Anti-siglec-8 antibody and use thereof

Abstract

Provided are an anti-Siglec-8 antibody or antigen-binding fragment, the use thereof in the preparation of a drug for treating a condition mediated by eosinophils and / or mast cells, and a composition and kit containing the anti-Siglec-8 antibody or antigen-binding fragment.

Description

Anti-Siglec-8 antibody and its application Technical Field

[0001] This invention belongs to the field of biomedicine and relates to anti-Siglec-8 antibodies and their applications. Background Technology

[0002] Sialic acid-binding immunoglobulin-like lectins (Siglec) are a class of classic immunoglobulin-like lectins whose ligands are glycoconjugates (glycoproteins or glycolipids) containing sialic acid structures. Currently, 15 human and 9 mouse Siglec molecules have been identified (Pillai et al., Annu Rev Immunol., 2012, 30:357-392). Among them, Siglec-8 was initially discovered as a novel marker for identifying the surface of human eosinophils. It is expressed only in humans and apes, and is undetectable in eosinophils of rhesus monkeys or cynomolgus monkeys. The homolog of Siglec-8 in mice is Siglec-F. Siglec-8 is highly expressed in eosinophils and mast cells, and lowly expressed in some basophils (Bradford A et al., Int Arch Allergy Immunol., 2019, 180(2):91-102). Siglec-8 recognizes a sulfated polysaccharide, either 6'-sulfo-sialyl Lewis X or 6'-sulfo-sialyl-N-acetyl-S-lactosamine, which contains an intracellular immunoreceptor-based tyrosine-based inhibitory motif (ITIM) domain and exhibits inhibitory effects on mast cell function.

[0003] Along with mast cells, eosinophils promote inflammatory responses that play beneficial functional roles, such as controlling infection at specific tissue sites. During the inflammatory response, eosinophil apoptosis can be inhibited by suppressing the activity of survival-promoting cytokines such as IL-3 and GM-CSF. However, an increase in activated eosinophils not rapidly removed through apoptosis can lead to the release of eosinophil granule proteins at already inflamed sites, which can damage tissue and further exacerbate inflammation. Several diseases have been shown to be associated with eosinophil activation, such as Qiu Shier syndrome, rheumatoid arthritis, and allergic asthma. Therapies are currently needed to control the activity of immune cells involved in inflammation, such as eosinophils and mast cells. Summary of the Invention

[0004] In one aspect, the present invention provides an anti-Siglec-8 antibody or antigen-binding fragment comprising one or more selected from the following heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3):

[0005] (1)HCDR1, which contains an amino acid sequence selected from any one of SEQ ID NO:13,16,18,21,24,27,72-75;

[0006] (2)HCDR2, which contains an amino acid sequence selected from any one of SEQ ID NO:14,19,22,25,28,76-83,98;

[0007] (3)HCDR3, which contains an amino acid sequence selected from any one of SEQ ID NO:15,17,20,23,26,29,99-104;

[0008] (4) LCDR1, which contains an amino acid sequence selected from any one of SEQ ID NO:30,33,35,40,42,105-108;

[0009] (5) LCDR2, which contains an amino acid sequence selected from any one of SEQ ID NO:31,34,36,38,43;

[0010] (6) LCDR3, which contains an amino acid sequence selected from any one of SEQ ID NO:32,37,39,41,44.

[0011] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1 selected from the amino acid sequence shown in any one of SEQ ID NO:72-75.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises HCDR2 with an amino acid sequence selected from any one of SEQ ID NO:19,22,28,76-83,98.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises HCDR3 of an amino acid sequence selected from any one of SEQ ID NO:15,17,20,26,29,99-104.

[0014] In some embodiments, the antibody or antigen-binding fragment comprises LCDR1 selected from the amino acid sequence shown in SEQ ID NO:40.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises LCDR3 selected from the amino acid sequence shown in SEQ ID NO:44.

[0016] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, and / or the antibody or antigen-binding fragment comprises LCDR1, LCDR2, and LCDR3, wherein,

[0017] (1) HCDR1 of the amino acid sequence shown in SEQ ID NO:13, HCDR2 of the amino acid sequence shown in SEQ ID NO:14, and HCDR3 of the amino acid sequence shown in SEQ ID NO:15, LCDR1 of the amino acid sequence shown in SEQ ID NO:30, LCDR2 of the amino acid sequence shown in SEQ ID NO:31, and LCDR3 of the amino acid sequence shown in SEQ ID NO:32; or

[0018] (2) HCDR1 of the amino acid sequence shown in SEQ ID NO:16, HCDR2 of the amino acid sequence shown in SEQ ID NO:14, and HCDR3 of the amino acid sequence shown in SEQ ID NO:17; LCDR1 of the amino acid sequence shown in SEQ ID NO:33, LCDR2 of the amino acid sequence shown in SEQ ID NO:34, and LCDR3 of the amino acid sequence shown in SEQ ID NO:32; or

[0019] (3) HCDR1 of the amino acid sequence shown in SEQ ID NO:18, HCDR2 of the amino acid sequence shown in SEQ ID NO:19, and HCDR3 of the amino acid sequence shown in SEQ ID NO:20; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:36, and LCDR3 of the amino acid sequence shown in SEQ ID NO:37; or

[0020] (4) HCDR1 of the amino acid sequence shown in SEQ ID NO:21, HCDR2 of the amino acid sequence shown in SEQ ID NO:22, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23, LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or

[0021] (5) HCDR1 of the amino acid sequence shown in SEQ ID NO:24, HCDR2 of the amino acid sequence shown in SEQ ID NO:25, and HCDR3 of the amino acid sequence shown in SEQ ID NO:26; LCDR1 of the amino acid sequence shown in SEQ ID NO:40, LCDR2 of the amino acid sequence shown in SEQ ID NO:34, and LCDR3 of the amino acid sequence shown in SEQ ID NO:41; or

[0022] (6) HCDR1 of the amino acid sequence shown in SEQ ID NO:27, HCDR2 of the amino acid sequence shown in SEQ ID NO:28 and HCDR3 of the amino acid sequence shown in SEQ ID NO:29, LCDR1 of the amino acid sequence shown in SEQ ID NO:42, LCDR2 of the amino acid sequence shown in SEQ ID NO:43 and LCDR3 of the amino acid sequence shown in SEQ ID NO:44.

[0023] In some embodiments, the antibody or antigen-binding fragment comprises

[0024] (1) HCDR1, which contains an amino acid sequence selected from any one of SEQ ID NO: 13, 16, 18, 21, 24, 27, 72-75; (2) HCDR2, which contains an amino acid sequence selected from any one of SEQ ID NO: 14, 19, 22, 25, 28, 76-83, 98; (3) HCDR3, which contains an amino acid sequence selected from any one of SEQ ID NO: 15, 17, 20, 23, 26, 29, 99-104.

[0025] In some embodiments, the antibody or antigen-binding fragment comprises

[0026] (1) LCDR1, comprising an amino acid sequence selected from any one of SEQ ID NO:30,33,35,40,42,105-108; (2) LCDR2, comprising an amino acid sequence selected from any one of SEQ ID NO:31,34,36,38,43; (3) LCDR3, comprising an amino acid sequence selected from any one of SEQ ID NO:32,37,39,41,44.

[0027] In some embodiments, the antibody or antigen-binding fragment comprises

[0028] (1)HCDR1, which contains an amino acid sequence selected from any one of SEQ ID NO:13,16,18,21,24,27,72-75;

[0029] (2)HCDR2, which contains an amino acid sequence selected from any one of SEQ ID NO:14,19,22,25,28,76-83,98;

[0030] (3)HCDR3, which contains an amino acid sequence selected from any one of SEQ ID NO:15,17,20,23,26,29,99-104;

[0031] (4) LCDR1, which contains an amino acid sequence selected from any one of SEQ ID NO:30,33,35,40,42,105-108;

[0032] (5) LCDR2, comprising an amino acid sequence selected from any one of SEQ ID NO: 31, 34, 36, 38, 43; and

[0033] (6) LCDR3, which contains an amino acid sequence selected from any one of SEQ ID NO:32,37,39,41,44.

[0034] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, and HCDR3, and / or the antibody or antigen-binding fragment comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises the sequence shown in any one of SEQ ID NO:21 or 72-75, HCDR2 comprises the sequence shown in any one of SEQ ID NO:22, 98, or 76-83, HCDR3 comprises the sequence shown in any one of SEQ ID NO:23 or 99-104, LCDR1 comprises the sequence shown in any one of SEQ ID NO:35 or 105-108, LCDR2 comprises the sequence shown in SEQ ID NO:38, and LCDR3 comprises the sequence shown in SEQ ID NO:39.

[0035] In some embodiments, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, and / or the antibody or antigen-binding fragment comprises LCDR1, LCDR2, and LCDR3, wherein,

[0036] (1) HCDR1 of the amino acid sequence shown in SEQ ID NO:21, HCDR2 of the amino acid sequence shown in any one of SEQ ID NO:79-81, 76 or 98, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23, and / or LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or

[0037] (2) HCDR1 of the amino acid sequence shown in SEQ ID NO:72, HCDR2 of the amino acid sequence shown in SEQ ID NO:22 or 79, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or

[0038] (3) HCDR1 of the amino acid sequence shown in SEQ ID NO:73, HCDR2 of the amino acid sequence shown in any one of SEQ ID NO:77, 78 or 81, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or

[0039] (4) HCDR1 of the amino acid sequence shown in SEQ ID NO:74, HCDR2 of the amino acid sequence shown in SEQ ID NO:77, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or

[0040] (5) HCDR1 of the amino acid sequence shown in SEQ ID NO:75, HCDR2 of the amino acid sequence shown in SEQ ID NO:82 or 83, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or

[0041] (6) HCDR1 of the amino acid sequence shown in SEQ ID NO:21, HCDR2 of the amino acid sequence shown in SEQ ID NO:79, and HCDR3 of any one of the amino acid sequences shown in SEQ ID NO:99-104, LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or

[0042] (7) HCDR1 of the amino acid sequence shown in SEQ ID NO:21, HCDR2 of the amino acid sequence shown in SEQ ID NO:79 and HCDR3 of the amino acid sequence shown in SEQ ID NO:99, LCDR1 of the amino acid sequence shown in any one of SEQ ID NO:105-108, LCDR2 of the amino acid sequence shown in SEQ ID NO:38 and LCDR3 of the amino acid sequence shown in SEQ ID NO:39.

[0043] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain variable region comprising a sequence or a variant thereof as shown in any one of SEQ ID NO:1,3,5,7,9,11,45,47,51-52,55-56,59-71,84-93.

[0044] In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region comprising a sequence or a variant thereof as shown in any one of SEQ ID NO:2,4,6,8,10,12,46,48-50,53-54,57-58,94-97.

[0045] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region, wherein,

[0046] (1) The heavy chain variable region comprises the sequence shown in SEQ ID NO:1 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:2 or a variant thereof; or

[0047] (2) The heavy chain variable region comprises the sequence shown in SEQ ID NO:3 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:4 or a variant thereof; or...

[0048] (3) The heavy chain variable region comprises the sequence shown in SEQ ID NO:5 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:6 or a variant thereof; or...

[0049] (4) The heavy chain variable region comprises the sequence shown in SEQ ID NO:7 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:8 or a variant thereof; or...

[0050] (5) The heavy chain variable region comprises the sequence shown in SEQ ID NO:9 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:10 or a variant thereof; or...

[0051] (6) The heavy chain variable region comprises the sequence shown in SEQ ID NO:11 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:12 or a variant thereof; or...

[0052] (7) The heavy chain variable region comprises the sequence shown in SEQ ID NO:45 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:46 or a variant thereof; or...

[0053] (8) The heavy chain variable region comprises the sequence shown in SEQ ID NO:47 or a variant thereof, and the light chain variable region comprises the sequence shown in any one of SEQ ID NO:48-50 or a variant thereof; or...

[0054] (9) The heavy chain variable region comprises a sequence as shown in SEQ ID NO:51 or 52 or a variant thereof, and the light chain variable region comprises a sequence as shown in SEQ ID NO:53 or 54 or a variant thereof; or...

[0055] (10) The heavy chain variable region comprises a sequence as shown in SEQ ID NO:55 or 56 or a variant thereof, and the light chain variable region comprises a sequence as shown in SEQ ID NO:57 or 58 or a variant thereof; or

[0056] (11) The heavy chain variable region comprises a sequence as shown in any one of SEQ ID NO:59-71, 84-93 or a variant thereof, and the light chain variable region comprises a sequence as shown in SEQ ID NO:53 or a variant thereof; or

[0057] (12) The heavy chain variable region contains a sequence as shown in SEQ ID NO:88 or a variant thereof, and the light chain variable region contains a sequence as shown in SEQ ID NO:94-97 or a variant thereof.

[0058] In some embodiments, the variant is a sequence that differs from the corresponding variable region by 3, 2, or 1 amino acid (preferably a conserved amino acid substitution) or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, respectively, and the variant retains binding affinity to Siglec-8.

[0059] The variable region sequence is shown below:

[0060] The CDR sequence is shown below:

[0061] In some implementations, the antigen-binding fragment is Fab, F(ab')2, Fv, or a single-chain Fv (scFv).

[0062] In some embodiments, the antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region.

[0063] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain consists of a heavy chain variable region and a heavy chain constant region, and the light chain consists of a light chain variable region and a light chain constant region.

[0064] In some embodiments, the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4 or variants thereof.

[0065] In some embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:112 or 113.

[0066] In some implementations, the light chain constant region is selected from the κ or λ light chain constant region or variations thereof.

[0067] In some embodiments, the amino acid sequence of the light chain constant region is shown in SEQ ID NO:114.

[0068] In some embodiments, the antibody or antigen-binding fragment binds to human Siglec-8.

[0069] In some implementations, the antibody or antigen-binding fragment is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0070] In some embodiments, the antibody or antigen-binding fragment has an enhanced ADCC effect. In some embodiments, the Fc region of the antibody or antigen-binding fragment is unfucosylated or poorly fucosylated.

[0071] In some embodiments, the antibody or antigen-binding fragment is a separated antibody or antigen-binding fragment.

[0072] In some embodiments, the antibody or antigen-binding fragment is a bispecific antibody or a multispecific antibody.

[0073] On the one hand, the present invention provides a biomaterial, which is:

[0074] 1) A nucleic acid molecule encoding an antibody or antigen-binding fragment or a portion thereof of the present invention;

[0075] 2) An expression vector comprising the nucleic acid molecule of the present invention;

[0076] 3) Host cell containing the nucleic acid molecule or expression vector of the present invention.

[0077] In some embodiments, the nucleic acid molecule comprises the sequences shown in SEQ ID NO:115 and / or SEQ ID NO:116.

[0078] An example nucleic acid sequence is as follows:

[0079] The nucleic acid sequence encoding VH of antibody I:

[0080] The nucleic acid sequence encoding VL of antibody I:

[0081] On one hand, the present invention provides a method for generating antibodies or antigen-binding fragments, comprising culturing said host cells under conditions suitable for gene expression. In some embodiments, the method further includes purifying said antibody or antigen-binding fragments. Purification can be performed using conventional methods, such as centrifuging the cell suspension and collecting the supernatant, and methods such as filtration, protein A affinity columns, and ion exchange columns can be used to purify antibody proteins.

[0082] In some embodiments, the antibody or antigen-binding fragment is expressed by CHO cells with the FUT8 gene knocked out.

[0083] On one hand, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment of the present invention. In some embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, such as pharmaceutically acceptable excipients, diluents, or carriers.

[0084] On one hand, the invention provides the use of its antibodies or antigen-binding fragments or biological materials or pharmaceutical compositions in the preparation of medicaments for treating diseases. On another hand, the invention provides the use of its antibodies or antigen-binding fragments or biological materials or pharmaceutical compositions in the treatment of diseases. On yet another hand, the invention provides a method of treating a disease, comprising administering an effective dose of the antibody or antigen-binding fragment or biological material or pharmaceutical composition of the invention to a patient in need. In some embodiments, the disease is a cell-mediated condition expressing Siglec-8. In some embodiments, the disease is an eosinophil and / or mast cell-mediated condition, such as eosinophilia or mastocytosis. Attached Figure Description

[0085] Figure 1 shows the binding ability of hybridoma subclonal cell supernatant to Siglec-8 on the surface of CHO-Siglec-8 cells, analyzed by flow cytometry.

[0086] Figure 2 shows the binding ability of the anti-Siglec-8 chimeric antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells, analyzed by flow cytometry.

[0087] Figure 3 shows the binding ability of the anti-Siglec-8 humanized antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells, analyzed by flow cytometry.

[0088] Figure 4 shows the binding ability of the anti-Siglec-8 mutant antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells, analyzed by flow cytometry.

[0089] Figures 5a-5c show the binding ability of the anti-Siglec-8 site-directed mutant antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells, analyzed by flow cytometry.

[0090] Figure 6 shows the ADCC activity mediated by the anti-Siglec-8 antibody;

[0091] Figure 7 shows the ability of anti-Siglec-8 antibody to induce apoptosis in eosinophils. Detailed Implementation

[0092] the term

[0093] Unless otherwise stated, each of the following terms shall have the meaning described below.

[0094] definition

[0095] The term “a” refers to one or more of the same entity. For example, “an antibody” should be understood as one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably in this document.

[0096] As used herein, the terms “comprising” or “including” mean that antibodies, compositions, or methods include the listed elements, such as components or steps, but do not exclude others. “Substantially composed of” means that antibodies, compositions, or methods exclude other elements that fundamentally affect the characterization of the composition, but do not exclude elements that do not substantially affect the antibody, composition, or method. “Composed of” means excluding elements not specifically listed.

[0097] An "antibody" is a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody ("immunoglobulin, IgG") and any antigen-binding fragment thereof, or a single chain thereof, such as an antigen-binding domain. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Examples of antibodies, including but not limited to, include the heavy chain, the light chain, the complementarity-determining region (CDR) of its ligand-binding moiety, the heavy chain variable region (VH), the light chain variable region (VL), the heavy chain constant region (CH), the light chain constant region (CL), the framework region (FR), or any portion thereof, or at least a portion of the binding protein. The CDR region includes the CDR regions of the light chain (LCDR1-3) and the CDR regions of the heavy chain (HCDR1-3). Those skilled in the art will understand that the categories of immunoglobulin heavy chains include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), among which there are also some subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of immunoglobulin, namely IgG, IgM, IgA, IgD, or IgE, and subclasses (isotypes), such as IgG which can have subclasses such as IgG1, IgG2, IgG3, and IgG4. All types of immunoglobulins are within the scope of protection disclosed in this invention. In some embodiments, the immunoglobulin molecule is of the IgG type, whose two heavy chains and two light chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues to surround the heavy chain through a variable region.

[0098] Those skilled in the art will understand that the CDR region of an antibody is responsible for the antibody's binding specificity to the antigen. Given the known sequences of the antibody's heavy and light chain variable regions, several methods exist for determining the antibody's CDR region, including the Kabat, IMGT, Chothia, Contact, and AbM numbering systems. However, the application of each definition of the CDR for an antibody or its variants will be within the scope of the terminology defined and used herein. Given the amino acid sequence of the antibody's variable region, those skilled in the art can generally determine which residues are contained in a particular CDR without relying on any experimental data outside of the sequence itself.

[0099] The "heavy chain constant region" includes at least one of the following: CH1 domain, hinge (e.g., upper, middle, and / or lower hinge regions), CH2 domain, CH3 domain, or variants or fragments. The heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of an antibody may include a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4.

[0100] The "light chain constant region" consists of a single domain CL. In some implementations, the light chain constant region may be derived from either the κ constant region domain or the λ constant region domain.

[0101] An example of a constant region sequence is as follows:

[0102] "Apoptosis-inducing" or "apoptotic" antibodies refer to those antibodies that induce programmed cell death according to standard apoptosis assays, such as annexin V binding, DNA breakage, cell contraction, endoplasmic reticulum swelling, cell rupture, and / or formation of membrane vesicles (called apoptotic bodies). For example, the apoptotic activity of the anti-Siglec-8 antibody of the present invention can be demonstrated by staining cells with annexin V.

[0103] Antibody "effector function" refers to biological activities attributable to the antibody's Fc region (either the native Fc region or the Fc region of an amino acid sequence variant) and that vary across antibody isotypes. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0104] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which a secreted Ig binding to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxicity. The antibody "arms" the cytotoxic cells and is required by this mechanism to kill the target cells. The main cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. In some embodiments, the anti-Siglec-8 antibody described herein has enhanced ADCC. To assess the ADCC activity of the target molecule, an in vitro ADCC assay can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells that can be used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the target molecule can be assessed in vivo, for example, in animal models. Other Fc variants can be used to modify ADCC activity and other antibody properties by known methods, such as reducing or eliminating antibody fucosylation.

[0105] "Low-fucosylated" or "fucosylated" antibodies refer to glycosylated antibody variants containing an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced or absent fucose. In some embodiments, antibodies with reduced or absent fucose have enhanced ADCC function. In some embodiments, the antibodies with reduced or absent fucose are expressed by CHO cells with α-(1,6)-fucosyltransferase knocked out. In some embodiments, the antibodies with reduced or absent fucose are expressed by CHO-BAT-KF fut8(- / -) cells (the aforementioned cells are disclosed in patent WO2019029713A1).

[0106] The term "fucosylation" or "fucosylated" refers to the presence of fucose residues within the oligosaccharides attached to the peptide backbone of an antibody. Typically, fucosylated antibodies contain α-(1,6)-linked fucose at the innermost N-acetylglucosamine (GlcNAc) residue of one or two N-linked oligosaccharides attached to the antibody's Fc region, such as position Asn297 in the human IgG1 Fc domain (EU numbering of Fc region residues). Due to minor sequence variations in immunoglobulins, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300.

[0107] The antibodies disclosed in this invention can be derived from any animal, including but not limited to fish, birds, and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse, or chicken-derived antibodies. In another embodiment, the variable region can be of condricthoid origin (e.g., from sharks).

[0108] This invention covers humanized antibodies. Various methods for humanizing nonhuman antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced from a nonhuman source. These nonhuman amino acid residues are often referred to as “input” residues, and they are typically derived from the “input” variable region. Humanized antibodies include chimeric antibodies, in which less than a complete human variable region is replaced with the corresponding sequence of a nonhuman species. Humanized antibodies may have some hypervariable region residues and possibly some FR residues replaced with residues from rodent-like antibody-like sites.

[0109] The selection of variable regions for both the human light and heavy chains used in the preparation of humanized antibodies can be important for reducing antigenicity. Following the so-called "best-fit" approach, an entire library of known human variable region sequences is screened using the variable region sequences of rodent (e.g., mouse) antibodies. The human sequence closest to the rodent sequence is then selected as the human frame for the humanized antibody. Another approach uses a specific frame derived from a common sequence of all human antibodies from a particular light or heavy chain subgroup. The same frame can be used for several different humanized antibodies.

[0110] The term "bispecific antibody" refers to an antibody (including antibodies or antigen-binding fragments, such as single-chain antibodies and nanobodies) that can specifically bind to two different antigens or two different epitopes of the same antigen. Based on the integrity of the IgG molecule, they can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies. Based on the number of antigen-binding regions, they can be divided into bivalent, trivalent, tetravalent, or more valent bispecific antibodies. Based on structural symmetry, they can be divided into symmetrical and asymmetrical bispecific antibodies. Fragment-type bispecific antibodies, such as Fab fragments lacking the Fc fragment, form bispecific antibodies by combining two or more Fab fragments into one molecule. They have lower immunogenicity, smaller molecular weight, and higher tumor tissue penetration. Typical antibody structures of this type include F(ab)2, scFv-Fab, and (scFv)2-Fab. IgG-like bispecific antibodies (e.g., those with an Fc fragment) have a relatively larger molecular weight. The Fc fragment helps in antibody purification and improves its solubility and stability. The Fc portion may also bind to the receptor FcRn, increasing the antibody's serum half-life. Bispecific antibody structural models include KiH, CrossMAb, Triomab quadroma, FcΔAdp, ART-Ig, BiMAb, Biclonics, BEAT, DuoBody, Azymetric, XmAb, 2:1 TCBs, 1Fab-IgG TDB, FynomAb, two-in-one / DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F(ab)2-CrossMAb, IgG-(scFv)2, Bs4Ab, DVD-Ig, Tetravalent-DART-Fc, (scFv)4-Fc, CODV-Ig, mAb2, and F(ab)4-CrossMAb, among other bispecific antibodies.

[0111] As used herein, the twenty common amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd edition, edited by ESGolub and DRGren, Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of the twenty common amino acids (e.g., D-amino acids), non-natural amino acids (such as α-, α-disubstituted amino acids), N-alkyl amino acids, lactate, and other unconventional amino acids may also be components applicable to the polypeptides disclosed herein. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide representation used herein, the left-hand direction is the amino-terminal direction, and the right-hand direction is the carboxyl-terminal direction, consistent with standard usage and convention. Conventional (or natural) amino acids are L-amino acids, including alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V), etc.

[0112] "Identity or sequence identity" of a polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that, when sequence alignment is performed, that percentage of bases (or amino acids) are identical in the two compared sequences. This alignment and identity percentage or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignment is preferably performed using default parameters. One alignment procedure is BLAST using default parameters, such as BLASTN and BLASTP, which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of identity and encode polypeptides with the same or similar biological activities.

[0113] Minor variations in the amino acid sequence of antibody or immunoglobulin molecules are covered within this disclosure, provided that the amino acid sequence identity is maintained at least 90%, such as at least 92%, 95%, 98%, or 99%. In some embodiments, the variation is a conserved amino acid substitution. A conserved amino acid substitution is a substitution that occurs within the relevant amino acid family in its side chain. Genetically encoded amino acids are broadly classified into the following categories: (1) acidic amino acids, such as aspartate and glutamate; (2) basic amino acids, such as lysine, arginine, and histidine; (3) nonpolar amino acids, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, such as glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) serine and threonine from the aliphatic-hydroxy family; (ii) asparagine and glutamine from the amide family; (iii) alanine, valine, leucine, and isoleucine from the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine from the aromatic family. In some embodiments, the conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. For example, it is reasonable to predict that leucine can be replaced by isoleucine or valine alone, aspartate by glutamate, threonine by serine, or a structurally related amino acid can be replaced by another amino acid, without significantly affecting the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within the binding site. Whether an amino acid change results in a functional peptide can be readily determined by measuring the specific activity of the polypeptide derivative. This measurement is described in detail herein. Fragments or analogues of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art.

[0114] In some embodiments, amino acid substitution has the following effects: (1) reducing sensitivity to proteolytic activity, (2) reducing sensitivity to oxidation, (3) altering the binding affinity for forming protein complexes, (4) altering the binding affinity, or (5) imparting or improving other physicochemical or functional properties to such analogs. Analogs may include various mutant proteins with sequences different from naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conserved amino acid substitutions) may be made in the naturally occurring sequence (preferably in the polypeptide portion outside the domains forming intermolecular contacts). Conserved amino acid substitutions should not significantly alter the structural characteristics of the parental sequence (e.g., the substituted amino acid should not tend to disrupt the helical structure present in the parental sequence or disrupt other types of secondary structures characterizing the parental sequence).

[0115] The term "polypeptide" is intended to encompass both the singular and plural forms of "polypeptide" and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not imply a specific length of the product. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and "polypeptide" can be used in place of any of the foregoing terms or interchangeably with any of the foregoing terms. The term "polypeptide" is also intended to refer to products modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced through recombinant technologies, but they do not necessarily have to be translated from a specified nucleic acid sequence; they can be produced in any manner, including chemical synthesis.

[0116] The antibody and antigen-binding fragments disclosed in this invention include modified derivatives, i.e., modified by covalently linking any type of molecule to the antibody or antigen-binding fragment, wherein the covalent link does not prevent the antibody or antigen-binding fragment from binding to the epitope. The antibody or antigen-binding fragment can be glycosylated, acetylated, polyethylene glycol-modified, phosphorylated, amidated, derivatized by known protecting / blocking groups, cleaved by proteolytic hydrolysis, linked to cellular ligands or other proteins, etc. Any of the numerous chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin.

[0117] In some implementations, the antibody or antigen-binding fragment may be conjugated to a therapeutic agent, drug precursor, peptide, protein, enzyme, virus, lipid, biological response modifier, oligonucleotide (e.g., siRNA), or PEG.

[0118] As used herein, the terms "specific binding" or "immune response" refer to a non-covalent interaction that occurs between an immunoglobulin molecule and one or more antigenic determinants of its target antigen. The strength or affinity of an immunobinding interaction can be expressed in terms of the equilibrium dissociation constant (K0). D ) indicates that the smaller K DThis represents a higher affinity. The immunobinding properties of the selected peptide can be quantified using methods well known in the art. One such method measures the rates of antigen-binding site / antigen complex formation and dissociation, where those rates depend on the concentration of the complex's partner, the affinity of the interaction, and other geometric parameters that equally affect the rate. Both the "binding rate constant" (kon) and the "dissociation rate constant" (koff) can be determined by calculating the concentration and the actual association and dissociation rates. The koff / kon ratio eliminates parameters unrelated to affinity and is equal to the equilibrium dissociation constant K. D Specific binding can be measured by radioligand binding assays, surface plasmon resonance (SPR) assays, flow cytometry binding assays, or similar assays known to those skilled in the art.

[0119] The term "isolated" as used in this invention for cells, nucleic acids, peptides, antibodies, etc., such as "isolated" DNA, RNA, and peptides, refers to molecules isolated from one or more other components such as DNA or RNA, respectively, in the cellular natural environment. The term "isolated" as used in this invention also refers to nucleic acids or peptides that, when produced by recombinant DNA technology, are substantially free of cellular material, viral material, or cell culture medium, or chemical precursors or other chemicals used in chemical synthesis. Furthermore, "isolated nucleic acids" is intended to include nucleic acid fragments that are not naturally present and will not exist in their natural state. The term "isolated" is also used in this invention to refer to cells or peptides isolated from other cellular proteins or tissues. Isolated peptides are intended to include purified and recombinant peptides. Isolated peptides, etc., are typically prepared by at least one purification step. In one or more embodiments, the purity of the isolated nucleic acids, peptides, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range (including extreme values) between any two of these values, or any value therein.

[0120] When the term “encoding” is applied to polynucleotides, it refers to a polynucleotide called the “encoding” polypeptide that, in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce the polypeptide and / or fragments thereof.

[0121] "Approximately" refers to a typical error range for the corresponding value that is readily known to those skilled in the art. In some embodiments, "approximately" as used herein refers to the described value and its range of ±10%, ±5%, or ±1%.

[0122] EC 50 That is, the half-maximal concentration (EC50). 50 () refers to the concentration that can cause 50% of the maximum effect.

[0123] "Treatment" refers to therapeutic treatments and preventative or preventative measures aimed at preventing, mitigating, improving, or stopping adverse physiological changes or disorders, such as disease progression, including but not limited to the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, mitigation, reduction, or disappearance of the disease state (whether partial or complete), and prolongation of expected survival without treatment. Patients requiring treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those needing prevention of the condition or disorder, as well as those who can or are expected to benefit from the application of the antibody or pharmaceutical composition disclosed in this invention for detection, diagnostic procedures, and / or treatment.

[0124] The effective dosage and treatment regimen for a specific patient will depend on various factors, including the specific antibody, antigen-binding fragment or derivative used, the patient's age and weight, general health condition, sex and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors will be determined by a healthcare professional, including those skilled in the art. The dosage used can be determined using principles of pharmacology and pharmacokinetics well known in the art. In some embodiments, the antibody of the present invention is administered to the patient at a dose ranging from 0.01 mg / kg to 100 mg / kg of patient body weight per administration. In some embodiments, it is administered once weekly or monthly.

[0125] The term "patient" refers to any mammal requiring diagnosis, prognosis, or treatment, including but not limited to humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, etc. In some implementations, the patient is a human patient.

[0126] The term "pharmaceuticalally acceptable" refers to a substance approved by a government regulatory agency or listed in a recognized pharmacopoeia for use in animals, and especially in humans. Furthermore, "pharmaceuticalally acceptable excipients" generally refer to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0127] The term "excipient" refers to a diluent, adjuvant, excipient, or carrier that can be administered to the patient along with the active ingredient. Such drug carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the drug composition is administered intravenously. Saline, glucose, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid, chelating agents, and tonic agents such as dextran or dextrose are also foreseeable. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions will contain a clinically effective dose of antibody or antigen-binding fragments or fusion proteins, preferably in a purified form, along with an appropriate amount of excipients to provide a dosage form suitable for the patient. The formulation should be suitable for the dosing regimen. Parent formulations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0128] In some embodiments, the composition is formulated according to standard procedures to be suitable for intravenous or subcutaneous injection into the human body. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. In cases where the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline.

[0129] The antibody or antigen-binding fragment or fusion protein of the present invention may be in neutral or salt form. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0130] Antibody preparation

[0131] Various methods for preparing antibodies are known in this field, such as hybridoma technology, recombinant DNA technology, transgenic mouse technology, and phage display library methods.

[0132] Antibodies can be prepared using conventional recombinant DNA techniques. Vectors and cell lines for antibody production can be selected, constructed, and cultured using techniques well-known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as *Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells*, DL Hacker, FMWurm, in Reference Module in Life Sciences, 2017, the entire contents of which, including supplementary information, are incorporated herein by reference.

[0133] In some embodiments, antibody-encoding DNA can be designed and synthesized according to the antibody amino acid sequence described herein using conventional methods, placed into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in a culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody-encoding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters of long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as actin promoters. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian host cells include 293 cells, COS-1 cells, COS-7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0134] In some implementations, the inserted gene fragment needs to contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, and hygromycin resistance genes to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the aforementioned genes. After culturing in a selective medium, successfully transfected cells grow in large numbers, producing the desired target protein. The resulting antibody can be separated or purified using conventional techniques, such as protein A-agarose gel chromatography, ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0135] The technical solution of the present invention will be further illustrated below through specific embodiments. These specific embodiments do not represent a limitation on the scope of protection of the present invention, and any non-essential modifications or adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0136] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0137] Example 1

[0138] Construction of stable CHO-Siglec-8 cell lines: The nucleic acid sequence corresponding to the above-mentioned human Siglec-8 (from UniProt: Q9NYZ4) was synthesized and then constructed into the pcDNA3.1 expression vector (Invitrogen, catalog number V79020). This vector was then transfected into CHO-K1 cells (Life Technologies, catalog number A13696-01) via electroporation. After 48 hours, 50 μM MSX (methionine imino sulfone) was added for selection. Positive cells were selected after 2 weeks. High-expressing cell lines were screened using FACS detection. Cells were collected, washed once with PBS (phosphate-buffered saline), and then 10 μg / ml of anti-Siglec-8 antibody HEKA (sequence from patent CN105916519B) was added. After incubation at 4°C for 1 hour, the cells were washed twice with PBS. Then, 100 μl of 1:500 diluted goat anti-human IgG-Fc PE fluorescent secondary antibody (catalog number: 12-4998-82, eBioscience) was added, and the cells were incubated at 4°C for 1 hour. After washing twice with PBS, the cells were analyzed using a C6 flow cytometer (BD, model: C6 Flow cytometer). The resulting cells were named CHO-Siglec-8 cells.

[0139] The extracellular region of human Siglec-8 (Met17-Ala363) was recombinantly expressed with the Fc fragment of the heavy chain of an antibody (such as human IgG1) or a His tag, and was expressed and purified by HEK293F cells (product number: CRL-3249, ATCC) to obtain the antigen and the protein for detection. The sequence of the extracellular region (ECD) of human Siglec-8 is as shown in SEQ ID NO:109, where the amino acid residues at positions 1-16 are the signal peptide sequence, the amino acid residues at positions 40-123 are the "V-set" domain, the amino acid residues at positions 157-240 are the "C2-set1" domain, and the amino acid residues at positions 246-344 are the "C2-set2" domain. The amino acid sequence of the recombinant protein obtained by linking the extracellular region of human Siglec-8 with the Fc fragment of human IgG1 (abbreviated as hS8-hFc protein) is as shown in SEQ ID NO:110; the amino acid sequence of the protein obtained by linking the extracellular region of human Siglec-8 with the His tag sequence (hSiglec8-His protein) is as shown in SEQ ID NO:111.

[0140] The extracellular amino acid sequence of human Siglec-8 is as follows:

[0141] The amino acid sequence of hS8-hFc protein (human Siglec-8 ECD-IgG1 Fc) is as follows:

[0142] The amino acid sequence of hSiglec8-His protein (human Siglec-8 ECD-His) is as follows:

[0143] Example 2: Preparation and Identification of Anti-Siglec-8 Hybridoma Monoclonal Antibodies, Chimeric Antibodies and Humanized Antibodies

[0144] 1. Animal Immunization

[0145] Eight female Balb / c mice, 6-8 weeks old (Guangdong Provincial Center for Medical Laboratory Animals, animal production license number: SCXK(Guangdong)2013-0002), were selected as the immunized animals, and the feeding environment was SPF level. After the mice were purchased, they were first raised for one week and then immunized.

[0146] The immunization protocol is as follows:

[0147] Each mouse was immunized subcutaneously with 50 μg of hS8-hFc antigen once every two weeks. Ten days after the third immunization, blood was collected from the tail vein, and the titer of anti-Siglec-8 antibody in the mouse plasma was detected by ELISA to monitor the degree of immune response of the mice.

[0148] 2. ELISA method for determining the titer of anti-Siglec-8 antibody in mouse plasma.

[0149] Tail vein blood was collected from the immunized mice and diluted with PBS to 1:2000, 1:20000, and 1:200000. 100 μl of this diluted blood was added to each well of an ELISA plate coated with hS8-hFc antigen. The plate was incubated at 37°C for 1 hour and washed 5 times with PBST. Goat anti-mouse HRP (catalog number: 115-035-003, Jackson ImmunoResearch) (1:10000 dilution) was added to each well; the plate was incubated at 37°C for 1 hour and washed 5 times. 100 μl of TMB chromogenic buffer (catalog number: TMB-S-001, Biopanda) was added and incubated at 37°C in the dark for 10 minutes. The chromogenic reaction was stopped by adding 50 μl of 0.1 M H2SO4. The OD450 value was measured using a SpectraMax M3 Molecular Devices microplate reader. After three rounds of immunization, all mice produced antibodies against Siglec-8; therefore, fusion could be performed.

[0150] 3. Spleen cell fusion

[0151] One day prior to fusion, peritoneal macrophages from blank mice were used as feeder cells and resuspended in RPMI 1640 medium (Yuanpei Biotechnology, L210KJ) containing 10% FBS (fetal bovine serum, Novizan, catalog number: F101-01) + 1×HAT (Sigma-Aldrich, catalog number: H0262-1VL) + 1× penicillin and streptomycin (the penicillin and streptomycin). The cells were then seeded in 96-well cell culture plates at 100 μL / well. On the day of fusion, spleen cells from Balb / c-1, Balb / c-2, and Balb / c-3 mice were aseptically extracted. Myeloma cells SP2 / 0 (Shanghai Cell Bank, Chinese Academy of Sciences) were fused with spleen cells at a 1:10 ratio using polyethylene glycol (50% W / V PEG solution, catalog number: P7181, Sigma) to generate hybridoma cells. The confluent cells were resuspended in 100 mL of RPMI 1640 complete medium containing 10% FBS, 1×HAT, and 1× antibiotics. The medium was then seeded into 10 wells of 96-well plates containing 100 μL / well of feeder cells. Ten 96-well plates were prepared with 100 μL of confluent cells per well. The plates were incubated at 37°C with 5% CO2. ELISA was performed based on the size of the resulting colonies.

[0152] 4. Hybridoma cell screening

[0153] ELISA-screened positive clones were further subcloned, and flow cytometry was performed on the supernatant of the subcloned cells. Human CHO-Siglec-8 cells (1×10⁶) were used. 5Mix 100 μl of the supernatant of the fusion cells to be tested with 100 μl of the sample, incubate at 4 °C for 1 h, wash twice with PBS, then add 100 μl of 1:500 diluted goat anti-mouse IgG-A647 fluorescent secondary antibody (catalog number: bs-0296G-AF647, Bioss), incubate at 4 °C for 1 h, wash twice with PBS, and then analyze using a CytoFLEX flow cytometer (model: AOO-1-1102, Beckman Coulter). Plot a bar graph with the fluorescence intensity of the goat anti-mouse IgG-A647 label (APC channel) on the x-axis and Count on the y-axis to analyze the cell binding in the supernatant.

[0154] The test results showed that clones s8-1, s8-2, s8-3, s8-4, s8-5, and s8-6 could all significantly bind to human CHO-Siglec-8 cells (the results are shown in Figure 1).

[0155] Hybridoma cells in the logarithmic growth phase (s8-1, s8-2, s8-3, s8-4, s8-5, s8-6) were collected. cDNA sequencing yielded the amino acid sequences of the heavy and light chain variable regions of the corresponding monoclonal antibodies s8-1, s8-2, s8-3, s8-4, s8-5, and s8-6, as shown in Table 1-1.

[0156] Chimeric antibodies CHs8-1, CHs8-2, CHs8-3, CHs8-4, CHs8-5, and CHs8-6 were prepared. Their heavy chains consisted of VH and the human IgG1 constant region (amino acid sequence as shown in SEQ ID NO:112), and their light chains consisted of VL and the kappa constant region (amino acid sequence as shown in SEQ ID NO:114). VH and VL were composed of the sequences shown in Table 1-1, respectively.

[0157] Table 1-1: Variable region sequence and CDR composition of the antibody (SEQ ID NO:)

[0158] The framework regions of the variable regions of the above-mentioned CHs8-1, CHs8-3, CHs8-4, and CHs8-6 antibodies were humanized. The humanized antibodies are prefixed with "H" followed by a hybridoma number. The heavy chain consists of VH and the human IgG1 constant region (amino acid sequence as shown in SEQ ID NO: 112), and the light chain consists of VL and the kappa constant region (amino acid sequence as shown in SEQ ID NO: 114). The combinations of variable regions of the humanized antibodies are shown in Table 1-2. The CDR sequences of the humanized antibodies and the corresponding chimeric antibodies are identical.

[0159] Table 1-2: Variable region sequence combinations of humanized antibodies

[0160] HEK293F cells (Catalog No.: CRL-3249, ATCC) were transiently transfected with plasmids expressing the light and heavy chains of the antibody at a 1:1 ratio using PEI reagent (catalog number: 24765-1, Polysciences). After 5–7 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified using a Protein A column. The column was washed with PBS, and the antibody protein was eluted with acidic elution buffer (pH 3.0–3.5). The pH was adjusted to neutral with 1M Tris-HCl. The eluted sample was concentrated using an ultrafiltration tube, replaced with PBS buffer, aseptically filtered, and aliquoted for storage.

[0161] 1) Anti-Siglec-8 antibody binds to Siglec-8 expressed on the surface of CHO-Siglec-8 cells.

[0162] The binding ability of anti-Siglec-8 antibody to Siglec-8 expressing cells was detected by flow cytometry. The experimental procedure was as follows: Siglec-8 positive cells (CHO-Siglec-8) were collected, washed once with PBS, and then resuspended in PBS at a concentration of 1×10⁶ cells / mL. 5 50 μl / well of cells were seeded into 96-well V-plates (catalog number: 3897, Costa). The antibody was pre-diluted to 400 nM with PBS, then down-diluted 12 times in 3-fold increments. 50 μl / well of each antibody was added to the cells in the 96-well V-plates and mixed well (final antibody concentrations: 200, 66.7, 22.2, 7.4, 2.4, 0.82, 0.27, 0.09, 0.03, 0.01, 0.003, 0.001 nM). After incubation at 4°C for 1 h, the cells were washed twice with PBS, and then 100 μl of 1:500 diluted goat anti-human IgG-Fc was added. The PE fluorescent secondary antibody (catalog number: 12-4998-82, eBioscience) was incubated at 4°C for 1 hour, washed twice with PBS, and then analyzed using a CytoFLEX flow cytometer (model: AOO-1-1102, Beckman Coulter).

[0163] The detection of anti-Siglec-8 chimeric antibodies and Siglec-8-expressing cells is shown in Figure 2 and Table 2-1. Chimeric antibodies CHs8-1, CHs8-2, CHs8-3, CHs8-4, CHs8-5, and CHs8-6 all bound to Siglec-8 on CHO cells, with chimeric antibody CHs8-3 showing better binding activity. Compared to the positive antibody HEKA, the chimeric antibody CHs8-3 exhibited better binding ability to Siglec-8 on CHO cells: CHs8-3 and HEKA EC50... 50 The values ​​are 0.92 nM and 2.355 nM, respectively.

[0164] Table 2-1 The binding of anti-Siglec-8 chimeric antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells in EC 50

[0165] The detection of anti-Siglec-8 humanized antibodies and Siglec-8-expressing cells is shown in Figure 3 and Table 2-2. All anti-Siglec-8 humanized antibodies could bind to Siglec-8 on CHO cells and EC... 50 The values ​​ranged from 2.68 nM to 4.0 nM, which was comparable to the positive control antibody HEKA.

[0166] Table 2-2 Anti-Siglec-8 humanized antibody and ECMO of Siglec-8 expressed on the surface of CHO-Siglec-8 cells 50

[0167] 2) Kinetics and affinity constants of anti-Siglec-8 chimeric antibody

[0168] On a Biacore T200 (GE Healthcare), surface plasmon resonance (SPR) was used to further measure the kinetic constants (Kon & Koff) and dissociation constant (K) of the anti-Siglec-8 antibody using the 6His-tagged hSiglec8-his protein (SEQ ID NO:111). D In short, following the manufacturer's instructions (GE Healthcare), detection was performed using a protein A chip. 5 μg / mL antibody dilution was passed through the experimental flow paths (Fc2, Fc4) at a flow rate of 10 μL / min for 15 seconds. The flow rate was then adjusted to 30 μL / min, and different concentrations of hSiglec8-his antigen dilutions (0 nM, 1.235 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM) were analyzed sequentially, flowing through both the experimental flow paths (Fc2, Fc4) and the reference flow paths (Fc1, Fc3). Binding time was 90 s, dissociation time was 300 s, and the chip was regenerated in Glycine pH 1.5 buffer for 60 s before the next cycle. The kinetic binding constant was estimated by nonlinearly fitting the sensing data to a 1:1 binding model provided by the Biacore T200 evaluation software.

[0169] The results are shown in Table 3 below. Except for CHs8-2 and CHs8-5(K), which have weaker affinities... D Apart from 23.4 nM and 31.1 nM respectively, all antibodies showed affinity in the nM range, comparable to or superior to the control antibody. DIt ranges from 0.89 to 3.8 nM.

[0170] Table 3: Kinetics and affinity constants of anti-Siglec-8 chimeric antibodies as determined by Biacore.

[0171] 3) Kinetics and affinity constants of anti-Siglec-8 humanized antibody

[0172] The experimental results of the kinetics and affinity constants of the anti-Siglec-8 humanized antibodies are shown in Table 4 below. Except for Hs8-4-1 and Hs8-4-3, whose affinity after humanization is comparable to that of the chimeric antibody CHs8-4, the affinity of the other humanized antibodies is lower than that of their corresponding chimeric antibodies. The affinity of Hs8-4-3 is comparable to that of the control antibody.

[0173] Table 4: Kinetics and affinity constants of anti-Siglec-8 humanized antibodies as determined by Biacore.

[0174] Example 3: Affinity maturation and site-directed mutagenesis of anti-Siglec-8 humanized antibody and its identification

[0175] The HCDR3 clone of Hs8-4-1 was cloned into the scFv phage library of HCDR1 and HCDR2. After screening, the supernatant of the phage clones was analyzed. The supernatant was diluted 50 times, and the binding OD value of hSiglec8-His protein was measured by ELISA. The supernatant was serially diluted 3 times, and the titer of antigen binding was measured by ELISA. 50 The clones with higher OD values ​​were selected for sequencing to obtain the heavy chain variable region sequences of each clone as shown in Table 5, and the light chain variable region sequences as shown in SEQ ID NO:53.

[0176] Table 5: Composition of the heavy chain variable region and CDR sequence of the mutant antibody

[0177] Further site-directed mutagenesis was performed on the variable region of antibody 8-7-1, and the sequence combinations are shown in Tables 6 and 7:

[0178] Table 6: Variable region sequences of site-directed mutagenesis

[0179] Table 7: Sequence combinations of site-directed mutagenic antibodies (SEQ ID NO:)

[0180] The above-mentioned antibody VH and VL gene fragments were synthesized. The antibody preparation process is described in Example 2. The sequence combinations of the variable regions of the antibodies are shown in Tables 5 and 7. The heavy chain consists of VH and the human IgG1 constant region (amino acid sequence as shown in SEQ ID NO: 112), and the light chain consists of VL and the kappa constant region (amino acid sequence as shown in SEQ ID NO: 114).

[0181] 1) Anti-Siglec-8 antibody binds to Siglec-8 expressed on the surface of CHO-siglec-8 cells.

[0182] The experimental method is described in Example 2.

[0183] The detection of anti-Siglec-8 antibodies and Siglec-8-expressing cells is shown in Figure 4 and Tables 8-1 and 5a-c. All of these antibodies can bind to Siglec-8 on CHO cells. Table 5 shows the ECG activity of anti-Siglec-8 mutant antibodies. 50 The values ​​were between 0.74 nM and 1.57 nM, superior to the mother antibody Hs8-4-1 (1.80 nM) and comparable to the positive control HEKA. Table 7 shows the anti-Siglec-8 site-directed mutant antibody EC. 50 The values ​​are between 0.80 nM and 2.31 nM.

[0184] Table 8-1 The binding of anti-Siglec-8 mutant antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells in EC 50

[0185] Table 8-2a shows the binding of anti-Siglec-8 site-directed mutagen antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells in ECMO cells. 50

[0186] Table 8-2b shows the binding of anti-Siglec-8 site-directed mutagen antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells in ECMO cells. 50

[0187] Table 8-2c shows the binding of anti-Siglec-8 site-directed mutagen antibody to Siglec-8 expressed on the surface of CHO-Siglec-8 cells in EC. 50

[0188] 2) Kinetics and affinity constants of anti-Siglec-8 antibody

[0189] The experimental method is described in Example 2.

[0190] The results are shown in Table 8-3. Except for antibodies 8-1-5 and 8-12-3, whose affinity was comparable to that of maternal antibody Hs8-4-1 and positive control HEKA, the affinity of other antibodies increased by at least one order of magnitude after mutation.

[0191] Table 8-3: Kinetics and affinity constants of anti-Siglec-8 mutant antibodies as determined by Biacore

[0192] The results are shown in Table 9. In Table 6, the affinity of most of the site-directed mutation antibodies remained unchanged, or even increased. The affinity of 8-7-1mu2, 8-7-1mu4, antibody I, antibody L, antibody S, 7mu2-I-SD, and 7mu2-I-SN were all significantly better than the control antibody HEKA.

[0193] Table 9: Kinetics and affinity constants of anti-Siglec-8 site-directed mutant antibodies as determined by Biacore.

[0194] Example 4: ADCC activity (antibody-dependent cell-mediated cytotoxicity) of anti-Siglec-8 antibody

[0195] To investigate the effects of different antibody subclasses and fucose knockout on the ADCC of the anti-Siglec-8 antibody, anti-Siglec-8 IgG4 antibody (named I-G4, whose heavy chain consists of the variable region of the heavy chain of antibody I and the constant region of the IgG4 heavy chain, and whose light chain is the same as that of antibody I) and anti-Siglec-8 IgG1 ADCC enhancing antibody (named I-V1, whose heavy chain consists of the variable region of the heavy chain of antibody I and the constant region of the IgG1 heavy chain, and whose light chain is the same as that of antibody I) were prepared. The original anti-Siglec-8 IgG1 antibody I was named I-G1 (whose heavy chain consists of the variable region of the heavy chain of antibody I and the constant region of the IgG1 heavy chain, and whose light chain is the same as that of antibody I). The antibody preparation method is described in Example 4. The amino acid sequence of the constant region of the IgG4 heavy chain is shown in SEQ ID NO:113. Antibodies I-G4 and I-G1 were transiently expressed in HEK293F cells (catalog number: CRL-3249, ATCC), and anti-Siglec-8 IgG1 ADCC enhanced antibody I-V1 was transiently expressed in CHO-BAT-KF cells (source can be found in patent CN110157694B).

[0196] This experiment used the ADCC Reporter Bioassay to evaluate the ADCC activity of the anti-Siglec-8 antibody. This is a bioluminescent reporter gene assay system that uses artificially constructed effector cells instead of NK cells, combined with highly sensitive detection reagents, to quantify the bioactivity of therapeutic antibody drugs based on the ADCC mechanism of action in their activation pathway. It is a mechanism-of-action assay. Siglec-8 expressing cells (CHO-Siglec-8) were used as target cells, and Jurkat-NFAT-luc-FcγRIIIa cells were used as effector cells (FcγRIIIa (sequence from NCBI, NP_001121065.1) was transferred to Jurkat (Shanghai Cell Bank, Chinese Academy of Sciences, Clone) via lentiviral infection). Jurkat-FcγRIIIa cells were transfected with puromycin B (A11138-03, Gibco) at a concentration of 0.3 μg / ml. Positive clones were selected and named Jurkat-FcγRIIIa. Next, the plasmid pGL4.30 [luc2P / NFAT-RE / Hygro] (E848A, Promega) containing the luciferin reporter gene was transfected into Jurkat-FcγRIIIa cells via electroporation. Positive clones were then selected with hygromycin B (A600230-0001, Sangon Biotech). The resulting cells were named Jurkat-NFAT-luc-FcγRIIIa. TM The Luciferase Assay System (catalog number: E6120-100 ml, Promega) was used for detection. The specific steps are as follows: CHO-Siglec-8 cells were collected, washed once with RPMI 1640 medium, and then the cell density was adjusted to 1.2 × 10⁻⁶ cells with RPMI 1640 medium. 6 10 cells / ml, 25 μl / well seeded into 96-well white plates (catalog number: 3917, Costa), i.e. 3 × 10⁶ cells / ml. 4 Collect cells / well and gently tap to mix. Using RPMI 1640 medium with 1% FBS as antibody dilution buffer, pre-dilute the antibody to 40 nM, then further dilute it 10 times down-diluted (2-fold). Add 50 μl / well to the cell plate and gently tap to mix. Next, collect Jurkat-NFAT-luc-FcγRIIIa cells in logarithmic growth phase, wash once with RPMI 1640 medium, and then adjust the cell density to 2.4 × 10⁶ cells / well with RPMI 1640 medium. 6 Add 25 μl per well to the cell culture plate described above, i.e., 6 × 10⁶ cells / ml. 4Add cells per well and mix well. Incubate the culture plate in an incubator (37°C, 5% CO2) for 6 hours, then remove and allow to reach room temperature for 10 minutes. Add 50 μl of ONE-GLO substrate (catalog number: E6120-100ml, Promega) and read the Luminesecence relative fluorescence units (RLU) using a microplate reader (model: SpectraMax M3, Molecular Devices).

[0197] HEKA and HEKA-V1 (ADCC enhanced type, expressed in CHO-BAT-KF cells) were used as antibody positive controls, and IgG1 isotype (catalog number: BE0297, Bio cell) was used as antibody negative controls.

[0198] As shown in Figure 6, the EC corresponding to I-G1 and HEKA 50 The values ​​were 0.558 nM and 0.686 nM, respectively; the EC values ​​corresponding to I-V1 and HEKA-V1 were... 50 The values ​​were 0.099 nM and 0.156 nM, respectively. This indicates that antibody I-G1 has a strong ADCC effect, which is stronger than that of the positive antibody HEKA; at the same time, the ADCC effect is greatly improved after the antibody is made into an ADCC-enhancing antibody; when the antibody is converted into the IgG4 subclass, the ADCC effect is eliminated.

[0199] Example 5: Anti-Siglec-8 antibody induces apoptosis in eosinophils

[0200] To assess the ability of the anti-Siglec-8 antibody to induce apoptosis in purified eosinophils, fresh erythrocyte sedimentation rate (ESR) amber layer was used, collected less than 24 hours after harvest from blood samples or equivalent blood products. Eosinophil purification was performed following the manufacturer's instructions (MACS Eosinophil Isolation Kit, 130-104-446). Cells were in RPMI complete medium at 1 x 10⁻⁶ ppm. 6 The purified eosinophils were resuspended at 100 cells / mL and cultured overnight in the presence of IL-5 (Acro, IL5-H52H3) at a concentration of approximately 50 ng / mL. The next day, the eosinophils from the culture flasks were seeded into 96-well plates at a seeding density of 1.0 × 10⁶ cells / mL. 6 Cells / ml, seeded in 50 μL. Dilute the antibody with 50 μL of RPMI complete medium (final antibody concentration 10 μg / ml) and add to each well. Incubate overnight at 37°C. Annexin V staining was performed according to the manufacturer's instructions for the Annexin V-PE / 7-AAD fluorescent double staining apoptosis detection kit (Jeves, E-CK-A216), and apoptotic cells were analyzed by flow cytometry.

[0201] HEKA was used as a positive antibody control, and IgG1 isotype (catalog number: BE0297, Bio cell) was used as a negative antibody control.

[0202] As shown in Figure 7, under the induction of 10 μg / ml antibody, the percentage values ​​of 7AAD+ eosinophils corresponding to antibody I and HEKA were 37.27% and 30.28%, respectively, indicating that antibody I can significantly induce eosinophil apoptosis and is more effective than the positive antibody HEKA.

Claims

1. An anti-Siglec-8 antibody or antigen-binding fragment comprising one or more selected from the following: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: (1)HCDR1, which contains an amino acid sequence selected from any one of SEQ ID NO:13,16,18,21,24,27,72-75; (2)HCDR2, which contains an amino acid sequence selected from any one of SEQ ID NO:14,19,22,25,28,76-83,98; (3)HCDR3, which contains an amino acid sequence selected from any one of SEQ ID NO:15,17,20,23,26,29,99-104; (4) LCDR1, which contains an amino acid sequence selected from any one of SEQ ID NO:30,33,35,40,42,105-108; (5) LCDR2, which contains an amino acid sequence selected from any one of SEQ ID NO:31,34,36,38,43; (6) LCDR3, which contains an amino acid sequence selected from any one of SEQ ID NO:32,37,39,41,44.

2. An anti-Siglec-8 antibody or antigen-binding fragment comprising HCDR1, HCDR2, and HCDR3, and / or said antibody or antigen-binding fragment comprising LCDR1, LCDR2, and LCDR3, wherein, The HCDR1 contains the sequence shown in any one of SEQ ID NO:21 or 72-75, the HCDR2 contains the sequence shown in any one of SEQ ID NO:22, 98 or 76-83, the HCDR3 contains the sequence shown in any one of SEQ ID NO:23 or 99-104, the LCDR1 contains the sequence shown in any one of SEQ ID NO:35 or 105-108, the LCDR2 contains the sequence shown in SEQ ID NO:38, and the LCDR3 contains the sequence shown in SEQ ID NO:

39.

3. An anti-Siglec-8 antibody or antigen-binding fragment comprising HCDR1, HCDR2, and HCDR3, wherein the antibody or antigen-binding fragment comprises LCDR1, LCDR2, and LCDR3, wherein, (1) HCDR1 of the amino acid sequence shown in SEQ ID NO:21, HCDR2 of the amino acid sequence shown in any one of SEQ ID NO:79-81, 76 or 98, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or (2) HCDR1 of the amino acid sequence shown in SEQ ID NO:72, HCDR2 of the amino acid sequence shown in SEQ ID NO:22 or 79, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or (3) HCDR1 of the amino acid sequence shown in SEQ ID NO:73, HCDR2 of the amino acid sequence shown in any one of SEQ ID NO:77, 78 or 81, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or (4) HCDR1 of the amino acid sequence shown in SEQ ID NO:74, HCDR2 of the amino acid sequence shown in SEQ ID NO:77, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or (5) HCDR1 of the amino acid sequence shown in SEQ ID NO:75, HCDR2 of the amino acid sequence shown in SEQ ID NO:82 or 83, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or (6) HCDR1 of the amino acid sequence shown in SEQ ID NO:21, HCDR2 of the amino acid sequence shown in SEQ ID NO:79, and HCDR3 of any one of the amino acid sequences shown in SEQ ID NO:99-104, LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or (7) HCDR1 of the amino acid sequence shown in SEQ ID NO:21, HCDR2 of the amino acid sequence shown in SEQ ID NO:79 and HCDR3 of the amino acid sequence shown in SEQ ID NO:99, LCDR1 of the amino acid sequence shown in any one of SEQ ID NO:105-108, LCDR2 of the amino acid sequence shown in SEQ ID NO:38 and LCDR3 of the amino acid sequence shown in SEQ ID NO:

39.

4. An anti-Siglec-8 antibody or antigen-binding fragment comprising HCDR1, HCDR2, HCDR3, and / or said antibody or antigen-binding fragment comprising LCDR1, LCDR2, and LCDR3, wherein, (1) HCDR1 of the amino acid sequence shown in SEQ ID NO:21, HCDR2 of the amino acid sequence shown in SEQ ID NO:22, and HCDR3 of the amino acid sequence shown in SEQ ID NO:23, LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:38, and LCDR3 of the amino acid sequence shown in SEQ ID NO:39; or (2) HCDR1 of the amino acid sequence shown in SEQ ID NO:13, HCDR2 of the amino acid sequence shown in SEQ ID NO:14, and HCDR3 of the amino acid sequence shown in SEQ ID NO:15; LCDR1 of the amino acid sequence shown in SEQ ID NO:30, LCDR2 of the amino acid sequence shown in SEQ ID NO:31, and LCDR3 of the amino acid sequence shown in SEQ ID NO:32; or (3) HCDR1 of the amino acid sequence shown in SEQ ID NO:16, HCDR2 of the amino acid sequence shown in SEQ ID NO:14, and HCDR3 of the amino acid sequence shown in SEQ ID NO:17, LCDR1 of the amino acid sequence shown in SEQ ID NO:33, LCDR2 of the amino acid sequence shown in SEQ ID NO:34, and LCDR3 of the amino acid sequence shown in SEQ ID NO:32; or (4) HCDR1 of the amino acid sequence shown in SEQ ID NO:18, HCDR2 of the amino acid sequence shown in SEQ ID NO:19, and HCDR3 of the amino acid sequence shown in SEQ ID NO:20; LCDR1 of the amino acid sequence shown in SEQ ID NO:35, LCDR2 of the amino acid sequence shown in SEQ ID NO:36, and LCDR3 of the amino acid sequence shown in SEQ ID NO:37; or (5) HCDR1 of the amino acid sequence shown in SEQ ID NO:24, HCDR2 of the amino acid sequence shown in SEQ ID NO:25, and HCDR3 of the amino acid sequence shown in SEQ ID NO:26; LCDR1 of the amino acid sequence shown in SEQ ID NO:40, LCDR2 of the amino acid sequence shown in SEQ ID NO:34, and LCDR3 of the amino acid sequence shown in SEQ ID NO:41; or (6) HCDR1 of the amino acid sequence shown in SEQ ID NO:27, HCDR2 of the amino acid sequence shown in SEQ ID NO:28 and HCDR3 of the amino acid sequence shown in SEQ ID NO:29, LCDR1 of the amino acid sequence shown in SEQ ID NO:42, LCDR2 of the amino acid sequence shown in SEQ ID NO:43 and LCDR3 of the amino acid sequence shown in SEQ ID NO:

44.

5. The antibody or antigen-binding fragment according to any one of claims 1-4, comprising a heavy chain variable region and / or a light chain variable region, wherein, (1) The heavy chain variable region comprises the sequence shown in SEQ ID NO:1 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:2 or a variant thereof; or (2) The heavy chain variable region comprises the sequence shown in SEQ ID NO:3 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:4 or a variant thereof; or... (3) The heavy chain variable region comprises the sequence shown in SEQ ID NO:5 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:6 or a variant thereof; or... (4) The heavy chain variable region comprises the sequence shown in SEQ ID NO:7 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:8 or a variant thereof; or... (5) The heavy chain variable region comprises the sequence shown in SEQ ID NO:9 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:10 or a variant thereof; or... (6) The heavy chain variable region comprises the sequence shown in SEQ ID NO:11 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:12 or a variant thereof; or... (7) The heavy chain variable region comprises the sequence shown in SEQ ID NO:45 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:46 or a variant thereof; or... (8) The heavy chain variable region comprises the sequence shown in SEQ ID NO:47 or a variant thereof, and the light chain variable region comprises the sequence shown in any one of SEQ ID NO:48-50 or a variant thereof; or... (9) The heavy chain variable region comprises a sequence as shown in SEQ ID NO:51 or 52 or a variant thereof, and the light chain variable region comprises a sequence as shown in SEQ ID NO:53 or 54 or a variant thereof; or... (10) The heavy chain variable region comprises a sequence as shown in SEQ ID NO:55 or 56 or a variant thereof, and the light chain variable region comprises a sequence as shown in SEQ ID NO:57 or 58 or a variant thereof; or (11) The heavy chain variable region comprises a sequence as shown in any one of SEQ ID NO:59-71, 84-93 or a variant thereof, and the light chain variable region comprises a sequence as shown in SEQ ID NO:53 or a variant thereof; or (12) The heavy chain variable region comprises the sequence shown in SEQ ID NO:88 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO:94-97 or a variant thereof; The variant is a sequence that differs from the corresponding variable region by 3, 2, or 1 amino acid (preferably a conserved amino acid substitution) or has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, respectively, and the variant retains binding affinity to Siglec-8.

6. An anti-Siglec-8 antibody or antigen-binding fragment comprising a heavy chain variable region and / or a light chain variable region, wherein, The heavy chain variable region comprises a sequence as shown in any one of SEQ ID NO:1,3,5,7,9,11,45,47,51-52,55-56,59-71,84-93, and the light chain variable region comprises a sequence as shown in any one of SEQ ID NO:2,4,6,8,10,12,46,48-50,53-54,57-58,94-97.

7. An anti-Siglec-8 antibody or antigen-binding fragment comprising a heavy chain variable region and / or a light chain variable region, wherein, (1) The heavy chain variable region comprises the sequence shown in SEQ ID NO:88, and the light chain variable region comprises the sequences shown in SEQ ID NO:94-97; or... (2) The heavy chain variable region comprises the sequence shown in SEQ ID NO:3, and the light chain variable region comprises the sequence shown in SEQ ID NO:4; or... (3) The heavy chain variable region comprises the sequence shown in SEQ ID NO:5, and the light chain variable region comprises the sequence shown in SEQ ID NO:6; or... (4) The heavy chain variable region comprises the sequence shown in SEQ ID NO:7, and the light chain variable region comprises the sequence shown in SEQ ID NO:8; or... (5) The heavy chain variable region comprises the sequence shown in SEQ ID NO:9, and the light chain variable region comprises the sequence shown in SEQ ID NO:10; or, (6) The heavy chain variable region comprises the sequence shown in SEQ ID NO:11, and the light chain variable region comprises the sequence shown in SEQ ID NO:12; or, (7) The heavy chain variable region comprises the sequence shown in SEQ ID NO:45, and the light chain variable region comprises the sequence shown in SEQ ID NO:46; or... (8) The heavy chain variable region comprises the sequence shown in SEQ ID NO:47, and the light chain variable region comprises the sequence shown in any one of SEQ ID NO:48-50; or... (9) The heavy chain variable region comprises a sequence as shown in SEQ ID NO: 51 or 52, and the light chain variable region comprises a sequence as shown in SEQ ID NO: 53 or 54; or... (10) The heavy chain variable region comprises a sequence as shown in SEQ ID NO:55 or 56, and the light chain variable region comprises a sequence as shown in SEQ ID NO:57 or 58; or... (11) The heavy chain variable region comprises a sequence as shown in any one of SEQ ID NO:59-71, 84-93, and the light chain variable region comprises a sequence as shown in SEQ ID NO:53; or (12) The heavy chain variable region contains the sequence shown in SEQ ID NO:1, and the light chain variable region contains the sequence shown in SEQ ID NO:

2.

8. The antibody or antigen-binding fragment according to any one of claims 1-7, wherein the antigen-binding fragment is Fab, F(ab')2, Fv, or scFv.

9. The antibody or antigen-binding fragment according to any one of claims 1-8, wherein the antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region.

10. The antibody or antigen-binding fragment of claim 9, wherein the heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3 and IgG4 or variants thereof.

11. The antibody or antigen-binding fragment of claim 9 or 10, wherein the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 112 or 113.

12. The antibody or antigen-binding fragment of claim 9, wherein the light chain constant region is selected from the κ or λ light chain constant region or a variant thereof.

13. The antibody or antigen-binding fragment of claim 9 or 12, wherein the amino acid sequence of the constant region of the light chain is shown in SEQ ID NO:

114.

14. The antibody or antigen-binding fragment according to any one of claims 1-13, wherein the antibody or antigen-binding fragment is a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

15. The antibody or antigen-binding fragment according to any one of claims 1-14, wherein the antibody or antigen-binding fragment is a bispecific or multispecific antibody.

16. A biomaterial, comprising: 1) A nucleic acid molecule encoding an antibody or antigen-binding fragment or a portion thereof as described in any one of claims 1-15; 2) An expression vector comprising a nucleic acid molecule encoding an antibody or antigen-binding fragment or a portion thereof as described in any one of claims 1-15; 3) A host cell comprising a nucleic acid molecule or expression vector encoding an antibody or antigen-binding fragment or a portion thereof as described in any one of claims 1-15.

17. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of claims 1-15.

18. The pharmaceutical composition of claim 17 further comprises pharmaceutically acceptable excipients, such as pharmaceutically acceptable excipients, diluents, or carriers.

19. Use of the antibody or antigen-binding fragment of any one of claims 1-15, the biomaterial of claim 16, or the pharmaceutical composition of claim 17 or 18 in the preparation of a medicament for treating a disease.

20. The use as claimed in claim 19, wherein the disease is a cell-mediated condition expressing Siglec-8.

21. The use as described in claim 19 or 20, wherein the disease is an eosinophilic and / or mast cell-mediated condition.

22. The use according to any one of claims 19-21, wherein the disease is eosinophilia or mastocytosis.

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