) of the heavy chain variable region and the complementarity-determining regions (LCDR1, LCDR2, LCDR3) of the light chain variable region of each monoclonal antibody are as follows:

[0100] 1) The amino acid sequence of HCDR1 of antibody A1 is as follows: GIDLSIYV (SEQ ID NO: 41) ； The amino acid sequence of HCDR2 is as follows: ISSSGNK (SEQ ID NO: 42) ； The amino acid sequence of HCDR3 is as follows: ARLSGHTVRLDL (SEQ ID NO: 43) ； The amino acid sequence of LCDR1 is as follows: QSVYNNNW (SEQ ID NO: 44) ； The amino acid sequence of LCDR2 is as follows: SAS; The amino acid sequence of LCDR3 is as follows: LGGYSTADNTT (SEQ ID NO: 45) 。

[0101] The variable region sequences corresponding to the A1 antibody are as follows:

[0102] SEQ ID NO: 1A1 antibody heavy chain variable region amino acids

[0103] QSVEESGGRLVTPGGTLTLTCTVS GIDLSIYV MGWVRQAPGKGLEYIGI ISSSGNK YYANWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFC ARLSGHTVRLDL WGQGTLVTVSS;

[0104] SEQ ID NO: 21A1 antibody light chain variable region amino acids

[0105] AAVLTQTPSPVSAAVGGTVTISCQSS QSVYNNNW LGWYQQKPGQPPKLLIY SAS TLASGVPSRFKGSGSGTQFTLTISDLECDDAATYYC LGGYSTADNTT FGGGTEVVVK.

[0106] 2) The amino acid sequence of HCDR1 of the A3 antibody is as follows: GIDLSIFV (SEQ ID NO: 46) ； The amino acid sequence of HCDR2 is as follows: ISYSGNT (SEQ ID NO: 47) ； The amino acid sequence of HCDR3 is as follows: ARINGDTVRLDL (SEQ ID NO: 48) ； The amino acid sequence of LCDR1 is as follows: QSVYNANF (SEQ ID NO: 49) ； The amino acid sequence of LCDR2 is as follows: RAS; The amino acid sequence of LCDR3 is as follows: LGGYSSTSDNT (SEQ ID NO: 50) 。

[0107] The variable region sequences corresponding to the A3 antibody are as follows:

[0108] SEQ ID NO: 2A3 Antibody heavy chain variable region amino acids

[0109] QSVEESGGRLVTPGGSLTLTCTVS GIDLSIFV MGWVRQAPGKGLEYIGI ISYSGNT YYAN WAKGRFAISKTSSTTVDLKITSPTTEDTATYFC ARINGDTVRLDL WGQGTLVTVSS;

[0110] SEQ ID NO: 22A3 antibody light chain variable region amino acid

[0111] AAVMTQTPSSVSAAVGGTVTINCQSS QSVYNANF LSWYQQKPGQPPKLLIY RAS TLAS GVPSRFRGSGSGTQFTLTINDLECDDAATYYC LGGYSSTSDNT FGGGTEVVVK.

[0112] 3) The amino acid sequence of HCDR1 of the A5 antibody is as follows: GFSLSSYA (SEQ ID NO: 51) ； The amino acid sequence of HCDR2 is as follows: ISDGGGNT (SEQ ID NO: 52) ； The amino acid sequence of HCDR3 is as follows: ARGANSDGDYWDRLDL (SEQ ID NO: 53) ； The amino acid sequence of LCDR1 is as follows: QSVYNNNY (SEQ ID NO: 54) ； The amino acid sequence of LCDR2 is as follows: DAS; The amino acid sequence of LCDR3 is as follows: LGGYDDDADTT (SEQ ID NO: 55) 。

[0113] The variable region sequence of the A5 antibody is as follows:

[0114] SEQ ID NO: 3A5 antibody heavy chain variable region amino acids

[0115] QSVEESGGRLVTPGTPLTLTCTVS GFSLSSYA MSWVRQAPGKGLEWIGI ISDGGGNT YYAT WAKGRFTISKTSTTVDLKITSPTTEDTATYFC ARGANSDGDYWDRLDL WGQGTLVTVSS; SEQ ID NO: 23A5 Antibody light chain variable region amino acids

[0116] AAVLTQTPSPVSAAVGGTVTISCQSS QSVYNNNY LSWYQQKPGQPPKLLIY DAS KLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYC LGGYDDDADTT FGGGTEVVVK.

[0117] 4) The amino acid sequence of HCDR1 of the A8 antibody is as follows: GIDLSDYV (SEQ ID NO: 56) ； The amino acid sequence of HCDR2 is as follows: SSIVGSA (SEQ ID NO: 57) ； The amino acid sequence of HCDR3 is as follows: ARNPGYSDVI (SEQ ID NO: 58) ； The amino acid sequence of LCDR1 is as follows: QSIGSN (SEQ ID NO: 59) ； The amino acid sequence of LCDR2 is as follows: IAS;The amino acid sequence of LCDR3 is as follows: QSAYYSTTSSSDT (SEQ ID NO: 60) 。

[0118] The variable region sequence of the A8 antibody is as follows:

[0119] SEQ ID NO: 4A8 antibody heavy chain variable region amino acid

[0120] QSLEESGGRLVTPGTPLTLTCTVS GIDLSDYV MSWVRQAPGKGLEYIGW SSIVGSA YYA TWAKGRFTISRTSTTVDLKMTSLTTEDTATYFC ARNPGYSDVI WGPGTLVTVSS;

[0121] SEQ ID NO: 24A8 antibody light chain variable region amino acid

[0122] DIVMTQTPSSVEAAVGGTVTIKCQAS QSIGSN LAWYQQKPGQPPQLLIY IAS TLASGVSSRFKGSRSGTEYTLTISDLECADAATYYC QSAYYSTTSSSDT FGGGTEVVVK.

[0123] 5) The amino acid sequence of HCDR1 of the A9 antibody is as follows: GIDLSSYA (SEQ ID NO: 61) ； The amino acid sequence of HCDR2 is as follows: ISSSGST (SEQ ID NO: 62) ； The amino acid sequence of HCDR3 is as follows: ARAGAGGWAYGGYDI (SEQ ID NO: 63) ； The amino acid sequence of LCDR1 is as follows: QSIGSN (SEQ ID NO: 59) ； The amino acid sequence of LCDR2 is as follows: KAS; The amino acid sequence of LCDR3 is as follows: TO THE END (SEQ ID NO: 64) 。

[0124] The variable region sequence of the A9 antibody is as follows:

[0125] SEQ ID NO: 5A9 antibody heavy chain variable region amino acid

[0126] QSVEESGGRLVTPGGSLTLTCTVS GIDLSSYA MGWVRQAPGKGLQWIGI ISSSGST YYAS WANGRITISKTSTTVDLKITSPTTEDTATYFC ARAGAGGWAYGGYDI WGPGTLVTVSS;

[0127] SEQ ID NO: 25A9 antibody light chain variable region amino acid

[0128] DVVMTQTPASVSEPVGGTVTIKCQAS QSIGSN LAWYLQKPGQPPKLLIY KAS TLASGVS SRFKGSGSGTEFTLTISDLECADAATYYC TO THE END FGGGTEVVVK.

[0129] 6) The amino acid sequence of HCDR1 of the A11 antibody is as follows: GFSLSTSD (SEQ ID NO: 65) ； The amino acid sequence of HCDR2 is as follows: ISGGAIL (SEQ ID NO: 66) ； The amino acid sequence of HCDR3 is as follows: GRGYDHLDL (SEQ ID NO: 67) ； The amino acid sequence of LCDR1 is as follows: ATTITUDE (SEQ ID NO: 68) ； The amino acid sequence of LCDR2 is as follows: TVF; The amino acid sequence of LCDR3 is as follows: QGGDYDGSYVFA (SEQ ID NO: 69) 。

[0130] The variable region sequence of the A11 antibody is as follows:

[0131] SEQ ID NO: 6A11 antibody heavy chain variable region amino acid

[0132] QSVEESGGRLVTPGTPLTLTCTTSGFSLSTSDMTWVRQAPGKGLEWIGFISGGAILHYAT WAKGRFTISKTSTTVDLKMTSLTNEDTATYFCGRGYDHLDLWGQGTLVTVSS;

[0133] SEQ ID NO: 26A11 antibody light chain variable region amino acid

[0134] DVVMTQTPASVSEPVGGTVTIKCQASETIGNALAWYQQKPGQPPKLLIYTVFKVASGVP SRFFGSGSGTEFTLTISDLECDDAATYYCQGGDYDGSYVFAFGGGTEVVVK.

[0135] 7) The amino acid sequence of HCDR1 of the A13 antibody is: GFSLSSYY (SEQ ID NO: 70); the amino acid sequence of HCDR2 is: IGPGNT (SEQ ID NO: 71); the amino acid sequence of HCDR3 is: ARVYSNNDDFDP (SEQ ID NO: 72); the amino acid sequence of LCDR1 is: QSISSY (SEQ ID NO: 73); the amino acid sequence of LCDR2 is: RAS; the amino acid sequence of LCDR3 is: HQGYSYSDVDNA (SEQ ID NO: 74).

[0136] The variable region sequence of the A13 antibody is as follows:

[0137] SEQ ID NO: 7A13 Antibody heavy chain variable region amino acid

[0138] QSVEESGGRLVKPDESLTLTCTASGFSLSSYYMTWVRQAPGKGLEWIGIIGPGGNTYYA NWAKGRFTFSKTSTTVDLKITSPTTEDTATYFCARVYSNNDDFDPWGPGTLVTVSS;

[0139] SEQ ID NO: 27A13 Antibody light chain variable region amino acid

[0140] AYDMTQTPASVEVAVGGTVTIKCQASQSISSYLNWYQQKPGQPPKLLIYRASTLASGVS SRFKGSGSGTEYTLTISGVECADAATYYCHQGYSYSDVDNAFGGGTEVVVK.

[0141] 8) The amino acid sequence of HCDR1 of the A14 antibody is: GIDLRSNA ​​(SEQ ID NO: 75); the amino acid sequence of HCDR2 is: ISAGGSV (SEQ ID NO: 76); the amino acid sequence of HCDR3 is: ARAGYGGWSYGGFDP (SEQ ID NO: 77); the amino acid sequence of LCDR1 is: QSIGSN (SEQ ID NO: 59); the amino acid sequence of LCDR2 is: KAS; the amino acid sequence of LCDR3 is: QSTYYGVSYVGS (SEQ ID NO: 78).

[0142] The variable region sequence of the A14 antibody is as follows:

[0143] SEQ ID NO: 8A14 Antibody heavy chain variable region amino acid

[0144] QSVEESGGRLVTPGTPLTLTCTVSGIDLRSNAMSWVRQAPGEGLEWIGTISAGGSVYYA SWVNGRFTISKTSTTMDLKMTSLTAADTATYFCARAGYGGWSYGGFDPWGPGTLVTVSS;

[0145] SEQ ID NO: 28A14 Antibody light chain variable region amino acids

[0146] DVVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQKPGHPPKLLIYKASTLASGVP SRFKGSGSGTQFTLTISDLECADIATYYCQSTYYGVSYVGSFGGGTEAVVK.

[0147] 9) The amino acid sequence of HCDR1 of the B2 antibody is: GIDLSYYA (SEQ ID NO: 79); the amino acid sequence of HCDR2 is: LTSGGNT (SEQ ID NO: 80); the amino acid sequence of HCDR3 is: ARDVNQPTAWYFDI (SEQ ID NO: 81); the amino acid sequence of LCDR1 is: QNIYSN (SEQ ID NO: 82); the amino acid sequence of LCDR2 is: DAS; the amino acid sequence of LCDR3 is: QGGVYSSDVEIT (SEQ ID NO: 83).

[0148] The variable region sequence of the B2 antibody is as follows:

[0149] SEQ ID NO: 9B2 antibody heavy chain variable region amino acids

[0150] QSVEESGGRLVTPGTPLTLTCTVSGIDLSYYAMGWFRQAPGKGLEYIGILTSGGNTYYAT WAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCARDVNQPTAWYFDIWGPGTLVTVSS;

[0151] SEQ ID NO: 29B2 antibody light chain variable region amino acids

[0152] DVVMTQTPASVEVAVGGTVTINCQASQNIYSNLAWYQQKPGQGPKLLIYDASKLASGV SSRFSGSGSGTQFTLTINGVECADAATYYCQGGVYSSDVEITFGGGTEVVVK.

[0153] 10) The amino acid sequence of HCDR1 of the B3 antibody is: GFSLSSYY (SEQ ID NO: 70); the amino acid sequence of HCDR2 is: IGHSGTT (SEQ ID NO: 84); the amino acid sequence of HCDR3 is: ARVYDNSDRLDL (SEQ ID NO: 85); the amino acid sequence of LCDR1 is: QSISGY (SEQ ID NO: 86); the amino acid sequence of LCDR2 is: KAS; the amino acid sequence of LCDR3 is: QQGYSYTNVDNT (SEQ ID NO: 87).

[0154] The variable region sequence of the B3 antibody is as follows:

[0155] SEQ ID NO: 10B3 antibody heavy chain variable region amino acids

[0156] QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYYMSWVRQAPGKGLEWIGIIGHSGTTYYAS WVKGRFTISKTSTTVDLKITSPTTEDAATYFCARVYDNSDRLDLWGQGTLVTVSS;

[0157] SEQ ID NO: 30B3 antibody light chain variable region amino acid

[0158] AYDMTQTPASVEVAVGGTVTIKCQASQSISGYLNWYHQKPGQRPNLLIYKASTLASGV SSRFKGSGSGTEYTLTISGVECADAATYYCQQGYSYTNVDNTFGGGTEVVVK.

[0159] 11) The amino acid sequence of HCDR1 of the B5 antibody is: EFSLSSYV (SEQ ID NO: 88); the amino acid sequence of HCDR2 is: IKPSGNT (SEQ ID NO: 89); the amino acid sequence of HCDR3 is: ARDCYSSDTGYDV (SEQ ID NO: 90); the amino acid sequence of LCDR1 is: ESIGSR (SEQ ID NO: 91); the amino acid sequence of LCDR2 is: SAS; the amino acid sequence of LCDR3 is: QCSYDGSGYVGGP (SEQ ID NO: 92).

[0160] The variable region sequence of the B5 antibody is as follows:

[0161] SEQ ID NO: 11B5 antibody heavy chain variable region amino acids

[0162] QSVEESGGRLVTPGTPLTLTCTASEFSLSSYVMGWVRQAPGKGLEWIGIIKPSGNTHYAT WAKGRFTISKTSTTVELRITRPTTEDTATYFCARDCYSSDTGYDVWGPGTLVTVSS;

[0163] SEQ ID NO: 31B5 antibody light chain variable region amino acid

[0164] DPVLTQTPASVSEPVGGTVTIKCQASESIGSRLAWYQQKPGQPPKLLIYSASTLASGVSS RFKGSRSGTEYTLTISGVQCDDAATYYCQCSYDGSGYVGGPFGGGTEVVVK.

[0165] 12) The amino acid sequence of HCDR1 of C14 antibody is: GFTLSNNYW (SEQ ID NO: 93); the amino acid sequence of HCDR2 is: IYGDSSVST (SEQ ID NO: 94); the amino acid sequence of HCDR3 is: ARDLGGTSLYPDWLDL (SEQ ID NO: 95); the amino acid sequence of LCDR1 is: QNIYSN (SEQ ID NO: 82); the amino acid sequence of LCDR2 is: GAS; the amino acid sequence of LCDR3 is: QGYYYSSGSVDNA (SEQ ID NO: 96).

[0166] The variable region sequence of the C14 antibody is as follows:

[0167] SEQ ID NO: 12C14 antibody heavy chain variable region amino acid

[0168] QSGGGAEGGLVKPGGSLELCCKASGFTLSNNYWMCWVRQAPGKGLEWIGCIYGDSSV STYYASWVNGRFTLSRDIDQSTGCLQLNSLTVADTAMYYCARDLGGTSLYPDWLDLWGQG TLVTVSS;

[0169] SEQ ID NO: 32C14 antibody light chain variable region amino acid

[0170] AVVLTQTASPVSGAVGGTVTIKCQASQNIYSNLAWYQQKPGQPPKLLIYGASKLTSGVP SRFSGSRSGTEFTLTISDLECADAATYYCQGYYYSSGSVDNAFGGGTEVVVK.

[0171] 13) The amino acid sequence of HCDR1 of the C16 antibody is: GFSLSSYA (SEQ ID NO: 51); the amino acid sequence of HCDR2 is: ISSSGST (SEQ ID NO: 62); the amino acid sequence of HCDR3 is: ARGVGDNGYLNI (SEQ ID NO: 97); the amino acid sequence of LCDR1 is: ESISSW (SEQ ID NO: 98); the amino acid sequence of LCDR2 is: KAS; the amino acid sequence of LCDR3 is: QSVIVGTSFLGG (SEQ ID NO: 99).

[0172] The variable region sequence of the C16 antibody is as follows:

[0173] SEQ ID NO: 13C6 antibody heavy chain variable region amino acids

[0174] QSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIISSSGSTYYAS WVNGRFTISKTSTTVDLKMTSLTAADTATYFCARGVGDNGYLNIWGPGTLVTVSS

[0175] SEQ ID NO: 33C6 antibody light chain variable region amino acid

[0176] NIVLTQTPSPVSGAVGGTVTIKCQASESISSWLAWYQQKPGQPPKLLIYKASTLASGVSS RFEGSGSGTEFTLTISDLECDDAATYYCQSVIVGTSFLGGFGGGTEVVVK

[0177] 14) The amino acid sequence of HCDR1 of the C8 antibody is: GIDLSSNA (SEQ ID NO: 100); the amino acid sequence of HCDR2 is: VSGSGHT (SEQ ID NO: 101); the amino acid sequence of HCDR3 is: ARGAYASDNSYYDI (SEQ ID NO: 102); the amino acid sequence of LCDR1 is: QNIYRN (SEQ ID NO: 103); the amino acid sequence of LCDR2 is: TTA; the amino acid sequence of LCDR3 is: QSYYYDLFGTAYD (SEQ ID NO: 104).

[0178] The variable region sequence of the C8 antibody is as follows:

[0179] SEQ ID NO: 14C8 antibody heavy chain variable region amino acid

[0180] QSVEESGGRLVTPGTPLTLTCTASGIDLSSNAMGWVRQAPGKGLEYIGVVSGSGHTYYT KWAKGRFTISKASTTVDLKITSPTIEDTATYFCARGAYASDNSYYDIWGPGTLVTVSS

[0181] SEQ ID NO: 34C8 antibody light chain variable region amino acid

[0182] DVVMTQTPSSVSAAVGGTVTIKCQASQNIYRNLAWYQQKPGQRPKLLIYTTANLASGV PSRFKGSGSGTEYTLTISGVQCDDAATYYCQSYYYDLFGTAYDFGGGTEVVVK

[0183] 15) The amino acid sequence of HCDR1 of the C16 antibody is: GFSLSSYV (SEQ ID NO: 105); the amino acid sequence of HCDR2 is: ISASGNG (SEQ ID NO: 106); the amino acid sequence of HCDR3 is: AREVDYGDYFFNI (SEQ ID NO: 107); the amino acid sequence of LCDR1 is: QNIYSN (SEQ ID NO: 82); the amino acid sequence of LCDR2 is: AAS; the amino acid sequence of LCDR3 is: QSAYYSSSADTFA (SEQ ID NO: 108).

[0184] The variable region sequence of the C16 antibody is as follows:

[0185] SEQ ID NO: 16C16 antibody heavy chain variable region amino acid

[0186] QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYVLSWVRQAPGKGLEWIGIISASGNGYYAS WAKGRFTISKTSSTTVDLKMTSPTTEDTATYFCAREVDYGDYFFNIWGPGTLVTVSS

[0187] SEQ ID NO: 36C16 antibody light chain variable region amino acid

[0188] AIEMTQTPFSVSAAVGGTVTINCQASQNIYSNLAWYQQKPGQPPKLLIYAASLLESGVPS RFKGSGSGTQFTLTISGVQCADAATYYCQSAYYSSSADTFAFGGGTEVVVK

[0189] 16) The amino acid sequence of HCDR1 of the C17 antibody is: GFSLSSNA (SEQ ID NO: 109); the amino acid sequence of HCDR2 is: IYGRGDT (SEQ ID NO: 110); the amino acid sequence of HCDR3 is: ARTYGSNNDYYNI (SEQ ID NO: 111); the amino acid sequence of LCDR1 is: QSISSY (SEQ ID NO: 73); the amino acid sequence of LCDR2 is: KAS; the amino acid sequence of LCDR3 is: QQGYSETNVDNP (SEQ ID NO: 112).

[0190] The variable region sequence of the C17 antibody is as follows:

[0191] SEQ ID NO: 17C17 antibody heavy chain variable region amino acid

[0192] QSVEESGGRLVTPGGSLTLTCTVS GFSLSSNA MSWVRLAPGKGLEWIGIIYGRGDTYYP RWAKGRFTISKTSSTTVDLRLTSLTTEDTATYFCARTYGSNNDYYNIWGPGTLVTVSS

[0193] SEQ ID NO: 37C17 antibody light chain variable region amino acid

[0194] AYDMTQTPASVEVAVGGTVTIKCQASQSISSYLAWYQQKPGQPPKLLIYKASTLASGVP SRFKGSGSGTQFTLTISGVQCADAATYYCQQGYSETNVDNPFGGGTQVVVK

[0195] 17) The amino acid sequence of HCDR1 of C20 antibody is: GIDLSSYA (SEQ ID NO: 61); the amino acid sequence of HCDR2 is: ISSSGST (SEQ ID NO: 62); the amino acid sequence of HCDR3 is: ARCGGSNYFDSCAFNI (SEQ ID NO: 113); the amino acid sequence of LCDR1 is: ESISSW (SEQ ID NO: 98); the amino acid sequence of LCDR2 is: GAS; the amino acid sequence of LCDR3 is: QSYYYISSSDSNP (SEQ ID NO: 114).

[0196] The variable region sequence of the C20 antibody is as follows:

[0197] SEQ ID NO: 18C20 antibody heavy chain variable region amino acid

[0198] QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMGWVRQAPGKGLEYIGIISSSGSTYYSS WPKGRFTISKTSTTVDLRITSPTTEDTATYFCARCGGSNYFDSCAFNIWGPGTLVTVSS;

[0199] SEQ ID NO: 38C20 antibody light chain variable region amino acid

[0200] AVVLTQTASPVSGAVGGTVTINCQASESISSWLAWYQQKPGQPPKLLIYGASKLASGVS SRFSGSGSGTQFTLTISDLECADAATYYCQSYYYISSSDSNPFGGGTEVVVK.

[0201] 18) The amino acid sequence of HCDR1 of the C23 antibody is: GIDLDRYY (SEQ ID NO: 115); the amino acid sequence of HCDR2 is: INAYGST (SEQ ID NO: 116); the amino acid sequence of HCDR3 is: ARVGLVGVIHFLDI (SEQ ID NO: 117); the amino acid sequence of LCDR1 is: EDIYNL (SEQ ID NO: 118); the amino acid sequence of LCDR2 is: EAS; the amino acid sequence of LCDR3 is: QCAYYGSHYIFT (SEQ ID NO: 119).

[0202] The variable region sequence of the C23 antibody is as follows:

[0203] SEQ ID NO: 19C23 antibody heavy chain variable region amino acid

[0204] QSVEESGGRLVTPGTPLTLTCTASGIDLDRYYMSWVRQAPGKGLEWIGIINAYGSTYYA SWAKGRFTISKTSTTVDLKMTSPTPEDTATYFCARVGLVGVIHFLDIWGPGTLVTVSS;

[0205] SEQ ID NO: 39C23 antibody light chain variable region amino acid

[0206] DPVLTQTPPSASEAVGGTVTIKCQASEDIYNLLAWYQQKPGQPPKLLMYEASKLASGVP SRFKGSGSGTEFTLTISDLECADAATYYCQCAYYGSHYIFTFGGGTEVVVK.

[0207] 19) The amino acid sequence of HCDR1 of the C24 antibody is: GIDLSSYA (SEQ ID NO: 61); the amino acid sequence of HCDR2 is: TNIHDMT (SEQ ID NO: 120); the amino acid sequence of HCDR3 is: ARVFGYAGYGYGTGFYYYGLDL (SEQ ID NO: 121); the amino acid sequence of LCDR1 is: QSIGYN (SEQ ID NO: 122); the amino acid sequence of LCDR2 is: KAS; the amino acid sequence of LCDR3 is: QCTYYGDTYIDA (SEQ ID NO: 123).

[0208] The variable region sequence of the C24 antibody is as follows:

[0209] SEQ ID NO: 20C24 Antibody heavy chain variable region amino acids

[0210] QSVEESGGRLVTPGTPLTLTCTVSGIDLSSYAMSWVRQAPGRGLEWIGITNIHDMTYYAT WAKGRVTISKTSTTVDLKITSPTTEDTATYFCARVFGYAGYGYGTGFYYYGLDLWGPGTLVT VSS;

[0211] SEQ ID NO: 40C24 antibody light chain variable region amino acid

[0212] DVVMTQTPASVSEPVGGTVTIKCQASQSIGYNVAWYQQKPGQPPRLLIYKASNLASGV SSRFSGSGYGTEFTLTISGVQCEDAATYYCQCTYYGDTYIDAFGGGTEVVVK.

[0213] It should be noted that proteins or polypeptides having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) with the same or similar CD163 binding activity as SEQ ID NOs: 1-14 and 16-40 are also within the scope of this application.

[0214] In this application, homology refers to the “sequence identity” between two amino acid sequences, that is, the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For instance, it can be determined using the BLAST program of the NCBI database. For information on the determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Primers for Sequence Analysis, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991.

[0215] Amino acid residues can be represented by a three-letter or one-letter amino acid code according to standards known and agreed upon in the art. Conservative amino acid substitutions or replacements are known in the art. For example, a conserved amino acid substitution is preferably one amino acid residue from the following groups (1)-(5) replaced by another amino acid from the same group: (1) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (2) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (3) positively charged polar residues: His, Arg, and Lys; (4) larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (5) aromatic residues: Phe, Tyr, and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by lysine; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln, or Glu; Met is replaced by Leu, Tyr, or Ile; Phe is replaced by Met, Leu, or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; and Val is replaced by Ile or Leu.

[0216] Therefore, the modified sequences of SEQ ID NOs: 1-40 obtained by replacing the conventionally conserved amino acids (preferably the modified sequences with the 6 CDR sequences unchanged) are also within the scope of protection of this application.

[0217] In some embodiments, the anti-CD163 monoclonal antibody further includes a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is a constant region of human, mouse, monkey, rabbit, or sheep immunoglobulin, preferably a rabbit IgG constant region; and the light chain constant region is a rabbit κ-type constant region.

[0218] The structure and stability of immunoglobulin molecules depend on the type and subtype of the host animal. Compared to rodents, rabbits can also produce effective immune responses to small molecules and fine epitopes, resulting in higher antibody production and a greater chance of obtaining high-affinity antibodies. The stability of rabbit antibody molecules is inseparable from the diversity of their light chains. Rabbits have only one γ-immunoglobulin (IgG) isotype, with approximately 80-90% germline utilization of the VH1 variant in their heavy chain variable region. However, rabbit light chains are diverse, containing four κ isotypes besides the λ chain: b4, b5, b6, and b9. A disulfide bond exists between Cys80 and Cys170 of the rabbit κ chain, linking the variable region (Vκ) to the constant region (Cκ). This interdomain disulfide bond is unique to rabbit antibody structures. Therefore, the antibodies described in this application also possess the various advantages of rabbit-derived antibodies.

[0219] In some embodiments, the anti-CD163 monoclonal antibody is any one of the following: rabbit-derived antibody molecules, mouse-derived antibody molecules, humanized antibody molecules, or chimeric antibodies; preferably, the anti-CD163 monoclonal antibody is a full-length antibody, a single-chain antibody, Fab, Fab', Fab'-SH, or F(ab')2.

[0220] It should be noted that, depending on the research content and purpose, the anti-CD163 monoclonal antibody of this application can also be prepared into a labeling antibody.

[0221] Specific labeling methods can employ some of the methods reported previously. For example: 1) Enzyme labeling: For instance, using enzymes such as horseradish peroxidase (HRP) for labeling, a commonly used method is the sodium periodate method. This method involves oxidizing the glycosyl group of HRP to an aldehyde group, which then binds to the amino group of the antibody IgG to form a Schiff base, which is finally stabilized with sodium borohydride. Other commonly used enzyme labels include alkaline phosphatase and glucose oxidase. 2) Fluorescent labeling: Using fluorescent dyes such as fluorescein isothiocyanate (FITC) to label antibodies. These labeled antibodies can be used in techniques such as flow cytometry and immunofluorescence. 3) Isotope labeling: Using radioactive isotopes to label antibodies. This method is very sensitive for detecting protein synthesis but is not suitable for single-cell level studies. 4) Biotin labeling: Labeling is accomplished by a coupling reaction between biotin and the free lysine residues of the antibody molecule. The main steps include reacting the antibody with biotin succinimide ester, followed by dialysis to remove the free biotin. 5) Gold labeling: Antibodies are labeled using gold particles. This method can be used for immunolabeling under an electron microscope. 6) Chemiluminescent labeling: Antibodies are labeled using chemiluminescent substances. This method is suitable for research requiring the detection of luminescent signals.

[0222] Besides the methods mentioned above for forming labeled antibodies, fluorescent antibodies can also be formed by fusing with fluorescent proteins (such as GFP, RFP, or YFP). There are two common fusion methods. Taking GFP as an example, one is to place GFP after the target gene (i.e., anti-CD163 antibody gene-GFP), and the other is to place GFP before the target gene (GFP-anti-CD163 antibody gene). It is important to note that a linker is used between the two genes; a nucleotide segment (usually a multiple of 3 in length) is added at the junction of the two genes to minimize the spatial interference between the protein products of the two genes, which is beneficial for GFP luminescence; the gene before GFP must have a start codon, not a stop codon; the gene after GFP must have a stop codon; after constructing the fusion gene, sequencing verification is necessary to ensure the correct reading frame before further research. Fluorescently labeled antibodies formed by fusing with GFP can be used in various biological studies and diagnostics. They are commonly used for qualitative and quantitative detection or microscopic observation. Qualitative detection can be performed using conventional epifluorescence microscopy or confocal microscopy; quantitative detection can be performed using immunoblotting or enzyme-linked immunosorbent assay (ELISA); image analysis can be performed in live cells, and GFP in fixed cells can also be detected.

[0223] It should be noted that the types of fluorescent dyes mentioned above are very numerous, including but not limited to any one or more of the following: Alexa350, Alexa 405, Alexa 430, Alexa488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630 / 650, BODIPY 650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade The following are listed: Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, Dansyl chloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1,3-diazole), OregonGreen488, Oregon Green 500, Oregon Green514, Pacific Blue, Phthalic acid, Terephthalic acid, Isophthalic acid, Cresol Violet, Cresol Blue Violet, Brilliant Cresol Blue, p-Aminobenzoic acid, Erythrosine, Phthalocyanine, Azocyanine, Anthocyanin, Xanthine, Succinyl fluorescein, Rare earth metal cavitary compounds, Tribispyridyldiamine europium, europium cavitary compounds or chelates, Diamine, Dianthocyanin, Rhodamine Green, Rhodamine isothiocyanate, Rhodamine Red, ROX, TAMRA, TET, TRIT (Tetramethylrhodamine isothiol), Tetramethylrhodamine, and Texas Red.

[0224] Radioactive isotopes including but not limited to <110> In、 <111> In、 <177> Lu、 <18> F, <52> Fe、 <62> Cu、 <64> Cu、 <67> Cu、 <67> Ga、 <68> Ga、 <86> Y、 <90> Y、 <89> Zr、 <94> mTc, <94> Tc, <99> mTc, <120> I, <123> I, <124> I, <125> I, <131> I、<154-158>Gd、 <32> P, <11> C <13> N、 <15> O、 <186> Re、 <188> Re、 <51> Mn, <52> mMn, <55> Co、 <72> As、 <75> Br、 <76> Br、 <82> mRb and <83> Any of the following in Sr.

[0225] The labeling methods described above are all direct labeling methods, where the labeled molecules are covalently bound to the anti-CD163 antibody. In certain applications, the anti-CD163 antibody of this application can also be indirectly labeled. For example, the anti-CD163 antibody can be incubated as an unlabeled primary antibody, and then a labeled secondary antibody can be added for detection.

[0226] In other embodiments, the anti-CD163 antibody of this application can also be prepared as a bispecific antibody. The appropriate preparation method needs to be selected according to the specific application and the target antibody to be fused.

[0227] In a second typical embodiment of this application, an anti-CD163 antibody composition is provided, comprising any one or more of the aforementioned monoclonal antibodies. It should be noted that the antibody composition may also include other known or marketed anti-CD163 monoclonal or polyclonal antibodies. Specifically, it may be a combination of any one of the anti-CD163 monoclonal antibodies of this application with one or more existing antibodies. Alternatively, it may be a combination of any one or more anti-CD163 monoclonal antibodies of this application with one existing antibody.

[0228] When the antibody composition includes two or more monoclonal antibodies of this application, a reasonable selection can be made according to different application scenarios or actual needs. For example, a combination of two or more monoclonal antibodies with the same antigenic epitope can be selected, or a combination of monoclonal antibodies with different antigenic epitopes can be selected. Specific combinations can be any two or more combinations from any group of groups 1) to 5), or combinations of any two or more intergroup antibodies from groups 1) to 5). For example, it can be a combination of antibody A1 from group 1) with one, two, three, or four antibodies from group 2), or a combination of antibody A3 from group 1) with one, two, three, or four antibodies from group 2). Alternatively, it can be a combination of any antibody from groups 6) to 10) with any one or more antibodies from any group of groups 1) to 5).

[0229] 1) A1 antibody and A3 antibody;

[0230] 2) A13 antibody, B2 antibody, B3 antibody, and C17 antibody;

[0231] 3) A8 antibody and C24 antibody;

[0232] 4) B5 antibody, C8 antibody, C20 antibody, and C23 antibody;

[0233] 5) A9 antibody and A14 antibody;

[0234] 6) C14 antibody;

[0235] 7) C6 antibody;

[0236] 8) C16 antibody;

[0237] 9) Antibody A; and

[0238] 10) A11 antibody.

[0239] Because different applications have different requirements for half-life, stability, antibody purity, or antibody performance, the 19 pairs of antibodies may be used in mixtures of two or more. The proportions of these mixtures can vary depending on the application. For example, in some embodiments, in ELISA / immunofluorescence chromatography applications, to capture CD163 protein in different samples, or to apply the antibody solution under different environments (e.g., different temperature conditions: high temperature, room temperature, or low temperature; or different osmotic pressures or different salt ion concentrations in the sample solution), 1-4 antibodies from any of the 10 groups of the 19 antibodies can be mixed in different proportions to form a coating antibody or detection antibody. Alternatively, 1-4 antibodies from the same group can be mixed with 1-4 antibodies from other groups in different proportions to form a coating antibody or detection antibody.

[0240] In some embodiments, such as in the application of the Proximity Extension Assay (PEA) technique in proteomics, in order to capture soluble CD163 protein in serum or plasma, one antibody from any one of the above 19 antibody groups can be used in combination with one antibody from another group, or the antibodies in group 4) above can be used in pairs (except for C8 and C20).

[0241] In other embodiments, such as in IHC applications, to form a stable primary antibody reagent, or to achieve excellent specificity and non-specific background staining in different tissue samples, or to be compatible with automated staining instruments from various manufacturers, or to be compatible with secondary antibodies, DAB reagents, etc. from various manufacturers, the two IHC antibodies in this application can be used together to form a mixture, the three IHC antibodies can be used together to form a mixture, or any N (N≤3, for example, 1, 2, or 3) IHC antibodies can be used together with the 1-19 antibodies in this invention to form a mixture, thus preparing an IHC primary antibody product.

[0242] In a third typical embodiment of this application, a nucleic acid molecule is provided that encodes the aforementioned anti-CD163 monoclonal antibody.

[0243] Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence. DNA nucleic acids are preferred when ligated into a vector.

[0244] The term "recombinant expression vector," also simply called a vector, refers to a nucleic acid delivery vehicle into which nucleic acid molecules can be inserted. When a vector enables the expression of the protein encoded by the inserted nucleic acid molecule, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids, phage particles, Cos plasmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; or animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). In some embodiments, the vectors in this application contain regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0245] In a fourth typical embodiment of this application, a host cell is provided, which is transfected with the aforementioned recombinant expression vector. Preferably, the host cell is selected from prokaryotic cells or eukaryotic cells; preferably, the eukaryotic cell is selected from yeast cells, insect cells or mammalian cells; preferably, the mammalian cell is 293F cells, HEK293 cells, CHO cells or NSO cells.

[0246] In a fifth typical embodiment of this application, a kit is provided comprising any one or more of the above-described CD163 monoclonal antibodies or their antigen-binding fragments, or any one or more of the above-described antibody compositions, or the above-described nucleic acid molecules, recombinant expression plasmids, or host cells. Depending on the specific application, the kit can be a detection kit or a diagnostic kit. Specifically, it can be an ELISA kit, an immunohistochemical (IHC) kit, a flow cytometry (Fc) kit, or a Western blot (WB) kit. Accordingly, the kit may also include other reagents or antibodies related to ELISA, IHC, flow cytometry, or WB detection, such as secondary antibodies capable of indicating binding strength or specificity.

[0247] It should be noted that when performing ELISA, immunohistochemical (IHC), flow cytometry (Fc), or Western blotting, the aforementioned anti-CD163 monoclonal antibody can be used as a single antibody or in combination with multiple antibodies (preferably corresponding to multiple antigenic epitopes). Furthermore, the anti-CD163 monoclonal antibody can also exist in the form of a labeled antibody with a detection marker. Specific detection markers are as described above, including but not limited to any of the following markers commonly used in immunoassays: enzymes, fluorescent dyes, fluorescent proteins, isotopes, biotin, or colloidal gold.

[0248] In a sixth typical embodiment of this application, the use of the above-described anti-CD163 monoclonal antibody or the above-described CD163 antibody composition in the preparation of a detection product for detecting CD163 is provided. As described above, the use of the above-described anti-CD163 monoclonal antibody or the above-described CD163 antibody composition includes detecting CD163 protein or detecting cells expressing CD163 using the above-described methods such as ELISA, WB, Fc, or IHC.

[0249] In addition, the above applications also include combined or conjugated use with other reagents, such as antibodies, proteins, peptides, magnetic beads, chemiluminescent substances or nucleic acids, which can be rationally selected according to the different needs of the actual application.

[0250] It should be noted that, in addition to the applications mentioned above, the rabbit recombinant monoclonal antibody for CD163 developed in this application also has potential application value in basic research related to the CD163 target, disease diagnosis, and the development of novel treatment strategies.

[0251] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0252] Example 1: Gene Synthesis and Protein Expression

[0253] To prepare the recombinant CD163 protein as an immunogen, we constructed the CD163_pcDNA3.4 expression vector (gene synthesis was performed by the synthesis platform of BGI Changzhou Xinyisheng Life Technology Co., Ltd.). The 3' end of the coding region of the CD163 protein (Residues S42-S1045, UniProt accession number Q86VB7) in the expression vector is tagged with a Twin-Strep-Tag. Codon optimization was performed according to the codon preference of the Homo sapiens species, and the protein was cloned into the pcDNA3.4 expression vector using XbaI and HindIII restriction enzyme sites. After transforming the plasmid CD163_pcDNA3.4 into *E. coli* DH5α (TIANGEN), a large-scale extraction of the plasmid was performed, and then the plasmid was transfected into Expi293F using PEI (POLYSCIENCE). TM Protein expression was performed in Thermo cells. Cell supernatant containing the target protein was harvested 5 days after transfection. The cell supernatant was subjected to affinity chromatography (Strep-Tactin XT column, IBA; affinity chromatography) and gel filtration chromatography (Superose). TM 6. Increase 10 / 300GL, GE (i.e., size-exclusion chromatography) purification yields a high-purity target protein for subsequent use as an immunogen in animal immunization.

[0254] The results of protein purification and identification are attached. Figure 1A and Figure 1B As shown, the size identified by SDS-PAGE is 140 kDa. Among them, Figure 1A The left-middle figure shows the results of affinity chromatography using a Strep XT column. In the right figure, band A represents the elution peak and band B represents the elution peak. Figure 1B The left image shows the results of further purification of the protein after affinity chromatography by molecular sieve chromatography (where P1, P2, and P3 represent Peak 1, Peak 2, and Peak 3, respectively). The right image shows P2 (Peak 2) as the protein peak of sCD163, while P1 and P3 show no protein. SDS-PAGE electrophoresis results indicate that the prepared CD163 protein concentration is above 95%.

[0255] Example 2: Animal Immunization

[0256] In this embodiment, the immunogen is a recombinant CD163 protein expressed in a eukaryotic system. The immunogen was mixed with an adjuvant and injected into New Zealand white rabbits for immunization. After multiple rounds of immunization, blood was collected from the ears, serum was separated, and antibody titers in the serum were detected by ELISA. Once the serum titer reached 20w, B cells were sorted.

[0257] The serum titer test results are shown in Table 1 and Appendix. Figure 2 As shown, the serum titer reached 400,000 after four immunizations. Figure 2 The horizontal axis represents the 12 dilutions (from 1 / 200 to 1 / 409600) as shown in Table 1, and the vertical axis represents OD450.

[0258] Table 1:

[0259]

[0260] Example 3: Sorting of B cells in immunized rabbits

[0261] PBMCs were isolated from 10 mL of rabbit whole blood. First, PBMCs were labeled with biotin-T-lymphocyte antibody (Bio-Rad), biotin-IgM antibody (BD Pharmingen), and biotin-CD11b antibody (STEMCELL Technologies). Then, streptavidin magnetic beads (Miltenyi Biotec) were added, and the cells were incubated on ice for 15 min. Finally, B cells were obtained by negative sorting using a magnetic column.

[0262] One x 10^6 B cells were collected, and CD163 recombinant protein was added. The cells were incubated on ice for 30 min. After incubation, the cells were washed three times with PBS. Then, AF488 Donkey Anti-Rabbit IgG H&L (Biolegend) and StrepMAB-Immo DY-649 (IBA) antibodies were added. The cells were incubated on ice for 30 min, washed three times with PBS, and finally sorted by flow cytometry using FACSAria. TM II sorting CD163 + IgG + B cells were placed into 384-well plates, one B cell per well.

[0263] The results of the flow cytometry sorting are attached. Figure 3 As shown. First, the principal cell population was selected using forward scattering (FSC-A) and side scattering (SSC-A) light, and cell debris (P1) was removed. Then, adherent cells (P2 and P3) were removed using the area (A) and height (H) of FSC and SSC, respectively. Finally, FITC-positive IgG cells were selected. +Cells (P4) were then further selected for APC channel-positive Ag. + lgG + Cells (P5).

[0264] Example 4: Single B cell sequencing and antibody expression vector construction

[0265] The sorted B cells were subjected to Smart-seq single-cell sequencing. The B cells obtained in Example 3 were reverse transcribed using Alpha Reverse Transcriptase (BGI). The reverse transcription primer was Oligo-dT Primer (SEQ ID NO: 124: 5'-AAGCAGTGGTATCAACGCAGAGTACT). 30 The reaction procedure for VN-3' is as follows: 42℃ (90min) → (50℃ (2min), 42℃ (2min), 10 cycles in total) → 70℃ (15min) → 4℃ (HOLD).

[0266] The above reverse transcription product was pre-amplified by PCR. 2x Alpha High-Fidelity ReadyMix (BGI) and IS PCR Primer (SEQ ID NO: 125: 5'-AAGCAGTGGTATCAACGCAGAGT-3') were added. The reaction program was as follows: 98℃ (3min) → (98℃ (20s), 67℃ (15s), 72℃ (6min), 26 cycles in total) → 72℃ (5min) → 4℃ (HOLD).

[0267] Segmentase (BGI) was added to the PCR product to break it down, then T4 PNK (BGI) and T4 DNA polymerase (BGI) were added for end repair. Then, plate-type MGIeasy DNA Adapters-96 (BGI) was added to add adapters to the fragment product. Finally, 2x Alpha High-Fidelity ReadyMix (BGI) and Ad153_Primer (SEQ ID NO: 126: / 5Phos / GAACGACATGGCTACGA-3' and SEQ ID NO: 127: 5'-TGTGAGCCAAGGAGTTG-3') were added for pre-amplification. The reaction program was as follows: 72℃ (5min) → 95℃ (3min) → (98℃ (20s), 60℃ (15s), 72℃ (20s), 25 cycles) → 72℃ (5min) → 4℃ (HOLD).

[0268] The above products were mixed and added to VAHTSDNA Clean Beads (Vazyme). The magnetic beads were purified according to the instructions. The purified product underwent DNB circularization (BGI) using the MGIEasy circularization kit. Finally, sequencing was performed using the MGISEQ-2000RS high-throughput sequencing reagent (PE100).

[0269] After obtaining the transcriptome data, BCR analysis of the immune repertoire was performed to obtain antibody sequences (see the sequences of the aforementioned 19 antibodies). The antibody sequences with light and heavy chain pairing were selected and sent to BGI Changzhou New Life Technology Co., Ltd. to synthesize antibody expression vector plasmids.

[0270] Example 5: Expression and purification of recombinant antibodies

[0271] Recombinant antibody expression: The antibody sequence was cloned into the pCDNA3.4(+) expression vector. A large number of expression plasmids containing the light and heavy chain encoding genes of the specific antibody obtained in Example 4 were extracted and then co-transfected into 293F cells at a molar ratio of 3:2. The plasmid was mixed with PEI (POLYSCIENCE) at a ratio of 1:3. After mixing, the mixture was allowed to stand at room temperature for half an hour and then added dropwise to the cells. SMS293-SUPI feeding solution (Sino Biological) was added at 24 h and 72 h after transfection. The cell supernatant was collected 5 days later.

[0272] Recombinant antibody purification: Collect cell supernatant and add 1 mL of Protein A packing material (Essential Biotech). Incubate at room temperature for half an hour. Remove the packing material and load it into an empty purification column. Wash with 20 mL of PBS solution, then elute with 10 mL of 100 mM glycine solution (pH 3.0). Neutralize the eluent to pH 7.0 with 1 M Tris (pH 9.0) neutralization solution. Concentrate the neutralized eluent to 1 mL, dialyze into PBS, determine the protein concentration, and perform SDS-PAGE on a representative sample for purity assessment.

[0273] Some representative protein purity identification results are attached. Figure 4 As shown, most antibodies have high purity and single bands, where M represents a protein marker.

[0274] Example 6: Initial screening of binding antibodies and identification of antibody affinity using BLI

[0275] Antibodies binding to the CD163 recombinant protein were initially screened using biomembrane interferometry (BLI). BLI allows real-time monitoring of intermolecular interactions, with molecular changes displayed as relative shifts (nm) in the interference spectrum. The experiment used a protein A probe to capture antibodies (capture amount greater than 0.2 nM), flowing antigen, and PBST (0.2% Tween) as buffer. Antibodies were diluted to 5 μg / mL, and antigen to 200 nM. A Gator Primer was used to sequentially flow PBST, antibody, PBST, antigen, and PBST again for association and dissociation. PBST was added to each group as a control for association, used to subtract the background change in relative shifts in the interference spectrum during dissociation. Kinetic parameters were calculated using a 1:1 binding model in the analysis software to confirm antibody binding to the antigen and determine the antibody affinity K. D .

[0276] Representative experimental results are attached. Figure 5 As shown, it can be observed that most of the antibodies obtained through screening can bind to the immunogen. Figure 5 In the graph, the horizontal axis represents time (s), and the vertical axis represents relative displacement height (nm). The antibody affinity results are as follows: Figure 6 As shown.

[0277] Example 7: BLI typing antibody epitopes

[0278] Antibody binding epitope typing was performed using a tandem method in an antibody competitive binding assay. The experiment used a Strep probe to capture recombinant CD163 protein, and then immobilized Strep-... The XT probe underwent a sequential binding and dissociation process: baseline (PBST, 30s), loading (CD163-Strep antigen, 10μg / mL, 100s), baseline (PBST, 30s), association 1 (binding; association is a common term in molecular interaction experiments, referring to the step where the mobile phase flows into the chip / sensor and binds to the immobilized antibody; first antibody, concentration determined in pre-experiment, 300s), and association 2 (second antibody, concentration determined in pre-experiment, 180s). After the experiment, the inhibition index of the first antibody against the second antibody was calculated based on the ratio of the relative shift change in the interference spectrum of the second antibody binding to the antigen when the first antibody reached saturation to the relative shift change in the interference spectrum of the second antibody directly binding to the antigen when the first antibody was absent. The competitive relationship between the corresponding antibodies was analyzed by using the mutual inhibition index between the antibodies.

[0279] The grouping results for epitope competition are attached. Figure 6 As shown, the results reveal the affinity of 10 epitope groups identified through competition experiments for each of the 19 binding antibodies. Straight lines connecting two antibodies indicate inter-antibody competition; dashed lines indicate that the antibody in question is in complete competition with the representative antibody of that group, belonging to the same epitope group. Therefore, it did not compete with other antibodies within the group (e.g., in group 2, the representative antibody is C17; antibodies B2, B3, and A13 are in complete competition with C17, thus belonging to the same epitope group. Further verification of this antibody relationship by conducting a competition experiment with A13 and B3 showed that they were still in complete competition). The competitive relationships of antibodies B5 and C23 outside the circle in group 4 are relatively unique; they are in complete competition with the antibodies within the circle in group 4, but do not compete with each other.

[0280] Example 8: Flow cytometry assay using antibodies

[0281] 1. Cell preparation; In this example, the full-length CD163 sequence (M1-L1156) was constructed into the pEGFP-N1 vector by BGI Changzhou Xinyisheng Life Technology Co., Ltd. After plasmid extraction, the CD163_pEGFP-N1 plasmid was transfected into Expi293F cells via PEI. TM In cells. After culturing for 2 days, CD163-overexpressing cells were obtained, and untransfected 293F cells were prepared for the next step of flow cytometry testing.

[0282] 2. Cell washing: CD163 overexpressing cells with obvious green fluorescence can be used for flow cytometry. Collect cells into 15mL centrifuge tubes, centrifuge at 300g at room temperature for 5min, resuspend cells in PBS, and the cell density of each sample is 5E5.

[0283] 3. Primary antibody staining: Rabbit antibody was prepared at a concentration of 1 μg / mL to stain cells, and incubated at 37°C in the dark for 30 min; the positive control was Ibrance's CD163 rabbit antibody.

[0284] 4. Cell washing: Centrifuge at 300g at room temperature for 5 min, discard the supernatant, wash the cells of each sample with PBS, and repeat the above operation twice;

[0285] 5. Secondary antibody staining: Stain cells with anti-rabbit IgG / AF647 (Biolegend) at a ratio of 1:1000 and incubate at 37°C in the dark for 30 min;

[0286] 6. Cell washing: Centrifuge at 300g at room temperature for 5 min, discard the supernatant, wash the cells of each sample with 200μL of PBS, and repeat the above operation twice;

[0287] 7. Analyze the washed cells using a flow cytometer.

[0288] Flow cytometry results for all antibodies are as follows Figure 7 As shown, most antibodies exhibited positive flow cytometry results, but showed less non-specific binding to 293F antibodies that do not express CD163. Both the binding rate and non-specificity were superior to the positive control.

[0289] Example 9: Western Blot (WB) test with antibody

[0290] 1. Using CD163-overexpressing cells from Experiment 7, perform Western blotting (WB) testing of the antibody, using 3 × 10⁶ cells. 6 Cell samples were centrifuged at 800g to remove the supernatant, and then 200μL of reducing SDS loading buffer was added. The samples were then heated in a 100℃ metal bath for 10 min.

[0291] 2. Use 10% SDS-PAGE gel for electrophoresis. Load 15 μL of sample into each well and run the gel. Set the voltage to 120V. After 1 hour, turn off the power when the blue dye band reaches the bottom of the gel.

[0292] 3. The prepared NC membrane was treated with methanol and equilibration buffer respectively, and then the protein was transferred onto the NC membrane using an automated transfer apparatus.

[0293] 4. After the transfer is complete, the NC membrane is sealed with 5% skim milk at room temperature for 1 hour.

[0294] 5. After blocking, select one antibody with strong affinity from each antibody group, dilute it to 1 μg / mL with 3% skim milk, and then incubate with the primary antibody. Incubate at room temperature with shaking for 3 hours.

[0295] 6. After incubation with primary antibody, wash the membrane with PBST on a decolorizing shaker three times, for 10 minutes each time.

[0296] 7. Then, the NC membrane was incubated with goat anti-rabbit IgG (HRP) secondary antibody at room temperature for 1 hour. After the secondary antibody incubation was completed, the membrane washing step in step 5 was repeated.

[0297] 8. Develop the NC membrane with developing solution, and then take pictures for observation using a developing instrument after development is complete.

[0298] The results are attached. Figure 8 As shown, both A3 and C6 antibodies can be used in Western blot (WB) experiments.

[0299] Example 10: IHC test for positive antibodies

[0300] First, formalin-fixed paraffin-embedded tissue samples were prepared into 3-micrometer-thick paraffin sections. After being flattened in water at 40°C, the sections were baked in a 60°C oven for 1 hour. Dewaxing was achieved through xylene and a gradient alcohol treatment. Then, antigen retrieval was performed using EDTA microwave thermal retrieval, followed by blocking of tissue peroxidase with endogenous peroxidase blocking solution. Sheep serum was then added for further blocking, and the sections were incubated at 37°C for 30 minutes. Excess liquid was removed, and 0.1 μg / mL of primary antibody was added for incubation at room temperature for 1-2 hours. After primary antibody incubation, excess primary antibody was washed away with PBST, and HRP-labeled goat anti-rabbit secondary antibody was added and incubated at room temperature for 30 minutes. Excess secondary antibody was washed away with PBST. DAB chromogenic solution was then prepared for staining. The staining time was controlled under a microscope, and the reaction was terminated by washing with water. Cell nuclei were stained with hematoxylin and then treated with a bluing process. Finally, the staining was dehydrated by an alcohol gradient, cleared with xylene, dried, mounted with neutral resin, and the staining results were observed and photographed under a high magnification microscope.

[0301] Appendix Figure 9 The results showed that multiple rabbit recombinant antibodies produced significant positive staining on tonsil sections, with cell morphology consistent with macrophages and a pattern consistent with PC1 (Positive Control, purchased from Ibrex). Compared with the positive control antibody, the A1, A3, and C6 antibodies in this invention exhibited high staining intensity and no nonspecific background under the same experimental conditions, indicating that A1 and A3 showed stronger staining specificity in IHC experiments.

[0302] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0303] Recombinant antibodies are antibodies expressed through in vitro expression systems. The production methods for recombinant antibodies overcome the batch-to-batch inconsistency problems of traditional polyclonal and monoclonal antibodies, making antibody production controllable and reliable. Compared to mouse antibodies and polyclonal antibodies, rabbit antibodies have a single subtype, precise epitope recognition, and naturally higher affinity and stronger specificity, resulting in better experimental results in most experiments. The antibodies in this invention are all rabbit-derived recombinant antibodies, with controllable production, high batch-to-batch stability, and low concentrations used in various experiments, making costs easier to control. They also exhibit high antibody specificity and accuracy.

[0304] 1. The rabbit recombinant monoclonal antibodies disclosed in this invention can specifically bind to CD163, exhibiting very high affinity (picomolar and nanomolar levels), stronger specificity, and better batch-to-batch stability. They are more likely to achieve better experimental results in various applications such as Fc, WB, and IHC. The antibodies disclosed in this invention are all rabbit recombinant monoclonal antibodies that can be efficiently expressed in mammalian expression systems. They are non-animal-derived antibodies, comply with application standards in the EU and other regions, and have lower production costs.

[0305] 2. The A1, A3, and C6 rabbit monoclonal antibodies in this invention exhibit good efficacy in immunohistochemistry (IHC). Specifically, only 0.5 μg / mL of A1 rabbit monoclonal antibody is required for IHC, while only 0.25 μg / mL of A3 and C6 rabbit monoclonal antibodies are needed. Compared to the commonly used working concentration of 5 μg / mL for traditional antibodies, the concentration can be reduced by 10-20 times. Furthermore, A1 and A3 can both be used in flow cytometry (Fc), and A3 and C6 rabbit monoclonal antibodies can also be used in Western blot (WB). These three antibodies can be used as multi-purpose antibodies, and the commercialization of these antibodies can reduce purchasing costs for users.

[0306] 3. Based on different epitopes, the CD163 rabbit recombinant monoclonal antibody group disclosed in this invention can be divided into 10 antibody groups, which has unique advantages in discovering paired antibodies and subsequent paired applications. Moreover, the antibodies of this invention are the first disclosed antibody groups targeting CD163 with multiple epitope groups. Antibodies in the same antibody epitope group mostly have common applications. For example, antibodies in group 1 can all be used in IHC experiments. The epitope grouping can guide the application direction of subsequent CD163 antibodies and reduce the detection cost of newly discovered antibodies.

[0307] 4. The antibodies obtained by this invention can be used directly or labeled in various ways and packaged into commercial reagents or kits for application in multiple fields.

[0308] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An isolated monoclonal antibody against CD163 or its antigen-binding fragment, characterized in that, The monoclonal antibody or its antigen-binding fragment includes heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3. The amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from any one of the following groups: 1) SEQ ID NO: 41: GIDLSIYV, SEQ ID NO: 42: ISSSGNK, SEQ ID NO: 43: ARLSGHTVRLDL, SEQ ID NO: 44: QSVYNNNW, SAS and SEQ ID NO: 45: LGGYSSTADNT; 2) SEQ ID NO: 46: GIDLSIFV, SEQ ID NO: 47: ISYSGNT, SEQ ID NO: 48: ARINGDTVRLDL, SEQ ID NO: 49: QSVYNANF, RAS and SEQ ID NO: 50: LGGYSSTSDNT; 3) SEQ ID NO: 51: GFSLSSYA, SEQ ID NO: 52: ISDGGNT, SEQ ID NO: 53: ARAGANSDGDYWDRLDL, SEQ ID NO: 54: QSVYNNNY, DAS and SEQ ID NO: 55: LGGYDDDADTT; 4) SEQ ID NO: 56: GIDLSDYV, SEQ ID NO: 57: SSIVGSA, SEQ ID NO: 58: ARNPGYSDVI, SEQ ID NO: 59: QSIGSN, IAS and SEQ ID NO: 60: QSAYYSTTSSDT; 5) SEQ ID NO: 61: GIDLSSYA, SEQ ID NO: 62: ISSSGST, SEQ ID NO: 63: ARAGAGGWAYGGYDI, SEQ ID NO: 59: QSIGSN, KAS and SEQ ID NO: 64: QSTYYGISYVGA; 6) SEQ ID NO: 65: GFSLSTSD, SEQ ID NO: 66: ISGGAIL, SEQ ID NO: 67: GRGYDHLDL, SEQ ID NO: 68: ETIGNA, TVF and SEQ ID NO: 69: QGGDYDGSYVFA; 7) SEQ ID NO: 70: GFSLSSYY, SEQ ID NO: 71: IPGGGNT, SEQ ID NO: 72: ARVYSNNDDFDP, SEQ ID NO: 73: QSISSY, RAS and SEQ ID NO: 74: HQGYSYSDVDNA; 8) SEQ ID NO: 75: GIDLRSNA, SEQ ID NO: 76: ISAGGSV, SEQ ID NO: 77: ARAGYGGWSYGGFDP, SEQ ID NO: 59: QSIGSN, KAS and SEQ ID NO: 78: QSTYYGVSYVGS; 9) SEQ ID NO: 79: GIDLSYYA, SEQ ID NO: 80: LTSGGNT, SEQ ID NO: 81: ARDVNQPTAWYFDI, SEQ ID NO: 82: QNIYSN, DAS and SEQ ID NO: 83: QGGVYSSDVEIT; 10) SEQ ID NO: 70: GFSLSSYY, SEQ ID NO: 84: IGHSGTT, SEQ ID NO: 85: ARVYDNSDRLDL, SEQ ID NO: 86: QSISGY, KAS and SEQ ID NO: 87: QQGYSYTNVDNT; 11) SEQ ID NO: 88: EFSLSSYV, SEQ ID NO: 89: IKPSGNT, SEQ ID NO: 90: ARDCYSSDTGYDV, SEQ ID NO: 91: ESIGSR, SAS and SEQ ID NO: 92: QCSYDGSGYVGGP; 12) SEQ ID NO: 93: GFTLSNNYW, SEQ ID NO: 94: IYGDSSVST, SEQ ID NO: 95: ARDLGGTSLYPDWLDL, SEQ ID NO: 82: QNIYSN, GAS and SEQ ID NO: 96: QGYYYSSGSVDNA; 13) SEQ ID NO: 51: GFSLSSYA, SEQ ID NO: 62: ISSSGST, SEQ ID NO: 97: ARGVGDNGYLNI, SEQ ID NO: 98: ESISSW, KAS and SEQ ID NO: 99: QSVIVGTSFLGG; 14) SEQ ID NO: 100: GIDLSSNA, SEQ ID NO: 101: VSGSGHT, SEQ ID NO: 102: ARGAYASDNSYYDI, SEQ ID NO: 103: QNIYRN, TTA and SEQ ID NO: 104: QSYYYDLFGTAYD; 15) SEQ ID NO: 105: GFSLSSYV, SEQ ID NO: 106: ISASGNG, SEQ ID NO: 107: AREVDYGDYFFNI, SEQ ID NO: 82: QNIYSN, AAS and SEQ ID NO: 108: QSAYYSSSADTFA; 16) SEQ ID NO: 109: GFSLSSNA, SEQ ID NO: 110: IYGRGDT, SEQ ID NO: 111: ARTYGSNNDYYNI, SEQ ID NO: 73: QSISSY, KAS and SEQ ID NO: 112: QQGYSETNVDNP; 17) SEQ ID NO: 61: GIDLSSYA, SEQ ID NO: 62: ISSSGST, SEQ ID NO: 113: ARCGGSNYFDSCAFNI, SEQ ID NO: 98: ESISSW, GAS and SEQ ID NO: 114: QSYYYISSSDSNP; 18) SEQ ID NO:115: GIDLDRYY, SEQ ID NO:116:INAYGST, SEQ ID NO:117:ARVGLVGVIHFLDI, SEQ ID NO:118:EDIYNL, EAS and SEQ ID NO:119:QCAYYGSHYIFT; or 19) SEQ ID NO: 61: GIDLSSYA, SEQ ID NO: 120: TNIHDMT, SEQ ID NO: 121: ARVFGYAGYGYGTGFYYYGLDL, SEQ ID NO: 122: QSIGYN, KAS and SEQ ID NO: 123: QCTYYGDTYIDA.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are selected from any one of the following groups: 1) SEQ ID NO: 1 and 21; 2) SEQ ID NO: 2 and 22; 3) SEQ ID NO: 3 and 23; 4) SEQ ID NO: 4 and 24; 5) SEQ ID NO: 5 and 25; 6) SEQ ID NO: 6 and 26; 7) SEQ ID NO: 7 and 27; 8) SEQ ID NO: 8 and 28; 9) SEQ ID NO: 9 and 29; 10) SEQ ID NO: 10 and 30; 11) SEQ ID NO: 11 and 31; 12) SEQ ID NO: 12 and 32; 13) SEQ ID NO: 13 and 33; 14) SEQ ID NO: 14 and 34; 15) SEQ ID NO: 16 and 36; 16) SEQ ID NO: 17 and 37; 17) SEQ ID NO: 18 and 38; 18) SEQ ID NO: 19 and 39; 19) SEQ ID NO: 20 and 40; or An amino acid sequence that has at least 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the sequences in any of groups 1) to 19).

3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The monoclonal antibody or its antigen-binding fragment is selected from full-length antibodies, single-chain antibodies, Fab, Fab', Fab'-SH or F(ab')2.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The monoclonal antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region, wherein the heavy chain constant region is a constant region of human, mouse, monkey, rabbit or sheep immunoglobulin, preferably a rabbit IgG constant region; The light chain constant region is the constant region of the rabbit κ type.

5. An anti-CD163 antibody composition, characterized in that, The antibody composition comprises any one or more of the anti-CD163 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

6. The antibody composition according to claim 5, characterized in that, The antibody composition comprises a combination of two or more antibodies that bind to the same epitope; or a combination of two or more antibodies that bind to different epitopes.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CD163 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4.

8. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 7.

9. A host cell, characterized in that, The host cell is transfected with the recombinant expression vector of claim 8; Preferably, the host cell is selected from prokaryotic cells or eukaryotic cells; Preferably, the eukaryotic cells are selected from yeast cells, insect cells, or mammalian cells; preferably, the mammalian cells are 293F cells, 293T cells, HEK293 cells, CHO cells, or NSO cells.

10. A reagent kit, characterized in that, The invention comprises any one or more of the anti-CD163 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, or any one or more of the antibody compositions described in claims 5 or 6, the nucleic acid molecule described in claim 7, the recombinant expression vector described in claim 8, or the host cell described in claim 9.

11. The reagent kit according to claim 10, characterized in that, The anti-CD163 monoclonal antibody in the kit is a labeled antibody with a detection marker; Preferably, the detection marker is selected from any one of the following: polypeptide, protein, nucleic acid, chemiluminescent substance, isotope, biotin, or colloidal gold.

12. The use of the anti-CD163 monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 4, or the anti-CD163 antibody composition as described in claim 5 or 6, in the preparation of a detection product for detecting CD163.

13. The application according to claim 12, characterized in that, The applications include detecting CD163 protein or CD163-expressing cells using any one or more of the following methods: ELISA, proteoblotting, flow cytometry, or immunohistochemistry.

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