Anti-BDCA2 antibody-drug conjugates and uses thereof

Abstract

Anti-BDCA2 antibody-drug conjugates and uses thereof. The anti-BDCA2 antibody-drug conjugates comprise an antibody or antigen-binding fragment thereof that targets BDCA2, a linker fragment (L), and a glucocorticoid molecule. Methods for preparing the antibody-drug conjugates and uses of the antibody-drug conjugates are also provided.

Description

[Technical Field]

[0001] This application relates to anti-BDCA2 antibody-drug conjugates and uses thereof. The anti-BDCA2 antibody-drug conjugates comprise an antibody or antigen-binding fragment thereof that targets BDCA2, a linker fragment (L), and a glucocorticoid molecule. This application also provides methods for preparing the antibody-drug conjugates and uses thereof. [Background technology]

[0002] As medical research advances, various diseases are found to be associated with malfunctioning of the immune system, and research into the immune system provides a better understanding of pathogenesis and facilitates the development of better treatment regimens.

[0003] The immune system is a highly complex system composed of many cell types, including but not limited to T cells, B cells, natural killer cells, antigen-presenting cells, dendritic cells, monocytes, and macrophages. These cells have a complex and delicate system for controlling their interactions and responses. Cells utilize activation and inhibition mechanisms and feedback loops to maintain control of responses and possibly prevent the negative consequences of uncontrolled immune responses (e.g., autoimmune diseases). Among the multiple signal transduction pathways related to immunity, there are several signal transduction pathways and their related interactions.

[0004] Blood dendritic cell antigen 2 (BDCA2) is a C-type lectin expressed on human plasmacytoid dendritic cells (pDCs) (Dzionek et al., J. Immunol., 165:6037-6046 (2000)). BDCA2 consists of a single extracellular carbohydrate recognition domain (CRD) at its C-terminus (belonging to the type II C-type lectin group), a transmembrane region located from asparagine residue 45 to isoleucine residue 213, and a short cytoplasmic tail (without a signaling motif) at its N-terminus. BDCA2 transduces intracellular signals through the associated transmembrane adaptor FcεRIγ, triggering a B cell receptor (BCR)-like signaling cascade.

[0005] Antibody-drug conjugates (ADCs) consist of three parts: an antibody or its antigen-binding fragment (targeting), a linker, and a small molecule drug. The antibody or its antigen-binding fragment is conjugated to a biologically active small molecule drug via a cleavable or non-cleavable linker. This effectively utilizes the high efficacy of the small molecule drug along with the specificity of the antibody or its antigen-binding fragment to target cells of interest (target cells) or bind to highly expressed antigens, thereby reducing or avoiding toxic side effects on non-target cells.

[0006] Glucocorticoids (GCs) are small-molecule steroid compounds that bind to the glucocorticoid receptor (GR) and are used in anti-inflammatory and immunosuppressive therapies. However, glucocorticoid therapy is toxic in most organ systems due to the ubiquitous expression of glucocorticoid receptors in many cell types. Therefore, there is a need for the development of novel glucocorticoids and novel therapies that minimize the side effects caused by glucocorticoid administration, especially those caused by activation of the glucocorticoid receptor in non-target cells.

[0007] To date, no antibody-drug conjugates targeting BDCA2 have been developed, thus leaving significant room for and clinical demand for the development of related clinical drugs. Summary of the Invention [Problem to be solved by the invention]

[0008] The present application provides antibody-drug conjugates targeting BDCA2, which may have one or more effects selected from the group consisting of: (1) the ability to affect the activity of immune cells; (2) the ligand has a targeting effect; (3) the ability to be plasma stable; (4) the ability to be biologically safe; (5) the ability to affect cytokine release from immune cells; (6) the ability to affect the transcription of response genes in the IFN signaling pathway; (7) the ability to affect the degree of skin fibrosis; (8) the ability to affect the number and / or proportion of dendritic cells; (9) the ability to affect collagen content in the skin; (10) the ability to affect GRE expression levels; (11) the ability to affect cytokine release from peripheral blood mononuclear cells; (12) the ability to affect skin swelling; (13) the ability to affect arthritic symptoms; and (14) the ability to inhibit inflammation. [Means for solving the problem]

[0009] In one aspect, the present application provides an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof that targets BDCA2, a linker fragment (L), and a glucocorticoid molecule, wherein the antibody or antigen-binding fragment thereof targets BDCA2 and is (1) an HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 2, an HCDR3 having the amino acid sequence shown in SEQ ID NO: 3, an LCDR1 having the amino acid sequence shown in SEQ ID NO: 4, an LCDR2 having the amino acid sequence shown in SEQ ID NO: 5, and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 6; or (2) HCDR1 having the amino acid sequence shown in SEQ ID NO: 7, HCDR2 having the amino acid sequence shown in SEQ ID NO: 8, HCDR3 having the amino acid sequence shown in SEQ ID NO: 9, LCDR1 having the amino acid sequence shown in SEQ ID NO: 10, LCDR2 having the amino acid sequence shown in SEQ ID NO: 11, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 12 The present invention provides an antibody-drug conjugate comprising:

[0010] In some embodiments, in the antibody-drug conjugates described herein, the antibody or antigen-binding fragment thereof (1) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 13 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 14, or (2) a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 16, or (3) A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 18 Includes.

[0011] In some embodiments, in the antibody-drug conjugates described herein, the antibody is selected from a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

[0012] In some embodiments, in the antibody-drug conjugates described herein, the antibody or antigen-binding fragment thereof is selected from a humanized antibody or antigen-binding fragment thereof. In some embodiments, in the antibody-drug conjugates described herein, the antibodies described herein are monoclonal antibodies.

[0013] In some embodiments, in the antibody-drug conjugates described herein, the antibody is a multispecific antibody comprising the light chain variable region and the heavy chain variable region of an antibody described herein, or an antigen-binding fragment thereof.

[0014] In some embodiments, in the antibody-drug conjugates described herein, the antigen-binding fragment is selected from a Fab, a Fab', a F(ab')2, an Fv, a scFv, a Fab'-SH, a sdAb, a VHH, a bispecific antibody, and a linear antibody.

[0015] In some embodiments, in the antibody-drug conjugates described herein, the antibody or antigen-binding fragment thereof comprises an immunoglobulin constant region, and the immunoglobulin constant region is a human IgG constant region, e.g., a human IgG1 constant region.

[0016] In some embodiments, in the antibody-drug conjugates described herein, the antibody or antigen-binding fragment thereof has the following sequence: (1) a heavy chain having an amino acid sequence set forth in SEQ ID NO: 19 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO: 20 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto; or (2) a heavy chain having an amino acid sequence set forth in SEQ ID NO: 21 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO: 22 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto; or (3) a heavy chain having an amino acid sequence set forth in SEQ ID NO: 23 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO: 24 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto; Comprises or consists of

[0017] In some embodiments, in the antibody-drug conjugates described herein, the antibody or antigen-binding fragment thereof has the following sequence: (1) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 19 and a light chain having the amino acid sequence set forth in SEQ ID NO: 20; or (2) a heavy chain having the amino acid sequence set forth in SEQ ID NO: 21 and a light chain having the amino acid sequence set forth in SEQ ID NO: 22; or (3) A heavy chain having the amino acid sequence shown in SEQ ID NO: 23 and a light chain having the amino acid sequence shown in SEQ ID NO: 24 Comprises or consists of

[0018] In some embodiments, the amino acid sequences of the CDRs may have at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences set forth above. In some embodiments, the amino acid sequences of the variable regions may have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences set forth above.

[0019] As noted above, the addition, deletion, and / or substitution of at least one amino acid in the VH or VL region may not be within a CDR sequence, but may be within a framework (FRW) sequence. For example, an isolated antibody or antigen-binding portion thereof may contain one or more amino acid substitutions within a framework sequence, such as FRW1, FRW2, FRW3, and / or FRW4, of the VH or VL region.

[0020] In some embodiments, the six CDRs have a total of 0, 1, 2, 3, 4, or 5 amino acid modifications (complete amino acid substitutions), and can have multiple changes within the framework regions of the heavy and light chain variable regions, so long as the framework (excluding the CDRs) maintains at least about 80%, 85%, or 90% identity to the parent antibody. Thus, the same CDRs described herein can be combined with different framework sequences derived from human germline sequences, so long as the framework regions maintain at least 80%, 85%, or 90% identity to the human germline sequences.

[0021] In some embodiments, in the antibody-drug conjugates described herein, the antibody is an IgG antibody, e.g., an IgG1, IgG2, IgG3, or IgG4 antibody, or a modified form thereof as described below.

[0022] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thus generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (such as a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0023] In some embodiments, it may be necessary to generate cysteine-engineered antibodies, such as "sulfo-MAbs," in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0024] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art. Suitable moieties for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), glucan, or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol, etc.), polyvinyl alcohol, and mixtures thereof.

[0025] In some embodiments, in the antibody-drug conjugates described herein, the antibody is selected from anti-BDCA2 antibodies Hu033-03, Hu033-20, and Hu005-04.

[0026] In some embodiments, the antibodies Hu033-03, Hu033-20, and Hu005-04 used in the drug conjugates, compositions, uses, or methods of the disclosure are derived from the antibodies Hu033-03, Hu033-20, and Hu005-04 described in CN202210599888.X.

[0027] The anti-BDCA2 antibody Hu033-03 or antigen-binding fragment thereof described herein is prepared as described in CN202210599888.X. In some embodiments, the CDR sequences of an antibody used in a drug conjugate, composition, use, or method of the present disclosure comprise HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In some embodiments, the variable region sequences of an antibody used in a drug conjugate, composition, or use of the present disclosure comprise a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:13 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:14. In some embodiments, an antibody used in a drug conjugate, composition, use, or method of the present disclosure comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:19 and a light chain having the amino acid sequence set forth in SEQ ID NO:20.

[0028] The anti-BDCA2 antibody Hu033-20 or antigen-binding fragment thereof described herein is prepared as described in CN202210599888.X. In some embodiments, the CDR sequences of an antibody used in a drug conjugate, composition, use, or method of the present disclosure comprise HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In some embodiments, the variable region sequences of an antibody used in a drug conjugate, composition, or use of the present disclosure comprise a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:15 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, an antibody used in a drug conjugate, composition, use, or method of the present disclosure comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:21 and a light chain having the amino acid sequence set forth in SEQ ID NO:22.

[0029] The anti-BDCA2 antibody Hu005-04 or antigen-binding fragment thereof described herein is prepared as described in CN202210599888.X. In some embodiments, the CDR sequences of an antibody used in a drug conjugate, composition, use, or method of the present disclosure comprise HCDR1, HCDR2, and HCDR3 having the amino acid sequences set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively, and LCDR1, LCDR2, and LCDR3 having the amino acid sequences set forth in SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively. In some embodiments, the variable region sequences of an antibody used in a drug conjugate, composition, or use of the present disclosure comprise a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:17 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:18. In some embodiments, an antibody used in a drug conjugate, composition, use, or method of the present disclosure comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:23 and a light chain having the amino acid sequence set forth in SEQ ID NO:24.

[0030] In certain preferred embodiments of the present disclosure, certain groups within the structures represented by formulas (I), (II), (III-A), and (III-A1) through (III-A4) are defined as follows, and groups not recited are as recited in any one of the embodiments (or, briefly, "some embodiments") of this application.

[0031] In some embodiments, in the antibody-drug conjugates described herein, the glucocorticoid molecule comprises or is selected from the following structure: a structure represented by Formula (I), and tautomers, mesomers, racemates, enantiomers, diastereoisomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof:

[0032] [ka]

[0033] During the ceremony, X is -O-, -S-, and -N(R 1a )-, where R 1a is selected from H, C1-C6 alkyl, and —C1-C6 alkylhydroxy; R1 and R2 are each independently selected from H, protium, deuterium, tritium, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl; R3 is selected from C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C1-C6 alkylthio, halogenated C1-C6 alkylthio, -C1-C6 alkyl-OP(=O)(OC1-C6 alkyl)2, and -C1-C6 alkyl-OP(=O)(OH)2, wherein C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkylthio are each independently optionally substituted with one or more substituents selected from halogen, CN, OH, or SH; Ring B is selected from phenyl or 5-6 membered heteroaryl, and Ring B is optionally substituted with one or more substituents selected from halogen, OH, CN, NH, C1-C6 alkyl, C1-C6 alkoxy, and halogenated C1-C6 alkyl; W is a single bond, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, -N(C1-C6 alkyl)-C(O)-, -C1-C6 alkylene-, -N(C1-C6 alkyl)-, -C 1-6 selected from alkylene-NH-, -O-C1-C6 alkylene-, and -C1-C6 alkylene-O-, wherein the C1-C6 alkyl and C1-C6 alkylene are optionally substituted with one or more substituents selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, oxo, OH, CN, or NH2; V is a single bond or -(C(R 2a )(R 2b )) n - selected from, where R2a and R 2b are each independently selected from H, halogen, OH, NH, —CN, C1-C6 alkyl, and halogenated C1-C6 alkyl, or R 2a and R 2b together with the carbon atom to which they are attached form a carbonyl or C 3~6 forming a cycloalkyl, and n is selected from 1, 2, 3, 4, 5, or 6; Y1 is selected from H, halogen, OH, nitro, NH2, —NH(C1-C6 alkyl), —N(C1-C6 alkyl)2, C1-C6 alkyl, —CN, C1-C6 alkoxy, —C1-C6 alkylhydroxy, and halogenated C1-C6 alkyl; m is selected from 1, 2, or 3.

[0034] In some embodiments, in the antibody-drug conjugates described herein, X is selected from -O-, -S-, and -N(R 1a )-, where R 1a is selected from H, —CH3, and —CH2CH3.

[0035] In some embodiments, in the antibody-drug conjugates described herein, X is selected from -O-. In some embodiments, in the antibody-drug conjugates described herein, X is selected from —NH—.

[0036] In some embodiments, in the antibody-drug conjugates described herein, X is selected from -S-. In some embodiments, in the antibody-drug conjugates described herein, R1 and R2 are each independently selected from H, F, Cl, and -CH3.

[0037] In some embodiments, in the antibody-drug conjugates described herein, R1 and R2 are each independently selected from H or F. In some embodiments, in the antibody-drug conjugates described herein, R3 is -CH2Cl, -CH2SH, -CH2OH,

[0038] [ka]

[0039] Selected from —OCH3, —OCH2F, —OCH2Cl, —OCH2CN, —OCH2CH3, —SCH2F, —SCH2Cl, —SCH2CF3, and —SCH2CN. In some embodiments, in the antibody-drug conjugates described herein, R3 is selected from -CH2OH, -SCH2F, and

[0040] [ka]

[0041] is selected from. In some embodiments, in the antibody-drug conjugates described herein, R3 is -CH2OH and

[0042] [ka]

[0043] is selected from. In some embodiments, in the antibody-drug conjugates described herein, Ring B is selected from phenyl, pyridinyl, thienyl, pyrrolyl, furanyl, pyrazolyl, imidazolyl, and thiazolyl, and Ring B is optionally substituted with one or more substituents selected from halogen, OH, CN, NH, C1-C6 alkyl, C1-C6 alkoxy, and halogenated C1-C6 alkyl.

[0044] In some embodiments, in the antibody-drug conjugates described herein, Ring B is selected from phenyl and thienyl, and Ring B is optionally substituted with one or more substituents selected from halogen, OH, CN, NH, -CH, -CHCH, or -OCH.

[0045] In some embodiments, in the antibody-drug conjugates described herein, ring B is

[0046] [ka]

[0047] is selected from. In some embodiments, in the antibody-drug conjugates described herein, ring B is

[0048] [ka]

[0049] is. In some embodiments, in the antibody-drug conjugates described herein, ring B is

[0050] [ka]

[0051] is. In some embodiments, in the antibody-drug conjugates described herein, W is selected from -C1-C6 alkylene-, where C1-C6 alkylene is optionally substituted with one or more substituents selected from halogen, C1-C3 alkyl, C1-C3 alkoxy, halogenated C1-C3 alkyl, C3-C6 cycloalkyl, oxo, OH, CN, or NH2.

[0052] In some embodiments, in the antibody-drug conjugates described herein, W is

[0053] [ka]

[0054] is selected from. In some embodiments, in the antibody-drug conjugates described herein, W is

[0055] [ka]

[0056] is selected from. In some embodiments, in the antibody-drug conjugates described herein, V is a single bond or -(C(R 2a )(R 2b )) n - selected from, where R 2a and R 2b are each independently selected from H, halogen, OH, NH2, —CN, C1-C3 alkyl, and halogenated C1-C3 alkyl; and n is selected from 1, 2, or 3.

[0057] In some embodiments, in the antibody-drug conjugates described herein, V is selected from a single bond, —CH 2 —, or —CH 2 CH 2 —. In some embodiments, in the antibody-drug conjugates described herein, Y1 is selected from H, halogen, OH, NH2, -CN, C1-C3 alkyl, C1-C3 alkoxy, -C1-C3 alkylhydroxy, and halogenated C1-C3 alkyl.

[0058] In some embodiments, in the antibody-drug conjugates described herein, Y is selected from H, halogen, OH, NH, -CN, -CH, -CHCH, -OCH, and -CHOH.

[0059] In some embodiments, m is 1 in the antibody-drug conjugates described herein. In some embodiments, in the antibody-drug conjugates described herein, the structure of the glucocorticoid molecule is:

[0060] [Table 1]

[0061] is selected from During the ceremony, R1 and R2 are as defined in any one of the embodiments herein; R3 is as defined in any one of the embodiments herein; W is as defined in any one of the embodiments herein; Y1 is as defined in any one of the embodiments herein.

[0062] In some embodiments, in the antibody-drug conjugate described herein, the glucocorticoid molecule is

[0063] [Table 2-1]

[0064] [Table 2-2]

[0065] is selected from. In some embodiments, in the antibody-drug conjugates described herein, the linker fragment (L) comprises Tr, L1, L2, and L3, and the antibody-drug conjugate comprises a structure represented by formula (II):

[0066] [ka]

[0067] During the ceremony, Tr is absent or is any group; L3 is selected from polypeptide fragments; L2 is absent or selected from linker fragments; L1 is selected from the coupling unit, X, R1, R2, R3, B, W, V, Y1, and m are as defined in any one of the embodiments of the present application.

[0068] In some embodiments, in the antibody-drug conjugates described herein, Tr is not present or

[0069] [ka]

[0070] is selected from L3 is composed of glycine-glycine-phenylalanine-glycine (GGGF), alanine-alanine-alanine-glycine (AAAG), glycine-glycine-glycine-glycine (GGGG), valine-alanine-glycine (VAG), valine-citrulline-glycine (VCG), alanine-alanine-glycine (AAG), alanine-alanine-alanine (AAA), valine-alanine (VA), valine-citrulline (VC), alanine-alanine (AA), glutamic acid-alanine lysine-glycine (EAGG), glycine-glutamic acid-alanine-glycine (GEAG), glycine-glutamic acid-glycine-glycine (GEGG), glutamic acid-glycine-glycine (EGG), glutamic acid-alanine-glycine (EAG), valine-lysine-glycine (VKG), glycine-glutamic acid-glycine (GEG), glutamic acid-alanine (EA), glutamic acid-glycine (EG), and glycine-glutamic acid (GE); L2 is not present, or

[0071] [ka]

[0072] [ka]

[0073] wherein q is selected from any integer from 1 to 30, and p is any integer from 1 to 20; (1) When L1 is coupled to Ab via a sulfhydryl, L1 has the following structure:

[0074] [ka]

[0075] where R L1c is selected from hydrogen, optionally substituted alkyl, and optionally substituted aryl; (2) When L1 is coupled to Ab via an amino, L1 has the following structure:

[0076] [ka]

[0077] is selected from (3) When L1 is coupled to Ab via click chemistry, L1 has the following structure:

[0078] [ka]

[0079] is selected from. In some embodiments, in the antibody-drug conjugates described herein, the structural unit -Tr-L3-L2-L1- is

[0080] [ka]

[0081] is selected from. In some embodiments, the antibody-drug conjugate described herein has formula (III-A):

[0082] [ka]

[0083] and an antibody-drug conjugate having a structure represented by the formula: During the ceremony, Ab is as defined in any one of the embodiments herein, and N a-I is any number between 1 and 10, L is as defined in any one of the embodiments herein; X is -O-, -S-, and -N(R 1a )-, where R 1a is selected from H, C1-C6 alkyl, and C1-C6 alkylhydroxy; R1 and R2 are each independently selected from H, protium, deuterium, tritium, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl; R3 is selected from halogenated C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and —C1-C6 alkyl-OP(═O)(OH)2, wherein C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkylthio are each optionally substituted with one or more substituents independently selected from halogen, CN, OH, or SH; Ring B is selected from phenyl, or 5-6 membered heteroaryl, and Ring B is optionally substituted with one or more substituents selected from halogen, OH, CN, NH, C1-C6 alkyl, C1-C6 alkoxy, and halogenated C1-C6 alkyl; W is a single bond, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, -N(C1-C6 alkyl)-C(O)-, -C1-C6 alkylene-, -N(C1-C6 alkyl)-, -C 1-6 selected from alkylene-NH-, -O-C1-C6 alkylene-, and -C1-C6 alkylene-O-, wherein the C1-C6 alkyl and C1-C6 alkylene are optionally substituted with one or more substituents selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, oxo, OH, CN, or NH2; V is a single bond or -(C(R 2a )(R 2b )) n - selected from, where R 2a and R 2b are each independently selected from H, halogen, OH, NH, —CN, C1-C6 alkyl, and halogenated C1-C6 alkyl, or R 2a and R 2b are carbonyls or C together with the carbon atoms to which they are attached. 3~6 forming a cycloalkyl, and n is selected from 1, 2, 3, 4, 5, or 6; Y1 is selected from H, halogen, OH, NH2, C1-C6 alkyl, -CN, C1-C6 alkoxy, -C1-C6 alkylhydroxy, and halogenated C1-C6 alkyl; m is selected from 1, 2, or 3.

[0084] In some embodiments, the antibody-drug conjugates described herein have the formula (III-A1) to (III-A4):

[0085] [ka]

[0086] and an antibody-drug conjugate having a structure represented by the formula: During the ceremony, Ab is as defined in any one of the embodiments herein; and N a-I is any number between 1 and 10, L is

[0087] [ka]

[0088] is selected from X is -O-, -S-, and -N(R 1a )-, where R 1a is selected from H, C1-C6 alkyl, and C1-C6 alkylhydroxy; R1 and R2 are each independently selected from H, protium, deuterium, tritium, halogen, C1-C6 alkyl, and halogenated C1-C6 alkyl; R3 is selected from halogenated C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylthio, and -C1-C6 alkyl-OP(=O)(OH)2, wherein C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 alkylthio are each independently optionally substituted with one or more substituents selected from halogen, CN, OH, or SH; Ring B is selected from phenyl or 5-6 membered heteroaryl, and Ring B is optionally substituted with one or more substituents selected from halogen, OH, CN, NH, C1-C6 alkyl, C1-C6 alkoxy, and halogenated C1-C6 alkyl; W is a single bond, -O-, -S-, -C(O)-, -NH-, -C(O)NH-, -NHC(O)-, -C(O)-N(C1-C6 alkyl)-, -N(C1-C6 alkyl)-C(O)-, -C1-C6 alkylene-, -N(C1-C6 alkyl)-, -C1~6 selected from alkylene-NH-, -O-C1-C6 alkylene-, and -C1-C6 alkylene-O-, wherein the C1-C6 alkyl and C1-C6 alkylene are optionally substituted with one or more substituents selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, C3-C6 cycloalkyl, oxo, OH, CN, or NH2; V is a single bond or -(C(R 2a )(R 2b )) n - selected from, where R 2a and R 2b are each independently selected from H, halogen, OH, NH, —CN, C1-C6 alkyl, and halogenated C1-C6 alkyl, or R 2a and R 2b are carbonyls or C together with the carbon atoms to which they are attached. 3~6 forming a cycloalkyl, and n is selected from 1, 2, 3, 4, 5, or 6; Y1 is selected from H, halogen, OH, NH2, C1-C6 alkyl, -CN, C1-C6 alkoxy, -C1-C6 alkylhydroxy, and halogenated C1-C6 alkyl; m is selected from 1, 2, or 3.

[0089] In some embodiments, in formulas (III-A), and (III-A1) to (III-A4), Ab, N a-I , L, X, R1, R2, R3, ring B, W, V, and Y1 are as described in any one of the embodiments of the present application.

[0090] In some embodiments, the antibody-drug conjugates described herein have the following structure:

[0091] [ka]

[0092] [ka]

[0093] is selected from wherein each Ab is as defined in any one of the embodiments herein; and N a-I is any number between 1 and 10.

[0094] In some embodiments, the antibody-drug conjugates described herein have the following structure:

[0095] [ka]

[0096] is selected from In the formula, N a-I is any number from 1 to 10, and preferably, N a-I is an integer or decimal number of 2 to 8, and preferably, N a-I is an integer or decimal number between 3 and 8, and preferably, N a-I is an integer or decimal number between 3 and 4 or between 4 and 5, for example, 4.01, 4.34, and 4.46, and Hu033-03, Hu033-20, and Hu005-04 are anti-BDCA2 antibodies.

[0097] In some embodiments, the antibody-drug conjugates described herein have the following structure:

[0098] [ka]

[0099] is selected from In the formula, the linkage number q is an integer of 1 to 10, preferably the linkage number q is an integer of 2 to 8, preferably the linkage number q is an integer of 4 to 6, preferably the linkage number q is 4 or 6, preferably 4; Hu033-03, Hu033-20, and Hu005-04 are anti-BDCA2 antibodies.

[0100] The amino acid sequences of the light and heavy chains of the anti-BDCA2 antibodies Hu033-03, Hu033-20, and Hu005-04 of the present disclosure are shown below: The antibody CDRs of the present disclosure are numbered using the Kabat numbering system. Amino acid sequence of Hu033-03 (underlined parts are CDRs) Heavy chain variable region VH SEQ ID NO: 13 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSTISSGDSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQIYYDYAYYFDFWGQGTTVTVSS HCDR1: SYGMS (SEQ ID NO: 1) HCDR2: TISSGDSYTYYPDSVKG (SEQ ID NO: 2) HCDR3: QIYYDYAYYFDF (SEQ ID NO: 3) Light chain variable region VL SEQ ID NO: 14 DIVLTQSPASLAVSPGQRATICRASESVSFRTSHLMHWYQQKPGQPPKLLIYGASNLESGVPARFSGSGSETDFTLTINPVEAEDTANYFCQQSIEDPPTFGGGTKVEIK LCDR1: RASESVSFRTSHLMH (SEQ ID NO: 4) LCDR2: GASNLES (SEQ ID NO: 5) LCDR3: QQSIEDPPT (SEQ ID NO: 6) Amino acid sequence of Hu033-20 (underlined parts are CDRs) Heavy chain variable region VH SEQ ID NO: 15 EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYGMSWVRQAPGKRLEWVSTISSGDSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLKAEDTAVYYCARQIYYDYAYYFDFWGQGTTVTVSS HCDR1: SYGMS (SEQ ID NO: 1) HCDR2: TISSGDSYTYYPDSVKG (SEQ ID NO: 2) HCDR3: QIYYDYAYYFDF (SEQ ID NO: 3) Light chain variable region VL SEQ ID NO: 16 DIVMTQSPDSLAVSLGERATINCRASESVSFRTSHLMHWYQQKPGQPPKLLIYGASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSIEDPPTFGGGTKVEIK LCDR1: RASESVSFRTSHLMH (SEQ ID NO: 4) LCDR2: GASNLES (SEQ ID NO: 5) LCDR3: QQSIEDPPT (SEQ ID NO: 6) Amino acid sequence of Hu005-04 (underlined parts are CDRs) Heavy chain variable region VH SEQ ID NO: 17 QVQLQQSGPGLVKPSETLSLTTCTVSGGSLTNYGVHWIRQPPGKGLEWIGVIWSGESTDYDAAFISRVTISKDNSKNQVSLKLSSVTAADTAVYYCARRRSHYYGYVMDYWGQGTTVTVSS HCDR1: NYGVH (SEQ ID NO: 7) HCDR2: VIWSGESTDYDAAFIS (SEQ ID NO: 8) HCDR3: RRSHYYGYVMDY (SEQ ID NO: 9) Light chain variable region VL SEQ ID NO: 18 DIVLTQSPASLAVSPGQRATITCKASQSIDYdaIGYLNWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQQSNEDPPTFGQGTKLEIK LCDR1: KASQSIDYDAIGYLN (SEQ ID NO: 10) LCDR2: AASNLES (SEQ ID NO: 11) LCDR3: QQSNEDPPT (SEQ ID NO: 12) Hu033-03-HC-IgG1: SEQ ID NO: 19 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSTISSGDSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQIYYDYAYYFDFWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Hu033-03-LC: SEQ ID NO: 20 DIVLTQSPASLAVSPGQRATICRASESVSFRTSHLMHWYQQKPGQPPKLLIYGASNLESGVPARFSGSGSETDFTLTINPVEAEDTANYFCQQSIEDPPTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Hu033-20-HC-IgG1: SEQ ID NO: 21 EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYGMSWVRQAPGKRLEWVSTISSGDSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLKAEDTAVYYCARQIYYDYAYYFDFWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Hu033-20-LC: SEQ ID NO: 22 DIVMTQSPDSLAVSLGERATINCRASESVSFRTSHLMHWYQQKPGQPPKLLIYGASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSIEDPPTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Hu005-04-HC-IgG1: SEQ ID NO: 23 QVQLQQSGPGLVKPSETLSLTTCTVSGGSLTNYGVHWIRQPPGKGLEWIGVIWSGESTDYDAAFISRVTISKDNSKNQVSLKLSSVTAADTAVYYCARRRSHYYGYVMDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Hu005-04-LC: SEQ ID NO: 24 DIVLTQSPASLAVSPGQRATITCKASQSIDYdaIGYLNWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQQSNEDPPTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In some embodiments, the average number of links N a-I can be an integer or a decimal number between 1 and 10. For example, the average number of connections N a-I can be an integer or decimal number between 2 and 8. For example, the average number of connections N a-1 can be an integer or decimal number between 3 and 8. For example, the average number of connections N a-1 can be an integer or decimal number between 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, or 9 and 10.

[0101] In some embodiments, the linkage number q described herein can be an integer from 1 to 10. For example, the linkage number q can be an integer from 2 to 8. For example, the linkage number q can be an integer from 3 to 8. For example, the linkage number q can be any integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0102] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0103] It is a further object of the present disclosure to provide a method for preparing a pharmaceutical composition of the present disclosure, comprising combining a compound described herein, or a pharmaceutically acceptable form thereof, or a mixture thereof, with one or more pharmaceutically acceptable carriers.

[0104] Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present disclosure are pharmaceutically acceptable carriers, and suitable examples of pharmaceutically acceptable carriers are as described in Remington's Pharmaceutical Sciences (2005).

[0105] The pharmaceutical composition may be administered in any form so long as it achieves the prevention, alleviation, inhibition, or cure of symptoms in a human or animal patient. For example, the pharmaceutical composition may be formulated into a variety of suitable dosage forms based on the route of administration.

[0106] In other embodiments, administration of the compounds or pharmaceutical compositions of the present disclosure can be combined with an additional treatment modality, which may be selected from, but is not limited to, radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

[0107] The present disclosure also relates to pharmaceutical formulations or pharmaceutical compositions of the present disclosure that include, as an active ingredient, a compound of the present disclosure, or a pharmaceutically acceptable form thereof, or a mixture thereof. In some embodiments, the formulation is in the form of a solid, semi-solid, liquid, or gaseous formulation.

[0108] In yet another aspect, the disclosure provides for the use of an antibody-drug conjugate described herein or a pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing a disease or disorder mediated by BDCA2 or plasmacytoid dendritic cells.

[0109] In yet another aspect, the present disclosure provides a method for treating and / or preventing a disease or disorder mediated by BDCA2 or plasmacytoid dendritic cells, comprising administering to a subject in need thereof an antibody-drug conjugate described herein or a pharmaceutical composition described herein.

[0110] In yet another aspect, the disclosure provides an antibody-drug conjugate or pharmaceutical composition described herein for use in treating and / or preventing a disease or disorder mediated by BDCA2 or plasmacytoid dendritic cells.

[0111] In some embodiments, the disease and / or disorder may include a disease and / or disorder associated with glucocorticoid receptor signaling. In some embodiments, the disease and / or disorder is selected from the group consisting of a proliferative disease and / or condition, a metabolic disease and / or condition, an inflammatory disease and / or condition, and a neurodegenerative disease and / or condition.

[0112] In some embodiments, the disease and / or disorder is selected from an autoimmune disease and / or disorder. In some embodiments, the disease and / or disorder is selected from inflammatory diseases and / or disorders.

[0113] In some embodiments, the disease and / or disorder is selected from systemic lupus erythematosus, discoid lupus erythematosus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

[0114] In yet another aspect, the present disclosure provides a pharmaceutical combination comprising an antibody-drug conjugate described herein or a pharmaceutical composition described herein and one or more additional therapeutic agents, including, but not limited to, radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

[0115] In yet another aspect, the present disclosure provides a kit comprising an antibody-drug conjugate described herein or a pharmaceutical composition described herein. The dosage regimen can be adjusted to provide the optimal desired response. For example, when administration is performed in the form of an injection, a single bolus injection, a bolus injection, and / or a continuous infusion can be performed. For example, several separate doses can be administered over time, or the dose can be proportionally increased or decreased as indicated by the exigencies of the treatment situation. It should be noted that the numerical dosage can vary depending on the type and severity of the condition to be alleviated and can include single or multiple administrations. Generally, the therapeutic dose will vary depending on considerations such as the age, sex, and overall health of the patient being treated; the frequency of treatment and the nature of the desired effect; the extent of tissue damage; the duration of symptoms; and other variables that can be adjusted by the physician. It is further understood that for any particular individual, the specific dosage regimen should be adjusted over time based on the individual's needs and the professional judgment of the person administering or supervising the administration of the composition. The amount and regimen of administering the pharmaceutical composition can be easily determined by those skilled in the clinical arts. For example, the compositions or compounds of the present disclosure may be administered in separate doses, for example, four times a day to once every three days, with the dosage being, for example, 0.01 to 1000 mg per dose. The desired dose may be administered one or more times to achieve the desired result. Pharmaceutical compositions according to the present disclosure may also be provided in unit dosage forms. Definition of Terms Unless otherwise indicated, the present disclosure will be practiced using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the skill of the art.

[0116] To facilitate understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless specifically defined otherwise herein, all technical and scientific terms or expressions used herein have the meanings commonly understood by those skilled in the art to which the present disclosure pertains. For definitions and terminology in the art, those skilled in the art may refer, at least in part, to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues conform to the standard three-letter and / or one-letter codes used in the art and represent one of the 20 commonly used L-amino acids. Unless otherwise expressly dictated by the context, singular forms used in this specification (including the claims) include their plural forms.

[0117] The term "about," when used in conjunction with a numerical value, is intended to encompass numerical values ​​within a range from a lower limit of 5% less than the stated numerical value to an upper limit of 5% greater than the stated numerical value, including, but not limited to, ±5%, ±2%, ±1%, and ±0.1%, where such variations are suitable for practicing the disclosed methods.

[0118] The term "and / or" should be understood to refer to any one of multiple options or a combination of any two or more of multiple options. The term "or," as used herein, should be understood to have the same meaning as "and / or," as defined above. For example, when items in a list are separated, "or" or "and / or" should be interpreted as being inclusive, i.e., including not only at least one number or one of the list of elements, but also two or more, and optionally additional, unlisted items. Only when counterexample terms, such as "only one," "exactly one," or, as used in the claims, "consisting of," explicitly appear, refer to only one listed number or one element in the list.

[0119] Unless a counterexample explicitly appears in the context, the words "a / an" and "one" as used herein should be interpreted as "at least one." The term "BDCA2" as used herein refers to a type II C-type lectin specifically expressed on plasmacytoid dendritic cells (pDCs). BDCA2 consists of a single extracellular carbohydrate recognition domain (CRD) located at the C-terminus, a transmembrane domain located between the asparagine residue at position 45 and the isoleucine residue at position 213, and a short cytoplasmic tail (without a signaling motif) located at the N-terminus. BDCA2 transmits intracellular signals through the associated transmembrane adaptor FcεRIγ. Antibody-mediated binding of BDCA2 triggers recruitment of spleen tyrosine kinase (SYK) to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) of FcεRIγ. Activation of Syk triggers activation of B-cell linker (BLNK), Bruton's tyrosine kinase (BTK), and phospholipase Cγ2 (PLCγ2), which results in Ca2+ / Ca ... 2+ This brings about a flow of

[0120] Unless otherwise specified, the term "BDCA2" includes human BDCA2, or variants, subtypes, and interspecies homologs of BDCA2 from other species, as well as analogs containing at least one shared epitope of BDCA2. The term encompasses full-length, unprocessed BDCA2 and any form of BDCA2 resulting from intracellular processing. The term encompasses "full-length" unprocessed BDCA2 and any form of BDCA2 resulting from intracellular processing, or any fragment thereof, such as a splice variant or allelic variant. In one embodiment, BDCA2 refers to full-length BDCA2 from human or cynomolgus monkey, or a fragment thereof (e.g., a mature fragment thereof lacking the signal peptide). The terms "anti-BDCA2 antibody," "anti-BDCA2," "BDCA2 antibody," or "antibody targeting BDCA2" refer to an antibody capable of binding to the BDCA2 protein or a fragment thereof with sufficient affinity, e.g., the antibody can be used as a diagnostic and / or therapeutic agent targeting BDCA2.

[0121] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by such cells or the liver, which results in the selective damage, destruction, or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues (in the case of autoimmunity), or pathological inflammation in the human body.

[0122] The term "plasmacytoid dendritic cells (pDCs)" refers to a specialized population of bone marrow-derived cells that secrete type I interferon (IFN) in response to toll-like receptor (TLR) ligands. pDCs are important mediators of cell-mediated immunity, involved in the induction and initiation of immune responses and antigen tolerance. pDCs differ from conventional DCs in that they acquire antigens, for example, via receptor-mediated internalization. Type I interferon produced by pDCs activated by pattern recognition receptors can also present antigens, thereby linking innate and adaptive immune responses. However, excessive activation of pDCs can have adverse effects on the immune response process, potentially leading to, for example, autoimmune diseases.

[0123] In this application, the terms "antibody-drug conjugate" and "ADC" are used interchangeably and generally refer to a binding protein (e.g., an antibody or antigen-binding fragment thereof) chemically linked to one or more chemical drugs (which may optionally be therapeutic or cytotoxic agents). In this application, an antibody may include an antibody or antigen-binding fragment thereof. In this application, a payload moiety may include a glucocorticoid molecule. In certain embodiments, a ligand may be linked to a glucocorticoid molecule via a linker fragment.

[0124] In this application, the term "antibody" generally refers to an immunoglobulin reactive with a specific protein or peptide, or a fragment thereof. The antibody can be of any class, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). The antibody can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibody of the present application can be derived from any species. The term "antibody" can include intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies and any other modified immunoglobulin molecules, so long as the antibody exhibits the desired biological activity.

[0125] In this application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that contains amino acids involved in the specific binding of the antibody to the antigen. The portion of the antigen that is specifically recognized by and binds to an antibody is referred to as an "epitope," as described above. As described above, an antigen-binding domain may generally contain an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, an antigen-binding domain does not necessarily have to contain both. An Fd fragment, for example, has two VH regions and generally retains some of the antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include: (1) Fab fragment: a monovalent fragment having a VL, VH, constant light chain (CL), and CH1 domain; (2) F(ab')2 fragment: a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) Fd fragment: having two VH and CH1 domains; (4) Fv fragment: having the VL and VH domains consisting of a single arm of an antibody; (5) dAb fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli," Nature, Vol. 341:544-546 (1989), incorporated herein by reference in its entirety): having a VH domain; (6) isolated complementarity-determining regions (CDRs); and (7) single-chain Fv (scFv): derived, for example, from an scFV library.However, the two domains (VL and VH) of an Fv fragment are encoded by separate genes and can be linked by recombinant methods using a synthetic linker, which allows them to be prepared as a single protein chain (the pair of VL and VH domains forms a monovalent molecule (known as single-chain Fv (scFv))) (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988)); and (8) "VHH": relates to the variable antigen-binding domain of heavy chain antibodies from the Camelidae family (camel, dromedary, llama, alpaca, etc.) (Nguyen VK et al., 2000, The EMBO Journal, 19, pp. 921-930; Muyldermans S., 2001, J Biotechnol., vol. 74, pp. 277-302; and Vanlandschoot P. et al., 2011, Antiviral Research, vol. 92, pp. 389-407 (see reviews). VHHs are also called nanobodies (Nbs).

[0126] In this application, the term "variable region" or "variable domain" generally refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. In this application, the term "variable" generally means that certain portions of the sequence of antibody variable domains vary significantly, conferring on certain different antibodies the binding and specificity for their particular antigens. The variability is not evenly distributed throughout the antibody variable region. The variability is known as the complementarity-determining regions (CDRs) or hypervariable regions (HVRs), and is concentrated in three segments within the light and heavy chain variable regions, respectively: LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. The more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of naturally occurring heavy and light chains each contain four FRs (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, and L-FR2, L-FR3, L-FR4) that largely adopt a β-sheet configuration. The FRs are connected by three CDR structural loop regions. The CDRs within each chain are held together in close proximity by the FRs and, together with the CDRs from the other chain, form the antigen-binding site of the antibody.

[0127] Various methods have been used in the art to identify antibody numbering schemes, such as the Kabat numbering scheme and definition rules based on sequence variation (see Kabat et al., Sequences of Proteins of Immunological Interest, fifth edition, National Institutes of Health, Bethesda, Md. (1991)), the Chothia numbering scheme and definition rules based on the location of structural loop regions (A1 - see Lazikani et al., J Mol Biol, 273:927-48, 1997), the IMGT numbering scheme by efranc et al., based on the alignment of germline V gene amino acid sequences, and the Honneger (AHo) numbering scheme, the Martin numbering scheme, the Gelfand numbering scheme (see Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue The variable region of the antibody may be encoded by a fragment thereof, or the CDR of the antibody may be split by a fragment thereof (see, for example, "Definition," Front. Immunol. October 16, 2018).

[0128] A "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations (which may be present in minor amounts). Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" reflects the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as referring to production of the antibody by any particular method.

[0129] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally occurring, contains at least four peptide chains, including two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into highly variable complementarity-determining regions (CDRs) and more conserved regions (called framework regions (FRs)) separated by the CDRs. Each VH or VL region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen, and the constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (Clq) of the classical complement system.

[0130] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide also contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0131] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the heavy or light chain variable region. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody may target the same antigen or different antigens.

[0132] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. However, a bivalent antibody can be bispecific. The term "bivalent antibody" refers to a small antibody fragment having two antigen-binding sites and comprising a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains in one chain, the domains are forced to pair with the complementary domains of the other chain to form two antigen-binding sites.

[0133] The term "humanized antibody" refers to forms of antibodies that contain sequences derived from both human and non-human (e.g., murine, rat, etc.) antibodies. Generally, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the framework regions (FR) being those of a human immunoglobulin sequence. The humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

[0134] The "isotype" of an antibody refers to the type of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, and IgG (e.g., IgG1, IgG2, or IgG4, etc.)). Isotypes also include modified forms of one of these types (modified to alter Fc function, e.g., enhanced or weakened effector function or binding to Fc receptors).

[0135] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. In some embodiments, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. (The numbering in this paragraph is based on the EU numbering system, also known as the EU index (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).)

[0136] The term "cross-reactivity" refers to binding to antigenic fragments of the same target molecule originating from humans, monkeys, and / or rodents (mouse or rats). Thus, "cross-reactivity" should be understood as the interspecies reaction between an antigen-binding molecule (e.g., an antibody) and a similar molecule (e.g., BDCA2) expressed in a different species. The cross-reactivity specificity of a monoclonal antibody that recognizes human BDCA2 and monkey and / or rodent (mouse or rat) BDCA2 can be determined by FACS analysis.

[0137] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y is determined by the dissociation rate constant (k dis ) association rate constant (k on ) to the equilibrium dissociation constant (K D) Affinity can be measured by common methods known in the art. In some embodiments of the present disclosure, for example, the affinity between an antibody of the present disclosure and an antigen is measured using surface plasmon resonance (SPR) technology. In some preferred embodiments of the present disclosure, one particular method for measuring affinity is the BIAcore method herein.

[0138] The term "does not bind" to a protein or cell means that it does not bind to the protein or cell or does not bind with high affinity, i.e., 1.0 x 10 -6 M or more, preferably 1.0 × 10 -5 M or more, preferably 1.0 × 10 -4 M or more and 1.0×10 -3 M or more, and more preferably 1.0 x 10 -2 K over M D This means that the molecule binds to a protein or cell through the above structure.

[0139] The term "high affinity" for IgG antibodies refers to a concentration of 1.0 × 10 -6 M or less, preferably 5.0 × 10 -8 M or less, preferably 1.0 × 10 -8 M or less and 5.0 x 10 -9 M or less, preferably 1.0 × 10 -9 K for antigens less than M D For other antibody subtypes, "high affinity" binding may be different. For example, "high affinity" binding for IgM subtypes is defined as 10 -6 M or less, preferably 10 -7 M or less, preferably 10 -8 K below M D Refers to...

[0140] The term "percent amino acid sequence identity" or simply "identity" is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to those in a reference amino acid sequence after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the aligned sequences.

[0141] In this application, the term "glucocorticoid" refers generally to naturally occurring or synthetic steroid hormones that interact with the glucocorticoid receptor.

[0142] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine, and may be, for example, fluorine or chlorine. In this application, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. The alkyl may be substituted or unsubstituted, or may be substituted or unsubstituted. The term "alkyl" generally refers to a straight-chain or branched saturated aliphatic hydrocarbon group having a residue derived from a parent alkane by removing hydrogen atoms from the same carbon atom or from two different carbon atoms, and may be a straight-chain or branched group containing 1 to 20 carbon atoms, e.g., 1 to 12 carbon atoms, e.g., alkyl containing 1 to 6 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. The alkyl may be substituted or unsubstituted, or may be substituted or unsubstituted. For example, when substituted, alkyl can be substituted at any available linkage site with a substituent that can be independently and optionally selected from one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, where the substituent can be, for example, hydrogen, protium, deuterium, tritium, halogen, —NO, —CN, —OH, —SH, —NH, —C(O)H, —COH, —C(O)C(O)H, —C(O)CHC(O)H, —S(O)H, —S(O)H, —C(O)NH, —SONH, —OC(O)H, —N(H)SOH, or C 1~6 It may be an aliphatic group.

[0143] In this application, the term "alkylene" generally refers to a straight-chain or branched saturated aliphatic hydrocarbon group having two residues derived from a parent alkane by removing two hydrogen atoms from the same carbon atom or two different carbon atoms, and alkylene can be a straight-chain or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" can refer to a residue derived from a single carbon atom group by removing two hydrogen atoms. The methylene can be substituted or unsubstituted, or replaced or unsubstituted. For example, alkylene contains 1 to 12 carbon atoms; for example, alkylene contains 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH-), 1,1-ethylene (-CH(CH)-), 1,2-ethylene (-CHCH-), 1,1-propylene (-CH(CHCH)-), 1,2-propylene (-CHCH(CH)-), 1,3-propylene (-CHCHCH-), 1,4-butylene (-CHCHCHCHCH-), 1,5-butylene (-CHCHCHCHCHCH-), etc. Alkylene may be substituted or unsubstituted, or may be substituted or unsubstituted. For example, when substituted, alkylene can be substituted at any available linkage site with a substituent that can be independently and optionally selected from one or more of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, e.g., the substituent can be hydrogen, protium, deuterium, tritium, halogen, —NO, —CN, —OH, —SH, —NH, —C(O)H, —COH, —C(O)C(O)H, —C(O)CHC(O)H, —S(O)H, —S(O)H, —C(O)NH, —SONH, —OC(O)H, —N(H)SOH, or C 1~6It can be an aliphatic group. The methylene or alkylene can be substituted or unsubstituted.

[0144] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where alkyl or cycloalkyl are as defined herein. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. An alkoxy can be optionally substituted or unsubstituted, and if substituted, the substituents are preferably one or more of these groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0145] The term "alkylthio" refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), where alkyl or cycloalkyl are as defined herein. Non-limiting examples of alkylthio include methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, and cyclohexylthio. Alkylthio can be optionally substituted or unsubstituted, and if substituted, the substituents are preferably one or more of these groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0146] In this application, the term "aryl" generally refers to a group having a residue derived from an aromatic ring by removing a hydrogen atom. The term "aromatic ring" can refer to a 6- to 14-membered all-carbon monocyclic ring or a fused polycyclic ring (i.e., rings sharing adjacent pairs of carbon atoms) having a conjugated π-electron system, which can be 6- to 10-membered, such as benzene and naphthalene. The aromatic ring can be fused to a heteroaryl ring, heterocyclyl ring, or cycloalkyl ring, and the ring connected to the parent structure is an aryl ring. The aryl can be substituted or unsubstituted, and if substituted, the substituents can be one or more of these groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. The aryl can be substituted or unsubstituted.

[0147] In this application, the term "heteroaryl" generally refers to a group having a residue derived from a heteroaromatic ring by removing a hydrogen atom from a carbon atom. The term "heteroaryl ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. Heteroaryls can be 5 to 10-membered, or 5 or 6-membered, such as furanyl, thienyl, pyridinyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. Heteroaryl rings can be fused to aryl, heterocyclyl, or cycloalkyl rings, and the ring connected to the parent structure is a heteroaryl ring. Heteroaryl can be optionally substituted or unsubstituted, and if substituted, the substituents can be one or more of these groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Heteroaryl can be substituted or unsubstituted.

[0148] In this application, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyls include spirocycloalkyls, fused cycloalkyls, and bridged cycloalkyls. Cycloalkyls can be substituted or unsubstituted. When a cycloalkyl is substituted, the substitution with a substituent may occur at any available linkage site, and the substituents are preferably independently selected from one or more of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0149] In this application, the term "ring atom" generally refers to an atom included in a ring structure. For example, a ring atom may be a carbon atom in a benzene ring or a nitrogen atom in a pyridine ring. When a hydrogen atom is connected to a ring atom, the ring atom may be substituted or unsubstituted.

[0150] In this application, the terms "independently of each" or "each is independently" generally mean that a variable applies in either case, whether or not there are variables with the same or different definitions in the same compound. For example, a variable can refer to the type or number of substituents in a compound, the type of atom in a compound, etc. For example, if R occurs twice in a compound and R is defined as "independently carbon or nitrogen," then both R can be carbon, both R can be nitrogen, or one R can be carbon while the other R is nitrogen.

[0151] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance can, but does not necessarily, occur, and that the description includes instances when the event or circumstance occurs or does not occur. For example, "a heterocyclyl group is optionally substituted with alkyl" means that alkyl can, but does not necessarily, be present, and that the description can include instances when the heterocyclyl group is substituted with alkyl or is not substituted.

[0152] In this application, the term "substituted" generally means that one or more hydrogen atoms in a group, for example, up to five (e.g., 1 to 3) hydrogen atoms, are each independently replaced with a corresponding number of substituents. Substituents are present only at their possible chemical positions, and those skilled in the art can determine (by experiment or theory) whether substitution is possible or not without undue effort. For example, if an amino or hydroxyl having free hydrogen is bonded to a carbon atom having an unsaturated bond (e.g., an olefin, etc.), the substitution may be unstable.

[0153] In this application, terms such as "alkyl," "alkenyl," and "cycloalkyl," as known to those skilled in the art, may be preceded by a designation that indicates the number of atoms present in the group under certain circumstances, such as C1-C4 alkyl, C3-C7 cycloalkoxy, and C1-C4 alkylcarbonylamino, where the subscript following the "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having 3 carbon atoms (e.g., n-propyl or isopropyl), and C 1~10 In the formula: the group members can have any number of carbon atoms in the range of 1 to 10.

[0154] In this application, "linking" a group X to a group Y generally can be in any orientation, but when group X is used in reference to a linker Y and a group Z, it generally means that two or more linking sites of group X can optionally be linked to either group Y or group Z.

[0155] In this application, the compounds or antibody-drug conjugates of the present application include their tautomers, mesomers, racemates, enantiomers, and / or diastereoisomers. In this application, the term "diastereoisomer" generally refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereoisomers may have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. In this application, the terms "tautomer" and "tautomeric form" are used interchangeably and generally refer to structural isomers that differ in energy and can be interconverted by overcoming a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via recombination of some bonding electrons. In this application, the term "mesomer" generally means that the molecule contains an asymmetric atom, but the overall optical rotation is zero due to the presence of symmetry factors. The terms "racemate" or "racemic mixture" refer to a composition consisting of equimolar amounts of two enantiomeric substances.

[0156] In this application, certain atoms of the compounds of this application may exist in one or more isotopic forms. For example, hydrogen is present in protium ( 1 H), deuterium ( 2 H), and tritium ( 3 H), and carbon can occur as three different isotopes ( 12 C. 13 C, and 14 Examples of isotopes that can be incorporated into the compounds of the present application include: 15 N, 18 O.17 O. 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125 I, or similar isotopes. Thus, one or more of such isotopes may be enriched in the compounds of the present application relative to the natural abundance of such isotopes. Such isotopically enriched compounds can be used for various purposes, as known to those skilled in the art. For example, heavy isotopes, such as deuterium ( 2 Substitution with, for example, deuterium (H) may offer certain therapeutic advantages, possibly due to increased metabolic stability. 2 The natural abundance of hydrogen (H) is about 0.015%. Therefore, about 1 in 6500 hydrogen atoms is a deuterium atom. Therefore, the deuterium abundance at one or more sites (as the case may be) within the deuterium-containing compounds of the present disclosure is greater than 0.015%. Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds having the same structure as those disclosed herein, except that a hydrogen atom is replaced by deuterium or tritium, or a carbon atom is replaced by carbon-13 or carbon-14, are also within the scope of this application.

[0157] In this application, the term "linker" generally refers to any chemical moiety capable of linking a protein (e.g., an antibody, antibody fragment (e.g., antigen-binding fragment), or functional equivalent) to a glucocorticoid. The moiety may be a chemical moiety. In certain embodiments, the linker may be cleavable, thereby facilitating release of the glucocorticoid molecule. For example, the linker may be susceptible to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the glucocorticoid and / or antibody retain activity. In certain embodiments, the linker may be substantially resistant to cleavage.

[0158] The term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds described herein, or physiologically / pharmaceutically acceptable salts or prodrugs thereof, and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism and facilitate absorption of the active ingredient, thereby exerting its biological activity. For the preparation of conventional pharmaceutical compositions, the Chinese Pharmacopoeia can be consulted. Pharmaceutical compositions can be in the form of sterile aqueous injections or oily suspensions for intramuscular and subcutaneous administration. Suspensions can be prepared according to known techniques using suitable dispersing or wetting agents and suspending agents as described above. Sterile preparations for injection can also be sterile solutions or suspensions for injection prepared in non-toxic parenterally acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Additionally, sterile, fixed oils can be conveniently used as solvents or suspending media. For example, any bland fixed oil can be employed including synthetic mono- or diglycerides.In addition, fatty acids such as oleic acid can be used in the preparation of injectables.

[0159] The term "pharmaceutically acceptable salt" generally refers to a salt of a compound or ligand-drug conjugate of the present application, or a salt of a compound described herein. Such salts may be safe and / or effective when used in mammals and may have the required biological activity, and the antibody-drug conjugate compounds of the present application may form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0160] The term "pharmaceutically acceptable carrier" generally refers to a carrier or carrier agent that provides a therapeutic agent, such as an antibody or polypeptide, a gene, or other therapeutic agent. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without causing undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, poly(amino acids), amino acid copolymers, lipid aggregates, and inactivated virus particles. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifying agents, or pH buffering substances, can also be present in such carriers.

[0161] The terms "treatment" and "treating" generally refer to a method of achieving a beneficial or desired result, including, but not limited to, a therapeutic benefit. A therapeutic benefit includes, but is not limited to, eradication, inhibition, reduction, or amelioration of the underlying disorder being treated. Additionally, a therapeutic benefit is achieved by eradicating, inhibiting, reducing, or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, although the patient may still be afflicted with the underlying disorder.

[0162] The terms "prevention" and "preventing" generally refer to a method of achieving a beneficial or desired result, including, but not limited to, a prophylactic benefit. For prophylactic benefit, a pharmaceutical composition may be administered to a patient at risk of developing a particular disease, or to a patient who complains of having one or more physiological symptoms of a disease, even if the patient has not yet been diagnosed with the disease.

[0163] The term "subject" or "patient" generally refers to humans (i.e., male or female within any age group, e.g., a pediatric subject (e.g., an infant, child, or adolescent), or an adult subject (e.g., a young, middle-aged, or elderly)), and / or other primates (e.g., cynomolgus or rhesus monkeys); commercially relevant mammals, including, for example, cattle, pigs, horses, sheep, goats, cats, and / or dogs; and / or commercially relevant birds, including, for example, chickens, ducks, geese, quail, and / or turkeys.

[0164] The terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a ligand-drug conjugate of the present disclosure that, when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents, is effective in preventing or ameliorating one or more symptoms of a disease or condition, or the progression of a disease or condition. A therapeutically effective dose also refers to a dose sufficient to cause an improvement in symptoms, e.g., an amount that treats, cures, prevents, or ameliorate the associated condition, or an amount that promotes the treatment, cure, prevention, or amelioration of such a condition. When an active ingredient is administered alone to an individual, a therapeutically effective amount refers to the amount of the ingredient alone. In the case of co-administration, a therapeutically effective dose refers to the combined amount of the active ingredients that produces a therapeutic effect, regardless of whether such active ingredients are co-administered sequentially or simultaneously. An effective amount of a therapeutic agent causes an increase in a diagnostic index or parameter of at least 10%, generally at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.

[0165] As used herein, the term "disease mediated by BDCA2 or plasmacytoid dendritic cells" refers to a disease in which plasmacytoid dendritic cells are involved in the onset, progression, delay, etc. of the disease, for example, a disease in which activation (e.g., abnormal activation or excessive activation) of plasmacytoid dendritic cells is involved, and in which the direct or indirect involvement of BDCA2 or plasmacytoid dendritic cells results in at least one of the following effects: onset / development of the disease; increase / deepening of pathological changes in the disease; expansion of the site / extent affected by the disease; exacerbation of physical damage caused by the disease; increase in pain due to the disease; insensitivity / resistance of the disease to treatment measures; decreased tendency for self-healing and poor prognosis of the disease. In some embodiments of the present disclosure, the disease mediated by BDCA2 or plasmacytoid dendritic cells is an inflammatory disease, and in certain embodiments of the present disclosure, the disease mediated by BDCA2 or plasmacytoid dendritic cells is systemic lupus erythematosus, discoid lupus erythematosus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type 1 diabetes, dermatomyositis, and / or polymyositis.

[0166] As used herein, the terms "treat," "treating," or "treatment" of any disease or disorder refer, in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting, or reducing the progression of, the disease or at least one of its clinical symptoms). In another embodiment, "treat," "treating," or "treatment" refers to the alleviation or amelioration of at least one physical parameter (including physical parameters that may not be discernible by the patient). In another embodiment, "treat," "treating," or "treatment" refers to modulating the disease or disorder physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both. Except as expressly stated herein, methods for assessing disease treatment and / or prevention are generally known in the art. [Brief explanation of the drawings]

[0167] [Figure 1a] This figure shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human CHOK1-hBDCA2 cells expressing the human BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 1b] This figure shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human CHOK1-hBDCA2 cells expressing the human BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 1c] This figure shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human CHOK1-hBDCA2 cells expressing the human BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 1d] This figure shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human CHOK1-hBDCA2 cells expressing the human BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 1e] This figure shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human CHOK1-hBDCA2 cells expressing the human BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 2a] FIG. 1 shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human 293F-cynoBDCA2 cells expressing the cynomolgus monkey BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 2b]FIG. 1 shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human 293F-cynoBDCA2 cells expressing the cynomolgus monkey BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 2c] FIG. 1 shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human 293F-cynoBDCA2 cells expressing the cynomolgus monkey BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 2d] FIG. 1 shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human 293F-cynoBDCA2 cells expressing the cynomolgus monkey BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 2e] FIG. 1 shows the results of a FACS binding assay of hybridoma antibodies to stably transfected human 293F-cynoBDCA2 cells expressing the cynomolgus monkey BDCA2 molecule, where the antibodies are derived from different hybridomas as indicated in the legend. [Figure 3a] FIG. 1 shows the results of a binding assay by FACS of anti-BDCA2 chimeric antibodies to human CHOK1-hBDCA2 cells, where the antibodies relate to different chimeric antibodies as indicated in the legend. [Figure 3b] FIG. 1 shows the results of a binding assay by FACS of anti-BDCA2 chimeric antibodies to human CHOK1-hBDCA2 cells, where the antibodies relate to different chimeric antibodies as indicated in the legend. [Figure 3c] FIG. 1 shows the results of a binding assay by FACS of anti-BDCA2 chimeric antibodies to human CHOK1-hBDCA2 cells, where the antibodies relate to different chimeric antibodies as indicated in the legend. [Figure 4a]Figure 4 shows internalization capacity assay for humanized antibodies. Figure 4a shows the results for humanized antibodies within the hu005 group assayed at a concentration of 100 nM. [Figure 4b] Figure 4b shows internalization capacity assay for humanized antibodies. Figure 4b shows the results for humanized antibodies within the hu005 group assayed at a concentration of 10 nM. [Figure 4c] Figure 4c shows the internalization capacity assay for humanized antibodies. Figure 4c shows the results for humanized antibodies within the hu033 group assayed at a concentration of 100 nM. [Figure 4d] Figure 4d shows the internalization capacity assay for humanized antibodies. Figure 4d shows the results for humanized antibodies within the hu033 group assayed at a concentration of 10 nM. [Figure 5] Figures 5a and 5b show the inhibition by BDCA2-ADC of CpG-A-induced production of IFN-α and TNF-α from human peripheral blood mononuclear cells. Figure 5a shows the inhibition by BDCA2-ADC of CpG-A-induced IFN-α from human peripheral blood mononuclear cells. Figure 5b shows the inhibition by BDCA2-ADC of CpG-A-induced TNF-α from human peripheral blood mononuclear cells. [Figure 6a] Figure 6a shows the inhibition of R848-induced production of IFN-α and TNF-α from human peripheral blood mononuclear cells by BDCA2-ADC. Figure 6a shows the inhibition of R848-induced IFN-α from human peripheral blood mononuclear cells by BDCA2-ADC. [Figure 6b] Figure 6b shows the inhibition by BDCA2-ADC of R848-induced production of IFN-α and TNF-α from human peripheral blood mononuclear cells. Figure 6b shows the inhibition by BDCA2-ADC of R848-induced TNF-α from human peripheral blood mononuclear cells. [Figure 6c] Figure 6c shows the inhibition of R848-induced production of IFN-α and TNF-α from human peripheral blood mononuclear cells by BDCA2-ADC. Figure 6c shows the inhibition of R848-induced IFN-α production from human peripheral blood mononuclear cells by BDCA2-ADC. [Figure 6d]Figure 6d shows the inhibition by BDCA2-ADC of R848-induced production of IFN-α and TNF-α from human peripheral blood mononuclear cells. Figure 6d shows the inhibition by BDCA2-ADC of R848-induced IFN-α from human peripheral blood mononuclear cells. [Figure 6e] Figure 6e shows the inhibition of R848-induced production of IFN-α and TNF-α from human peripheral blood mononuclear cells by BDCA2-ADC. Figure 6e shows the inhibition of R848-induced TNF-α production from human peripheral blood mononuclear cells by BDCA2-ADC. [Figure 6f] Figure 6f shows the inhibition by BDCA2-ADC of R848-induced production of IFN-α and TNF-α from human peripheral blood mononuclear cells. Figure 6f shows the inhibition by BDCA2-ADC of R848-induced TNF-α from human peripheral blood mononuclear cells. [Figure 7] Figures 7a and 7b show the results of a binding assay of Hu003-03-ADC to cells by FACS. Figure 7a shows the results of a binding assay of Hu003-03-ADC to CHOK1-human BDCA2 cells by FACS. Figure 7b shows the results of a binding assay of Hu003-03-ADC to 293F-cynomolgus monkey BDCA2 cells by FACS. [Figure 8a] Figure 8 shows an internalization ability assay of antibody-drug conjugates: Figure 8a shows the results for Hu003-03-ADC assayed at a concentration of 100 nM. [Figure 8b] Figure 8b shows an internalization ability assay of antibody-drug conjugates. Figure 8b shows the results of Hu003-03-ADC assayed at a concentration of 10 nM. [Figure 8c] Figure 8c shows the internalization ability assay of antibody-drug conjugates. Figure 8c shows the results for Hu003-03-ADC assayed at a concentration of 1 nM. [Figure 8d] Figure 8d shows the internalization ability assay of antibody-drug conjugates, and shows the results for Hu003-03-ADC assayed at a concentration of 100 nM. [Figure 8e] Figure 8e shows the internalization ability assay of antibody-drug conjugates. Figure 8e shows the results for Hu003-03-ADC assayed at a concentration of 10 nM. [Figure 8f] Figure 8f shows the internalization ability assay of antibody-drug conjugates. Figure 8f shows the results for Hu003-03-ADC assayed at a concentration of 10 nM. [Figure 9] Figures 9a and 9b show assays for the bystander killing effect of antibody-drug conjugates. Figure 9a shows the results of fluorescent signal detection in HEK293-human BDCA2 cells and GR reporter gene cells. Figure 9b shows the results of fluorescent signal detection in HEK293 cells that do not express BDCA2 and GR reporter gene cells. [Figure 10] Figures 10a-10c show the percentage of pDC concentration in the peripheral blood of cynomolgus monkeys assayed by FACS. Figure 10a shows the percentage of pDC concentration in the peripheral blood of cynomolgus monkeys in the blank control group. Figure 10b shows the percentage of pDC concentration in the BDCA2-ADC group. Figure 10c shows the percentage of pDC concentration in the peripheral blood of cynomolgus monkeys in the BIIB059 group. [Figure 11] Figures 11a-c show BDCA2 expression levels found on the surface of pDCs in cynomolgus monkey peripheral blood, as assayed by FACS. Figure 11a shows the BDCA2 expression levels found on the surface of pDCs in the blank control group, Figure 11b shows the BDCA2 expression levels found on the surface of pDCs in the BDCA2-ADC group, and Figure 11c shows the BDCA2 expression levels found on the surface of pDCs in the BIIB059 group. [Figure 12]Figures 12a to 12c show the inhibition of CpG-A-induced IFN-α production from cynomolgus monkey peripheral blood mononuclear cells by BDCA2-ADC or BIIB059. Figure 12a shows CpG-A-induced IFN-α from cynomolgus monkey peripheral blood mononuclear cells in the blank control group. Figure 12b shows the inhibition of CpG-A-induced IFN-α from cynomolgus monkey peripheral blood mononuclear cells by BDCA2-ADC. Figure 12c shows the inhibition of CpG-A-induced IFN-α from cynomolgus monkey peripheral blood mononuclear cells by BIIB059. In the figures, *: p<0.05, and **: p<0.01. [Figure 13] Figures 13a-13c show the inhibition of CpG-A-induced TNF-α production from cynomolgus monkey peripheral blood mononuclear cells by BDCA2-ADC or BIIB059. Figure 13a shows CpG-A-induced TNF-α from cynomolgus monkey peripheral blood mononuclear cells in the blank control group. Figure 13b shows the inhibition of CpG-A-induced TNF-α from cynomolgus monkey peripheral blood mononuclear cells by BDCA2-ADC. Figure 13c shows the inhibition of CpG-A-induced TNF-α from cynomolgus monkey peripheral blood mononuclear cells by BIIB059. DETAILED DESCRIPTION OF THE INVENTION

[0168] The present disclosure includes all combinations of the specific embodiments described. Further embodiments and the full scope of applicability of the present disclosure will become apparent from the Detailed Description provided below. However, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from the Detailed Description, it should be understood that the Detailed Description and specific examples (which illustrate preferred embodiments of the disclosure) are provided for illustrative purposes only. All publications, patents, and patent applications cited herein, including the cited references, are hereby incorporated by reference in their entirety for any purpose.

[0169] In the following examples, if no specific conditions are described for the experimental procedures, they were carried out according to conventional procedures and conditions or according to the product instructions. [Example]

[0170] The present disclosure is described in more detail below by reference to examples and experimental examples which are not intended to limit the scope of the disclosure and which can be modified without departing from the scope of the disclosure. Example 1. Anti-BDCA2 antibody In this study, mice were immunized with the BDCA2 antigen to obtain hybridoma cells, and suitable single-cell clones were screened to generate mouse antibodies with high affinity for BDCA2. After sequencing the obtained mouse antibodies, the hybridoma-derived immunoglobulin heavy and light chain variable regions were operably linked to human IgG constant regions to obtain chimeric antibodies, which were further humanized and subjected to post-translational modification removal to obtain anti-BDCA2 antibodies (Hu033-03, Hu033-20, and Hu005-04).

[0171] Example 1.1. Preparation of Recombinant Proteins to Assay for Anti-BDCA2 Antibody Activity The cDNA sequence encoding the extracellular domain (Asn45-Ile213) of human BDCA2 (hBDCA2) was synthesized using Genscript® based on the reference sequence in Genbank. The amplified fragment was cloned into a eukaryotic expression plasmid (pCP-EF1a-CMV-FcεRIγ) containing a human Fc fragment (hIgG1) constructed in-house using conventional cloning techniques, resulting in the recombinant fusion protein expression plasmid, human BDCA2Fc (pCP-EF1a-BDCA2-CMV-FcεRIγ). The expression plasmid was expressed in 293E cells and purified using a particle size exclusion chromatography column to obtain the human BDCA2Fc recombinant protein (containing the Fc region located N-terminally to the BDCA2 extracellular domain fused via a linker). The recombinant protein was identified by polyacrylamide gel electrophoresis.

[0172] A cDNA sequence encoding the extracellular domain (Asn45-Ile212) of cynomolgus monkey BDCA2 (CynoBDCA2) was synthesized using Genscript based on the reference sequence in Genbank, and a six-His sequence was added to the N-terminus of the cDNA. The fragment was cloned into an in-house constructed eukaryotic expression plasmid system (pCP-EF1a-CMV-FcεRIγ) using conventional cloning techniques, thereby generating the recombinant fusion protein expression plasmid CynoBDCA2His (pCP-EF1a-CynoBDCA2-CMV-FcεRIγ). The expression plasmid was expressed in 293E cells and purified using a particle size exclusion chromatography column to obtain the CynoBDCA2His recombinant protein, which was then identified by polyacrylamide gel electrophoresis. After identification, the concentration, total amount, and endotoxin content of the two proteins obtained were suitable for further use.

[0173] The amino acid sequence of human BDCA2Fc is represented by SEQ ID NO: 27, and the protein sequence of human BDCA2 is set forth under NCBI accession number Q8WTT0; the amino acid sequence of CynoBDCA2His is represented by SEQ ID NO: 28, and the protein sequence of CynoBDCA2 is set forth under NCBI accession number A0A2K5UWP4-1. All sequences were used in assays for antibody activity of the present disclosure.

[0174] The sequence of the reference antibody BIIB059, which specifically binds to BDCA2, was derived from US Patent No. 9902775B2, with the heavy chain sequence corresponding to patent SEQ ID NO: 4 and the light chain sequence corresponding to patent SEQ ID NO: 3. They were produced by using conventional recombinant antibody production processes.

[0175] Example 1.2. Construction of BDCA2-expressing cell lines to assay for activity of anti-BDCA2 antibodies The cDNA sequence encoding human BDCA2 was synthesized by Genscript based on the reference sequence in Genbank. The amplified fragment was cloned into an in-house constructed lentiviral packaging plasmid system (PLvx-EF1a-CMV-hFcεRIγ-IRES-puro) using conventional cloning techniques, thereby generating the lentiviral packaging plasmid human BDCA2 (PLvx-EF1a-hBDCA2-CMV-hFcεRIγ-IRES-puro).

[0176] The cDNA sequence encoding cynomolgus monkey CynoBDCA2 was synthesized by Genscript based on the reference sequence in Genbank. The amplified fragment was cloned into an in-house constructed lentiviral packaging plasmid system (PLvx-EF1a-CMV-hFcεRIγ-IRES-puro) using conventional cloning techniques, resulting in the lentiviral packaging plasmid CynoBDCA2 (PLvx-EF1a-cynoBDCA2-CMV-cynoFcεRIγ-IRES-puro). 293T cells were used as host cells, and the PLvx-EF1a-hBDCA2-CMV-hFcεRIγ-IRES-puro plasmid or PLvx-EF1a-cynoBDCA2-CMV-cynoFcεRIγ-IRES-puro plasmid was transiently transfected into 293T cells. Cell culture supernatants containing human BDCA2- or CynoBDCA2-expressing viruses were collected 48 and 72 hours after transfection and concentrated to 1E8 EU / mL.

[0177] CHOK1 cells, previously cultured to logarithmic growth phase, were digested and seeded into 6-well cell culture plates at 1E5 cells / well and cultured overnight to allow the cells to adhere to the cell walls. Subsequently, increasing infectious doses of concentrated human BDCA2 virus (100 μL and 200 μL) were added to the cells pre-adhered to the 6-well plates, and infected cells were screened using culture medium containing puromycin. Antigen expression levels were assayed by flow cytometry, and the cells were subcloned. Flow cytometry was performed using BIIB059 as the reference antibody for the assay, and the results showed significant binding to BDCA2 compared to the control hIgG. A single clone with a high antigen expression level was selected and subjected to expansion culture to serve as a cell line for subsequent experiments.

[0178] 293F cells pre-cultured to logarithmic growth phase were digested and seeded into 6-well cell culture plates at 1E5 cells / well and cultured overnight to allow cells to adhere to the cell walls. Subsequently, increasing infectious doses of concentrated CynoBDCA2 virus (100 μL and 200 μL) were added to the pre-adhered cells in the 6-well plates, and infected cells were screened using culture medium containing puromycin. Antigen expression levels were assayed by flow cytometry, and the cells were subcloned. Flow cytometry was performed using BIIB059 as the reference antibody for the assay, and the results showed significant binding to BDCA2 compared to the control hIgG. A single clone with a high antigen expression level was selected and subjected to expansion culture to serve as a cell line for subsequent experiments.

[0179] Example 1.3. Preparation of mouse hybridoma cells 1.3.1. Mouse Immunization and Serum Titer Assay 1.3.1.1. Mouse Immunization Regimen A total of three groups of mice (five mice per group) were immunized by two immunization routes, with a 14-day interval between immunizations. Group 1 included five Balb / c mice (8-week-old female BALB / c mice purchased from SLAC), and group 2 included five SJL mice (8-week-old female BALB / c mice purchased from SLAC). Mice were immunized five times with protein (human BDCA2Fc) using a combined gene gun (pCP-EF1a-BDCA2-CMV-FcεRIγ) regimen. The first four immunizations were gene gun immunizations at a dose of 4 μg / injection, and the fifth immunization was protein immunization at a dose of 25 μg / mouse. In Group 3, five SJL mice (purchased from SLAC, female, 8 weeks old) were immunized with pure protein (human BDCA2Fc) for a total of three immunizations, the first at a dose of 50 μg / mouse and the next two at a dose of 25 μg / mouse (see Table 1).

[0180] [Table 3]

[0181] 1.3.1.2. Mouse Immune Titer Assay Serum samples from immunized mice were collected at various time points and assayed by ELISA and FACS. Based on the results of several assays (the final mouse serum assay (TB) result was taken as the main reference factor, detailed results are not shown), four mice were selected and two fusions were carried out: for fusion #F0223, two mice were selected, Balb / c #9735 with the highest fluorescence intensity in group G1 and SJL #9740 with the highest fluorescence intensity in group G2; for fusion #F0511, two mice were selected, SJL #9947 and SJL #9948 with the highest fluorescence intensity in group G3. 1.3.2. Cell fusion Four days after the final booster immunization, the mice were sacrificed, and spleen cells were harvested and mashed in normal saline. The lymphocyte-rich suspension was collected and mixed with mouse myeloma cells Sp2 / 0 using a conventional electrotransfection method. The cells were fused by an efficient electrofusion method.

[0182] The fused cells were diluted in DMEM culture medium containing HT (hypoxanthine and thymidine), proportionally seeded into 96-well plates, and cultured overnight in a 37°C incubator containing 5% CO2. After 24 hours, DMEM culture medium containing 2x HAT (hypoxanthine, amethopterin, and thymidine) was added to screen for successfully fused cells (hybridoma cells). The MEM complete culture medium formulation was as follows: 15% FBS (fetal bovine serum) + 1:50 L-glutamine + 100 U / mL penicillin-streptomycin + 1:100 OPI (oxaloacetate, pyruvate, and insulin). The incubator conditions were 8% CO2 and 37°C.

[0183] Example 1.4. Screening of mouse hybridoma cells and purification of mouse anti-BDCA2 antibodies The fused cells were cultured and screened for approximately 10 days, after which the expression of BDCA2 antibody in the supernatant was assayed using an Acumen laser cytometer. In the assay, CHOK1-human BDCA2 was used as the positive cells, CHOK1-blank was used as the negative cells, and the reference antibody BIIB059 was used as the positive control antibody. Positive clones were selected based on the Acumen results and transferred to 24-well plates for expansion culture. After 3 days of culture, the growth culture supernatant in the 24-well plate was collected and assayed by the following method: 1) The binding activity to CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells was assayed by FACS; 2) The binding activity to human BDCA2Fc protein and cynoBDCA2His protein was assayed by ELISA; and 3) Inhibitory capacity (inhibition of IFNα production) was assayed in experiments in which PBMCs were stimulated with CpG-A to produce the cytokine IFNα.

[0184] Among the polyclonal hybridoma cells obtained by F0223 fusion, 189 hybridoma cell lines were screened using Acumen, and the antibodies secreted from them were found to specifically bind to hBDCA2. The 189 binding-positive hybridoma cell lines were further transferred to 24-well plates for expansion and rescreening. Antibodies expressed by 28 hybridoma cell lines were found to bind to hBDCA2 and cynoBDCA2 in ELISA assays and to cell surface-expressed hBDCA2 and cynoBDCA2 in FACS. In IFNα blocking experiments, the 28 polyclonal cell lines were found to inhibit CpG-induced IFNα secretion from PBMC cells. The 28 hybridoma cell lines were subcloned. Eighteen blocking monoclonal cell lines were obtained by screening the subclones and numbered MAb001-mAb018.

[0185] Among the polyclonal hybridoma cells obtained by F0511 fusion, 168 hybridoma cell lines were screened using Acumen, and the antibodies secreted from them were found to specifically bind to hBDCA2. The 168 binding-positive hybridoma cell lines were further transferred to 24-well plates for expansion and rescreening. Antibodies expressed by 35 hybridoma cell lines were found to bind to hBDCA2 and cynoBDCA2 in ELISA assays and to cell surface-expressed hBDCA2 and cynoBDCA2 in FACs. In IFNα blocking experiments, the 35 polyclonal cell lines were found to inhibit CpG-induced IFNα secretion from PBMC cells. The 35 hybridoma cell lines were subcloned. Sixteen blocking monoclonal cell lines were obtained by screening the subclones and numbered mAb019-mAb034.

[0186] A total of 34 positive single clones were finally obtained through two rounds of fusion and screening, and the secreted antibodies were purified and analyzed. The antibody concentration, quality, purity, and endotoxin content met the requirements for subsequent experiments.

[0187] Example 1.5. Determining the Performance of Anti-BDCA2 Mouse Antibodies The obtained mouse antibody had good binding activity to human BDCA2Fc protein, and the binding affinity EC 50 The murine antibodies had relatively good binding capacities to the cynoBDCA2His protein, and most of the antibodies had binding capacities ECs of less than 0.5 nM when assayed by ELISA. 50 had.

[0188] All mouse antibodies bound to CHOK1-human BDCA2 cells (see Figures 1a-1e for results) with EC values ​​superior to or comparable to the control antibody BIIB059.50 Most of the murine antibodies bound to 293F-cynoBDCA2 cells (see Figures 2a-e for results) and had EC values ​​that were superior to or comparable to the control antibody BIIB059. 50 had.

[0189] Thirty-two antibodies that bind to both CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells were assayed for their ability to inhibit the release of the cytokine IFNα in experiments in which PBMCs were stimulated with CpG-A. Each of these antibodies had a given inhibitory activity. Among them, the IC values ​​of mAb005, mAb019, mAb020, mAb021, mAb022, mAb024, mAb027, mAb030, and mAb033 for cytokine release inhibition were 0.01. 50 The values ​​were 0.08667 nM, 0.003323 nM, 0.006146 nM, 0.004047 nM, 0.006232 nM, 0.002744 nM, 0.07004 nM, 0.004762 nM, and 0.004083 nM, respectively.

[0190] The experimental procedures are as follows: among them, mAb001-mAb034 are mouse antibodies of the present disclosure, BIIB059 is the reference antibody, hIgG1 is a human IgG1 negative control, and mIgG1 is a mouse IgG1 negative control. 1.5.1. Assay of Binding Activity of Mouse Antibodies to Proteins by ELISA Human BDCA2Fc protein or cynoBDCA2His protein was diluted to 1 μg / mL in PBS and added to the ELISA plate at 100 μL / well. The plate was incubated overnight at 4°C. The plate was blocked for 2 hours at 37°C with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4; percentages are by weight). Antibodies were added at 10-fold dilutions to a total of eight concentration points (0.0001 nM to 100 nM, including a 0 nM control). The antibodies were added at 100 μL / well and the plate was incubated for 1 hour at 37°C. The plate was washed three times and anti-mouse IgG (Fab specific)-HRP (Sigma, A3682; 1:5000 dilution) secondary antibody was added at 100 μL / well. The plate was incubated for 1 hour at 37°C. The plate was washed three times, and 100 μL / well of TMB substrate (Huzhou InnoReagents Co., Ltd., EL0009) was added. The plate was incubated at 37°C for 10 minutes, and then 50 μL of 1N hydrochloric acid was added to stop the color reaction. The OD450nm values ​​were read on an ELISA plate reader, and curve fitting was performed using GraphPad Prism6 to calculate the EC 50 values ​​were calculated. 1.5.2. Assay of Binding Activity of Mouse Antibodies to Cells by FACS CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells cultured to logarithmic growth phase were digested with TrypLE trypsin. Cells were harvested and diluted to 2 × 10 cells in FACS buffer (PBS + 2% FBS). 6The cells were resuspended at 1:100 / mL. Cells were added to a 3799 cell plate (Corning) at 100 μL / well, centrifuged at 300 g, and the supernatant was discarded. The antibodies were serially diluted using FACS buffer (same as above), and the diluted antibodies were added to the centrifuged cells. The cells were resuspended and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. 100 μL of donkey anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Invitrogen, A21202; 1:1000) secondary antibody was added. The cells were incubated at 4°C for 1 hour, washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS (BD FACS Canto™ II) instrument. Curve fitting was performed using GraphPad Prism 6, and EC 50 values ​​were calculated. 1.5.3. Inhibitory Ability of Murine Antibodies to Release Cytokine IFN-α from Human PBMC Cells Stimulated with CpG-A 1) Cryopreserved human PBMC cells were thawed and incubated overnight for 18 hours in complete culture medium (1640 + 10% FBS + 1x NEAA + 1x L-glutamine). The next day, PBMC cells were collected, centrifuged at 600g for 8 minutes, and the supernatant was discarded. Cells were cultured at a concentration of 8x10 cells in culture medium (1640 + 10% FBS). 6 The antibody was resuspended at 100 μL / well in 3799 cell culture plates. The test antibody was diluted 10-fold in culture medium (1640 + 10% FBS) to a final concentration of 0.0001 nM to 10 nM, and 50 μL of the diluted antibody was added to PBMCs at 50 μL / well. The cells were resuspended and then incubated at 37°C for 6 hours. 50 μL of CpG-A (2 μL, Invitrogen, tlrl-2216-5) diluted in culture medium (1640 + 10% FBS) was added to each well, mixed, and then incubated at 37°C for an additional 20 hours. On day 3, the cell culture plate was centrifuged at 400 g for 5 minutes, and the cell culture supernatant was collected and assayed for the cytokine IFNα in the supernatant. 2) Cytokine IFNα assay by ELISA Antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) was diluted to 4 μg / mL in PBS and added to a CORNING ELISA plate at 100 μL / well. The plate was incubated and coated overnight at 4°C. The coating antibody was then discarded, and blocking solution (PBS + 1% BSA) was added to the plate at 300 μL / well, followed by incubation at 4°C overnight. The test supernatant was diluted to the appropriate concentration (selected from 1:3 to 1:7 as appropriate) in the blocking solution and added to the coated ELISA plate at 100 μL / well. IFNα assay standards were serially diluted two-fold in the blocking solution (up to 1000 pg / mL; 8 concentration points; the final concentration point was 0) and added to the coated ELISA plate at 100 μL / well. The ELISA plate was incubated in an incubator at 37°C for 1 hour. The plate was washed three times, and 100 μL / well of assay antibody MT2 / 4 / 6 (1:1000) was added. The plate was incubated for 1 hour at 37°C in an incubator. The plate was washed three times, and 100 μL / well of secondary antibody SA-HRP (1:1000) was added. The plate was incubated for 0.5 hours at 37°C in an incubator. The plate was washed three times, and 100 μL / well of TMB substrate solution was added. After the appropriate period of color development, the reaction was stopped by adding 50 μL / well of 1M HCl. The OD at 450 nm was read on an ELISA plate reader. Percent inhibition was calculated, and curve fitting was performed using GraphPad Prism 6 to determine the IC. 50 values ​​were calculated.

[0191] Example 1.6. Determination of variable region sequences of anti-BDCA2 mouse hybridoma (monoclonal antibody) (based on Kabat) Based on the results of the binding ability of the mouse antibodies to the antigen at the protein and cellular levels, and the ability of the antibodies to inhibit the release of the cytokine IFNα stimulated by CpG-A in a PBMC system, 16 hybridoma single clones were selected for VH / VL sequencing.

[0192] The DNA coding sequence corresponding to the variable region of the anti-BDCA2 mouse antibody was sequenced using a method based on degenerate primer PCR. Candidate hybridoma cells were cultured and collected by centrifugation at 1000 rpm, and total RNA was extracted with Trizol. First-strand cDNA was synthesized using total RNA as a template, and then the DNA coding sequence corresponding to the variable region was amplified by PCR using the first-strand cDNA as a template. The primer sequences used in the amplification reaction were complementary to the first framework region and constant region of the antibody variable region (Larrick, JW et al., 1990, Scand. J. Immunol., 32, 121-128; and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). To a 50-μL reaction mixture, 1 μL of cDNA, 5 μL of 10x PCR buffer, 1 μL (25 pmol) of each upstream and downstream primer, 1 μL of dNTPs, 1 μL of 25 mmol of PLMgCl2, and 39 μL of HO were added. The mixture was pre-denatured at 95°C for 10 minutes, and 1 μL of Taq enzyme was added before temperature cycling for PCR amplification. The reaction conditions were denaturation (94°C for 1 minute), annealing (58°C for 1 minute), and extension (72°C for 15 seconds), for a total of 32 cycles, followed by incubation (72°C for 10 minutes). The PCR product was recovered and purified. The amplified product was sequenced to obtain the amino acid sequences of the heavy and light chain variable regions of the anti-BDCA2 mouse antibody.

[0193] NCBI Ig-Blast (http: / / www.ncbi.nlm.nih.gov / project / igblast / ) was used to search for consensus sequences within germline and rearranged Ig variable region sequence databases. Amino acid sequences of complementarity-determining regions (CDRs) were identified by sequence annotation and internet-based sequence analysis (http: / / www.imgt.org / IMGT_vquest / vquest and http: / / www.ncbi.nlm.nih.gov / igblast / ) based on the Kabat system (Wu, TT and Kabat, EA 1970, J. Exp. Med., 132:211-250) and the IMGT system (Lefranc M.-P. et al., 1999, Nucleic Acids Research, 27:209-212).

[0194] The selected anti-BDCA2 murine antibodies were subjected to multiple sequence alignment and phylogenetic tree classification. Based on the results, the heavy chain variable region (VH) sequences of the 16 monoclonal antibodies were divided into five main categories: 1) mAb020, 022, and 026; 2) mAb021, 027, 028, 030, 032, and 034; 3) mAb019, 024, and 033; 4) mAb001 and 002; and 5) mAb005 and 016. mAb028 and mAb030 differ by only two amino acids in the heavy chain variable region, and mAb028 and mAb032 differ by only one amino acid. The light chain variable region VL can be divided into three categories: 1) mAb 019; 2) mAbs 021, 027, 028, 030, 032, 034; and 3) other antibodies, with the sequences of the light chain variable regions of mAbs 028, 030, and 032 being identical.

[0195] Example 1.7. Construction of anti-BDCA2 chimeric antibodies Based on the sequence detection results, 12 monoclonal antibody sequences were selected from the two fusions and used to construct human-mouse chimeric antibodies. The sequence of the hIgG1 Fc segment was selected as the human constant region sequence during plasmid construction. The heavy and light chain variable region coding sequences of the above-mentioned anti-BDCA2 mouse antibody were synthesized using Genscript. Expi293F cells were selected as host cells for antibody expression. The resulting chimeric antibodies were mab001c, mab002c, mab005c, mab016c, mab019c, mab020c, mab021c, mab022c, mab024c, mab027c, mab030c, and mab033c.

[0196] Various characterizations were performed on the expressed chimeric antibodies. Example 1.8. Screening for chimeric antibodies 1.8.1. Binding Activity of Anti-BDCA2 Chimeric Antibodies to Proteins Assayed by ELISA Human BDCA2Fc protein or cynoBDCA2His protein was diluted to 1 μg / mL in PBS and added to the ELISA plate at 100 μL / well. The plate was incubated overnight at 4°C. After coating, the plate was blocked for 2 hours at 37°C with ELISA blocking solution (PBS phosphate buffer containing 1% BSA, pH 7.4; percentages are by weight). A total of eight concentration points (0.0001 nM to 100 nM, including a 0 nM control) were added, with 100 μL / well of antibody. The plate was washed three times and anti-human IgG (Fab specific)-peroxidase antibody (Sigma, A0293; 1:5000 dilution) secondary antibody was added at 100 μL / well. The plate was incubated for 1 hour at 37°C. The plate was washed three times, and 100 μL / well of TMB substrate solution (Huzhou InnoReagents Co., Ltd., EL0009) was added. The plate was incubated at 37°C for 10 minutes, and then 50 μL of 1N hydrochloric acid was added to stop the reaction. OD450nm values ​​were read on an ELISA plate reader, and curve fitting was performed using GraphPad Prism6 to calculate EC 50 values ​​were calculated.

[0197] The results are as follows: the test chimeric antibodies bound to the human BDCA2Fc protein and the cynoBDCA2His protein, and the EC 50 values ​​ranged from 0.1 to 0.2 nM; most chimeric antibodies had an EC 50 The cynoBDCA2His protein was bound to the cynoBDCA2His protein. 1.8.2. Binding activity of chimeric antibodies to cells assayed by FACS.

[0198] CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells cultured to logarithmic growth phase were digested with TrypLE trypsin. Cells were harvested and diluted to 2 × 10 in FACS buffer (PBS + 2% FBS). 6 The cells were resuspended at 1:100 cells / mL. 100 μL / well of the cells were added to a 3799-cell plate, centrifuged at 300 g, and the supernatant was discarded. The antibodies were serially diluted in FACS buffer and added to the centrifuged cells. The cells were resuspended and incubated at 4°C for 1 hour. The incubated cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. 100 μL of goat anti-human IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Sigma, A11013; 1:1000) secondary antibody was added. The cells were incubated at 4°C for 1 hour, washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS (BD FACS Canto™ II) instrument. Curve fitting was performed using GraphPad Prism 6, and EC 50 values ​​were calculated.

[0199] As shown in Figure 3 (Figures 3a-c), none of the assayed chimeric antibodies showed EC2 activity on CHOK1-human BDCA2 cells that was superior to or comparable to the control antibody BIIB059. 50 On the other hand, all of the chimeric antibodies assayed bound with an EC of approximately 0.3-2 nM. 50 The antibody bound to 293F-cynoBDCA2 cells with values ​​(better than or comparable to the control antibody BIIB059). 1.8.3. Inhibitory Ability of Chimeric Antibodies on Cytokine IFNα Release from Human PBMC Cells Stimulated with CpG-A Cryopreserved human PBMC cells were thawed and incubated overnight for 18 hours in complete culture medium (1640 + 10% FBS + 1x NEAA + 1x L-glutamine). The next day, PBMC cells were collected, centrifuged at 600g for 8 minutes, and the supernatant was discarded. Cells were diluted to 8x10 cells in culture medium (1640 + 10% FBS). 6The antibody was resuspended at 100 cells / mL and the mixture was added to a 3799 cell culture plate at 100 μL / well. The test antibody was serially diluted in 10-fold increments (final concentrations of 0.00011 nM to 10 nM) with culture medium (1640 + 10% FBS), and the diluted antibody was added to PBMCs at 50 μL / well. The cells were resuspended and then incubated at 37°C for 6 hours. CpG-A (2 μM, Invivogen, tlrl-2216-5, 50 μL) diluted in culture medium (1640 + 10% FBS) was added to each well, mixed, and then incubated at 37°C for an additional 20 hours. On day 3, the cell culture plate was centrifuged at 400 g for 5 minutes, and the cell culture supernatant was collected and assayed for the cytokine IFNα in the supernatant.

[0200] Antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) was diluted to 4 μg / mL in PBS and added to Corning ELISA plates at 100 μL / well. The plates were incubated overnight at 4°C. The coated antibody was discarded, and blocking solution (PBS + 1% BSA) was added to the plates at 300 μL / well, followed by incubation at 4°C overnight. Test supernatants were diluted to the appropriate concentrations (typically 1:3 to 1:7) in blocking solution and added to the coated ELISA plates at 100 μL / well. IFNα assay standards were serially diluted 2-fold in blocking solution (up to 1000 pg / mL; 8 concentration points; the final concentration point was 0) and added to the coated ELISA plates at 100 μL / well. The ELISA plates were incubated in an incubator at 37°C for 1 hour. The plate was washed three times and the assay antibody MT2 / 4 / 6 (1:1000) was added at 100 μL / well. The plate was incubated in an incubator at 37°C for 1 hour. The plate was washed three times and the secondary antibody SA-HRP (1:1000) was added at 100 μL / well. The plate was incubated in an incubator at 37°C for 0.5 hours. The plate was washed three times and TMB substrate solution was added at 100 μL / well. After the appropriate period of color development, 1 M HCl was added at 50 μL / well. The OD450nm reading was read on an ELISA plate reader. Percent inhibition was calculated and curve fitting was performed using GraphPad Prism 6 to determine the IC. 50 was calculated.

[0201] In experiments in which PBMCs were stimulated with CpG-A to release cytokines, all 12 chimeric antibodies assayed inhibited the secretion of the cytokine IFNα, with IC values ​​of mab005c, mab019c, mab020c, mab021c, mab022c, mab024c, mab027c, mab030c, and mab033c being significantly higher than those of mab005c. 50The values ​​were 0.008359 nM, 0.01493 nM, 0.01853 nM, 0.01268 nM, 0.03239 nM, 0.01677 nM, 0.008976 nM, 0.01645 nM, and 0.008331 nM, respectively. 1.8.4. Assays for Internalization Ability of Chimeric Antibodies CHOK1-human BDCA2 cells cultured to logarithmic growth phase were digested with TrypLE trypsin. Cells were harvested and diluted to 2 × 10 cells in FACS buffer (PBS + 2% FBS). 6 The cells were resuspended at 100 μL / mL. 100 μL of the cells were added to a 3799-cell plate, centrifuged at 300 g, and the supernatant was discarded. The cells were placed in an ice-cooled box and cooled. The antibody was serially diluted with FACS buffer (3 concentrations: 100 nM, 10 nM, and 1 nM; for each concentration, three groups were set: control (0 h); 1 h at 4°C; and 1 h at 37°C). After dilution, the antibody was placed in an ice-cooled box and cooled for 15 minutes. After 15 minutes, the antibody was added to the centrifuged cells, resuspended, and incubated at 4°C for 40 minutes. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. Cells in the 0 h group were fixed with paraformaldehyde at room temperature for 10 minutes and washed twice with FACS buffer. The cells in the other two groups were resuspended in 100 μL of FACS buffer and incubated separately at 4°C and 37°C for 1 hour. The cells in the two groups, incubated at 4°C for 1 hour and at 37°C for 1 hour, were fixed with paraformaldehyde for 10 minutes at room temperature and washed twice with FACS buffer. 100 μL of secondary antibody, i.e., goat anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000), was added separately to the cells in group 3, and the cells were incubated at 4°C for 1 hour. The cells were washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS instrument. The percentage of surface signal was calculated.

[0202] All chimeric antibodies demonstrated internalization at concentrations of 100 nM, 10 nM, and 1 nM. The 12 chimeric antibodies showed slight differences in their ability to mediate internalization, with some chimeric antibodies being slightly better than the positive control antibody BIIB059. At an antibody concentration of 100 nM, the antibodies demonstrated 50% to 60% of the cell surface signal after 1 hour of internalization at 37°C.

[0203] Example 1.9. Humanization engineering of antibody variable regions For humanized engineering of antibody variable regions, human germline IgG genes homologous to the cDNA sequences of mouse antibodies were searched in the NCBI human immunoglobulin gene database (http: / / www.ncbi.nlm.nih.gov / igblast / ), and the amino acid sequences and precise boundaries of the variable region CDRs were defined using the Kabat or IMGT numbering system. In principle, human IGHV, which has high homology to mouse antibodies, was selected as a template for humanization, and the antibody variable regions were humanized by CDR grafting.

[0204] Based on the sequences of the mouse antibodies obtained above, mAb005 and mAb033 were selected for humanization. The humanization engineering process included the following steps: A. aligning the gene sequences of the mouse antibodies with those of human germline antibodies to find highly homologous sequences; B. analyzing and investigating HLA-DR affinity and selecting human germline framework sequences with low affinity; and C. analyzing the framework amino acid sequences of the variable regions and their surrounding areas by using computer simulation techniques and applying molecular docking to investigate their spatial and steric complexation modes. Each of the key amino acids that may interact with human BDCA2 and maintain the spatial framework within the gene sequences of the mouse antibodies was analyzed by calculating electrostatic forces, van der Waals forces, hydrophilicity and hydrophobicity, and entropy values, and then grafted onto the selected human germline gene framework. The amino acid sites in the framework regions that needed to be preserved were mapped. The humanized antibodies were then synthesized. Based on this, various humanized antibody variable region sequences were obtained, and various humanized anti-BDCA2 antibodies were obtained by combining the designed humanized anti-BDCA2 antibody variable regions in various ways.

[0205] Example 1.10. Screening for humanized anti-BDCA2 antibodies 1.10.1. Binding Activity of Humanized Antibodies to BDCA2 CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells cultured to logarithmic growth phase were digested with TrypLE trypsin. Cells were harvested and diluted to 2 × 10 cells in FACS buffer (PBS + 2% FBS). 6The cells were resuspended at a concentration of 100 μL / well. 100 μL of the resuspended cells were added to a 3799 cell plate, centrifuged at 300 g, and the supernatant was discarded. The test antibody was diluted 5-fold in FACS buffer (final concentrations of 0.0122 nM to 200 nM) and added to the centrifuged cells. The cells were resuspended and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. 100 μL of goat anti-human IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Sigma, A11013; 1:1000) secondary antibody was added. The cells were incubated at 4°C for 1 hour, washed twice with FACS buffer, and resuspended in PBS. The fluorescent signals of the antibodies for binding to the cell surface were analyzed using a FACS (BD FACS Canto™ II) instrument, and curve fitting was performed using GraphPad Prism 6 to determine the EC values ​​of the antibodies for binding to the BDCA2 antigen. 50 The values ​​were calculated. BIIB059 antibody was selected as a positive control and hIgG1 was selected as a negative control.

[0206] The binding activity of the candidate antibodies obtained by applying humanized engineering of the mAb033 sequence to CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells was superior to that of the positive control antibody BIIB059. The results are shown in Table 2. The affinity of the antibodies for human BDCA2 was determined by SPR, and the selected antibodies had stronger affinity for human BDCA2 than the positive control antibody BIIB059.

[0207] [Table 4]

[0208] EC of candidate antibodies obtained by applying human engineering of mAb005 sequence to CHOK1-human BDCA2 cells 50The values ​​were superior to those of the positive control antibody BIIB059. The results are shown in Table 3. Meanwhile, the candidate antibody obtained by humanization engineering of the mAb005 sequence also had relatively good binding activity to 293F-cynoBDCA2 cells.

[0209] [Table 5]

[0210] 1.10.2. Inhibitory Ability of Humanized Antibodies on Cytokine IFNα Release from Human PBMC Cells Stimulated with CpG-A A series of humanized candidate antibody molecules were selected and functionally tested for the inhibition of cytokine IFNα release from human PBMC cells stimulated with CpG-A. Cryopreserved human PBMC cells were thawed and incubated overnight for 18 hours in complete culture medium (1640 + 10% FBS + 1x NEAA + 1x L-glutamine). The next day, PBMC cells were collected and centrifuged at 600g for 8 minutes, and the supernatant was discarded. Cells were cultured at a concentration of 8x10 cells in culture medium (1640 + 10% FBS). 6 The antibody was resuspended at 100 μL / well and added to a 3799 cell culture plate. The test antibody was diluted 10-fold (0.00011 nM to 10 nM, and 0 concentration was used as a control) in culture medium (1640 + 10% FBS). 50 μL of the diluted antibody was added to PBMCs at 50 μL / well. The cells were resuspended and then incubated at 37°C for 6 hours. CpG-A (2 μM, Invitrogen, tlrl-2216-5, 50 μL) diluted in culture medium (1640 + 10% FBS) was added to each well, mixed, and then incubated at 37°C for an additional 20 hours. On day 3, the cell culture plate was centrifuged at 400 g for 5 minutes, and the cell culture supernatant was collected and assayed for the cytokine IFNα in the supernatant. BIIB059 antibody was selected as a positive control and hIgG1 was selected as a negative control.

[0211] Antibody MT1 / 3 / 5 (Mabtech, 3425-1H-20) was diluted to 4 μg / mL in PBS and added to Corning ELISA plates at 100 μL / well. The plates were incubated and coated overnight at 4°C. The coated antibody was then discarded, and blocking solution (PBS + 1% BSA) was added to the plates at 300 μL / well, followed by incubation at 4°C overnight. After blocking was complete, the test supernatants were diluted to the appropriate concentrations (typically 1:3 to 1:7) in blocking solution and added to the coated ELISA plates at 100 μL / well. IFNα assay standards were serially diluted 2-fold in blocking solution (up to 1000 pg / mL; 8 concentration points; the final concentration point was 0) and added to the coated ELISA plates at 100 μL / well. The ELISA plates were incubated in an incubator at 37°C for 1 hour. The plate was washed three times, and 100 μL / well of assay antibody MT2 / 4 / 6 (1:1000) was added. The plate was incubated for 1 hour at 37°C in an incubator. The plate was washed three times, and 100 μL / well of secondary antibody SA-HRP (1:1000) was added. The plate was incubated for 0.5 hours at 37°C in an incubator. The plate was washed three times, and 100 μL / well of TMB substrate solution was added. After the appropriate period of color development, 50 μL / well of 1 M HCl was added. OD values ​​at 450 nm were read on an ELISA plate reader, and curve fitting was performed using GraphPad Prism 6. The OD value corresponding to an antibody concentration of 10 nM was selected, and the maximum percent inhibition was calculated as follows: percent inhibition = (OD(IgG1) - OD(Ab)) / OD(IgG1). In experiments in which PBMCs were stimulated with CpG-A to release cytokines, the humanized antibody assayed was able to inhibit the secretion of the cytokine IFNα and inhibited the secretion of EC 50 The values ​​were lower than those of the positive control antibody BIIB059. The results are further shown in Table 4.

[0212] [Table 6]

[0213] 1.10.3 Assays for Internalization Ability of Humanized Antibodies CHOK1-human BDCA2 cells cultured to logarithmic growth phase were digested with TrypLE trypsin. Cells were harvested and diluted to 2 × 10 cells in FACS buffer (PBS + 2% FBS). 6 The cells were resuspended at 100 μL / mL. 100 μL / well of the cells were added to a 3799 cell plate, centrifuged at 300 g, and the supernatant was discarded. The cells were placed in an ice-cooled box and cooled. The antibody was serially diluted with FACS buffer (two concentrations: 100 nM and 10 nM; for each concentration, three groups were set: 0 h, 1 h at 4°C, and 1 h at 37°C). After dilution, the antibody was placed in an ice-cooled box and cooled for 15 minutes. After 15 minutes, the antibody was added to the centrifuged cells, resuspended, and incubated at 4°C for 40 minutes. After incubation, the cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. The cells in the 0 h group were fixed with paraformaldehyde at room temperature for 10 minutes and washed twice with FACS buffer. The cells in the other two groups were resuspended in 100 μL of FACS buffer and separately incubated at 4°C and 37°C for 1 hour. Cells in two groups, one at 4°C for 1 hour and the other at 37°C for 1 hour, were fixed with paraformaldehyde for 10 minutes at room temperature and washed twice with FACS buffer. 100 μL of secondary antibody, i.e., goat anti-human IgG (H+L) Cross-Adsorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000), was added individually to the cells in three groups, and the cells were incubated at 4°C for 1 hour. The cells were washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS instrument. The percentage of surface signal was calculated.

[0214] The results are shown in Figure 4. Both groups of humanized antibodies (groups Hu005 and Hu033) demonstrated internalization at concentrations of 100 nM and 10 nM, and the ability of the humanized antibodies to mediate internalization was comparable to that of the positive control antibody BIIB059. 1.10.4. Affinity Assays of Humanized Antibodies The humanized antibodies of the present disclosure were assayed for affinity using BIAcore experiments. The surface of a CM5 chip channel was activated with 50 mM NHS and 200 mM EDC (NHS and EDC from the amino coupling kit) mixed in a 1:1 ratio, and an anti-human IgG (Fc) antibody (diluted in sodium acetate solution at pH 5.0, resulting in a concentration of 25 μg / mL) was injected for 420 seconds. 1 M ethanolamine was then injected at a flow rate of 10 μL / min for 420 seconds to block excess active carboxyl groups on the chip. The humanized antibody was diluted to 1 μg / mL using running buffer and injected into the detection channel flow cell at a flow rate of 10 μL / min. Sample injections were performed for 60 seconds for capture. Recombinant human BDCA2 was diluted to 50 nM or 100 nM in 1x HBS-EP+ (pH 7.4) and then diluted in 1:2 increments down to 0.39 nM. The test samples of hBDCA2 with decreasing concentrations were individually injected into the detection channel at a flow rate of 30 μL / min, and two samples with a zero concentration were used to eliminate background signals. The antibody-antigen binding and dissociation times were 180 and 400 seconds, respectively. Data analysis was performed using Biacore Insight Evaluation Software (version 2.0.15.12933). After subtracting the signals from the reference channel (flow cell 1) and the zero concentration, a 1:1 binding model was selected for curve fitting, and kinetic parameters were calculated. The assay results are shown in Table 5 below.

[0215] [Table 7]

[0216] Reference antibody BIIB059: the light chain amino acid sequence is shown in SEQ ID NO: 25 and the heavy chain amino acid sequence is shown in SEQ ID NO: 26, and was generated with reference to CN105452295B. Example 2. Preparation of antibody-drug conjugates (ADCs) 2.1. Preparation of Compounds compound 1

[0217] [ka]

[0218] Step 1: Compound 1A (500 mg, 3.67 mmol) was dissolved in dichloromethane (10 mL), and then DIEA (1.42 g, 10.99 mmol) was added. TBSCl (830 mg, 5.51 mmol) dissolved in dichloromethane (5 mL) was added to the reaction solution, which was then stirred overnight for 17 hours. After the reaction was complete as detected by TLC (PE / EA = 20 / 1), the reaction solution was directly concentrated under reduced pressure by rotary evaporation and subjected to column chromatography (PE:EA = 100:0 to 100:1) to obtain a milky white oily liquid 1B (800 mg, yield: 87%).

[0219] Step 2: Compound 1B (125 mg, 0.50 mmol) and compound 1C (188 mg, 0.50 mmol) were added and dissolved in ultra-dry acetonitrile (8 mL). The reaction solution was purged with nitrogen, cooled to 0 °C, mixed with methanesulfonic acid (48 mg, 0.50 mmol), and MeCN (2 mL) was added via syringe, followed by stirring overnight for 16 hours. After the reaction was complete as detected by TLC (PE / EA = 3 / 1), sodium bicarbonate was added, the reaction solution was filtered, and concentrated by rotary evaporation to obtain the crude product, which was then purified by preparative chromatography to obtain compound 1 (62 mg, yield: 25%) as a white powder and 1S (7 mg, yield: 3%) as a white powder. Compound 1: MS-ESI: M / z 495.3 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 7.41 (d, J = 8.1 Hz, 2H), 7.34-7.28 (m, 3H), 6.16 (dd, J = 10.1, 1.9 Hz, 1H), 5.93 (s, 1H), 5.43 (s, 1H), 4.93 (d, J = 4.9 Hz, 1H), 4.79 (brs, 1H), 4.52 (d, J = 19.5 Hz, 1H), 4.48 (s, 2H), 4.34-4.25 (m, 1H), 4.19 (d, J = 19.5 Hz, 1H), 2.61-2.52 (m, 1H), 2.35-2.27 (m, 1H), 2.19-1.98 (m, 2H), 1.84-1.58 (m, 5H), 1.39 (s, 3H), 1.13-0.95 (m, 2H), 0.87 (s, 3H). Compound 1S: MS-ESI: M / z495.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.35-7.27 (m, 3H), 7.22 (d, J = 8.1 Hz, 2H), 6.17 (dd, J = 10.1, 1.9 Hz, 1H), 6.09 (s, 1H), 5.95 (s, 1H), 5.30 (d, J = 6.8 Hz, 1H), 4.81-4.76 (m, 1H), 4.48 (s, 2H), 4.30 (s, 1H), 4.25 (d, J = 19.2 Hz, 1H), 4.01 (d, J = 19.2 Hz, 1H), 2.59-2.53 (m, 1H), 2.36-2.28 (m, 1H), 2.12-1.97 (m, 2H), 1.90-1.69 (m, 5H), 1.39 (s, 3H), 1.26-1.13 (m, 1H), 1.06 (dd, J = 11.0, 3.4 Hz, 1H), 0.89 (s, 3H). Compound 2

[0220]

change

[0221] Step 1: Compound 2A (5.0 g, 25.4 mmol) and imidazole (2.59 g, 38.0 mmol) were dissolved in dichloromethane (50 mL), and then TBSCl (4.9 g, 32.5 mmol) was added. The reaction solution was reacted at 25 °C for 2 h. After the reaction was completed as detected by TLC, 100 mL of water was added, and the reaction solution was extracted twice with dichloromethane (100 mL × 2). The dichloromethane was combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The residue was directly stirred with silica gel and subjected to column chromatography (PE:EA = 20:1) to obtain a yellow solid 2B (3 g, yield: 38%). MS-ESI: M / z 312.2 [M+H] + .

[0222] Step 2: Compound 2B (1.0 g, 3.2 mmol) was dissolved in THF (10 mL), and the reaction solution was purged with N and cooled to 0 °C. BH3 / THF (6.4 mL, 6.4 mmol) was added, and the mixture was allowed to react for 17 h. After the reaction was complete as detected by LCMS, the reaction solution was added to methanol (40 mL) (kept in an ice bath at 0 °C), quenched with BH3, and then concentrated by rotary evaporation to give a colorless oil 2C (500 mg, yield: 52%). MS-ESI: M / z 298.1 [M+H] + .

[0223] Step 3: Compound 2C (500 mg, 1.68 mmol) was dissolved in dichloromethane (8 mL), and the reaction solution was cooled to 0° C. Dess-Martin reagent (1.43 g, 3.36 mmol) was added, and the mixture was allowed to react for 1.5 hours. After the reaction was complete as detected by LCMS, the reaction solution was diluted with water (100 mL) and extracted twice with ethyl acetate (100 mL + 50 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to give a white solid 2D (250 mg, yield: 50%). MS-ESI: M / z 296.0 [M+H] + .

[0224] Step 4: Compound 2D (100 mg, 0.34 mmol), compound 1C (127 mg, 0.34 mmol), and MgSO4 (250 mg, 2.08 mmol) were dissolved in acetonitrile (10 mL). The reaction solution was cooled to 0 °C and purged with nitrogen. Trifluoromethanesulfonic acid (150 mg, 1.0 mmol) was added, and after the addition was complete, the mixture was kept in an ice bath for 2 h. After the reaction was complete as detected by LCMS, the reaction solution was directly filtered, and the filter cake was rinsed three times with acetonitrile. The mother liquor was concentrated by rotary evaporation and subjected to column chromatography (DCM:MeOH = 30:1) to give a white solid 2E (100 mg, yield: 55%). MS-ESI: M / z 540.0 [M+H] + .

[0225] Step 5: Compound 2E (100 mg, 0.18 mmol) and iron powder (100 mg, 1.8 mmol) were added to ethanol (4 mL), and NHCl (96 mg, 1.8 mmol) was dissolved in water and added to ethanol. The reaction solution was purged with N, heated to 60 °C, and reacted for 2 h. After the reaction was completed as detected by LCMS, the reaction solution was filtered through Celite, and the filter cake was rinsed three times with ethanol. The mother liquor was concentrated by rotary evaporation, subjected to preparative chromatography, and lyophilized to obtain white solid powder 2 (40 mg, yield: 42%) and white solid powder 2S (5 mg, yield: 5%), respectively. Compound 2: MS-ESI: M / z 532.2 [M+Na] + . 11H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J = 10.1 Hz, 1H), 7.20 (d, J = 7.7 Hz, 1H), 6.94 (s, 1H), 6.89 (d, J = 7.7 Hz, 1H), 6.16 (dd, J = 10.1, 1.9 Hz, 1H), 5.93 (s, 1H), 5.34 (s, 1H), 4.95-4.89 (m, 1H), 4.80 (brs, 1H), 4.49 (d, J = 19.4 Hz, 1H), 4.44 (s, 2H), 4.33-4.27 (m, 1H), 4.18 (d, J = 19. Hz, 1H), 2.62-2.52 (m, 1H), 2.35-2.28 (m, 1H), 2.19-2.07 (m, 1H), 2.07-1.99 (m, 1H), 1.80-1.60 (m, 5H), 1.40 (s, 3H), 1.15-1.02 (m, 1H), 0.99 (dd, J = 11.2, 3.6 Hz, 1H), 0.86 (s, 3H). Compound 2S: MS-ESI:M / z532.3[M+Na] + . 1 1H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J = 10.1 Hz, 1H), 7.15-7.09 (m, 1H), 6.70 (s, 1H), 6.63 (d, J = 7.6 Hz, 1H), 6.17 (dd, J = 10.0, 1.9 Hz, 1H), 5.99 (s, 1H), 5.94 (s, 1H), 5.25 (d, J = 6.8 Hz, 1H), 4.78 (brs, 1H), 4.40 (s, 2H), 4.33-4.22 (m, 2H), 4.02 (d, J = 19.2 Hz,), 2.60-2.53 (m, 1H), 2.36-2.28 (m, 1H), 2.11-1.97 (m, 2H), 1.90-1.69 (m, 5H), 1.39 (s, 3H), 1.26-1.13 (m, 1H), 1.09-1.02 (m, 1H), 0.88 (s, 3H). compound 3

[0226] [ka]

[0227] Step 1: Compound 3A (6.58 g, 33.08 mmol, 1.0 equiv.) and compound 3B (5.03 g, 33.08 mmol, 1.0 equiv.) were added to a 250 mL three-neck flask, followed by THF (50 mL), water (5 mL), and K2CO3 (13.71 g, 99.24 mmol, 3.0 equiv.). Under a nitrogen atmosphere, Pd(dppf)Cl2 (1.21 g, 1.65 mmol, 0.05 equiv.) was added, and the reaction solution was purged with nitrogen three times, heated to reflux at 80 °C, and then stirred for 3–4 h. After the reaction was complete as detected by TLC (PE / EA = 5 / 1), the reaction solution was cooled to room temperature, filtered, and the filtrate was poured into 300 mL of water and extracted with EA (200 mL). The organic phase was washed with water (100 mL × 3), washed once with saturated NaCl, dried over anhydrous NaSO, and concentrated by rotary evaporation. The residue was stirred with silica gel and subjected to column chromatography (PE / EA = 7 / 1) to obtain the product, which was concentrated by rotary evaporation to give a white solid 3C (2.5 g, yield: 33%).

[0228] Step 2: Compound 1D (3 g, 7.97 mmol, 1.0 equiv.), compound 3C (1.8 g, 7.97 mmol, 1.0 equiv.), and MgSO4 (2.88 g, 23.91 mmol, 3.0 equiv.) were added to a 100 mL three-neck flask, and acetonitrile (30 mL) was added. Under a nitrogen atmosphere, trifluoromethanesulfonic acid (1.2 g, 7.97 mmol, 1.0 equiv.) was added to an ice-water bath, and the reaction solution was heated to room temperature and allowed to react for 2 h after the addition was complete. After completion of the reaction as detected by TLC (PE / EA = 3 / 1), the reaction solution was filtered, and the filtrate was concentrated by rotary evaporation. The residue was stirred with silica gel and subjected to column chromatography (PE / EA = 2 / 1) to obtain the product, which was concentrated by rotary evaporation to give a white solid 3 (3.7 g, yield: 79%). MS-ESI: M / z 585.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 10.1 Hz, 1H), 7.24 (d, J = 7.7 Hz, 2H), 7.20 (d, J = 7.5 Hz, 1H), 7.15 (s, 1H), 7.11 (d, J = 7.6 Hz, 1H), 7.07 (d, J = 7.6 Hz, 1H), 6.16 (dd, J = 10.1, 1.9 Hz, 1H), 5.92 (s, 1H), 5.39 (s, 1H), 4.91 (d, J = 4.8 Hz, 1H), 4.78 (brs, 1H), 4.49 (d, J = 19.4 Hz, 1H), 4.43 (s, 2H), 4.32-4.25 (m, 1H), 4.17 (d, J = 19.5 Hz, 1H), 3.90 (s, 2H), 2.59-2.51 (m, 1H), 2.35-2.26 (m, 1H), 2.18-1.95 (m, 2H), 1.82-1.56 (m, 5H), 1.39 (s, 3H), 1.11-0.96 (m, 4H), 0.85 (s, 3H). compound 4

[0229] [ka]

[0230] Step 1: Compound 4A (500 mg, 2.51 mmol) was dissolved in THF / HO (10:2 mL), followed by the addition of compound 4B (573 mg, 3.77 mmol) and KCO (1.04 g, 7.53 mmol). The reaction solution was purged with nitrogen, and then Pd(dppf)Cl (367 mg, 0.50 mmol) was added. The reaction solution was heated to 80 °C under a nitrogen atmosphere and reacted for 2 h. The reaction solution was cooled to room temperature, diluted with water (20 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was subjected to column chromatography (PE / EA = 1:0 to 3:1) to give a yellow solid, 4C (270 mg, yield: 48%).

[0231] Step 2: Compound 4C (150 mg, 0.66 mmol) was dissolved in MeCN (10 mL), and then compound 1C (250 mg, 0.66 mmol) and MgSO (160 mg, 1.32 mmol) were added. Trifluoromethanesulfonic acid (299 mg, 1.99 mmol) was added at 0° C. under a nitrogen atmosphere. The reaction solution was reacted at 0° C. for 1 hour, diluted with water (20 mL), extracted with dichloromethane (10 mL × 3), the organic phases were combined, washed with brine (10 mL), dried, and then concentrated by rotary evaporation to give the crude product, which was then subjected to preparative chromatography to give white powdery solid 4 (120 mg, yield: 31%) and white powdery solid 4S (13 mg, yield: 3%), respectively. Compound 4: MS-ESI: M / z 585.4 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 7.35 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 10.1 Hz, 1H), 7.24-7.16 (m, 4H), 7.14 (d, J = 8.1 Hz, 2H), 6.16 (dd, J = 10.1, 1.9 Hz, 1H), 5.92 (s, 1H), 5.38 (s, 1H), 4.90 (d, J = 4.8 Hz, 1H), 4.48 (d, J = 19.4 Hz, 1H), 4.42 (s, 2H), 4.31-4.25 (m, 1H), 4.16 (d, J = 19.4 Hz, 1H), 3.89 (s, 2H), 2.59-2.52 (m, 1H), 2.36-2.25 (m, 1H), 2.18-1.96 (m, 2H), 1.82-1.55 (m, 5H), 1.38 (s, 3H), 1.10-0.94 (m, 2H), 0.85 (s, 3H). Compound 4S: MS-ESI: M / z585.3 [M+H] + . 1 H NMR (400 MHz, クロロホルム-d) δ 7.31-7.26 (m, 3H), 7.21-7.11 (m, 6H), 6.31 (dd, J = 10.1, 1.9 Hz, 1H), 6.08 (s, 2H), 5.43-5.38 (m, 1H), 4.65 (s, 2H), 4.52-4.45 (m, 1H), 4.27 (d, J = 19.9 Hz, 1H), 4.05 (d, J = 19.9 Hz, 1H), 3.95 (s, 2H), 2.66-2.54 (m, 1H), 2.23-2.07 (m, 3H), 1.95-1.83 (m, 2H), 1.80-1.68 (m, 1H), 1.63 (dd, J = 14.1, 2.6 Hz, 1H), 1.46 (s, 3H), 1.31-1.13 (m, 2H), 0.99 (s, 3H). Compound 5

[0232]

change

[0233] Step 1: Compound 5A (50 g, 216 mmol, 1.0 equiv.) and imidazole (22 g, 323 mmol, 1.5 equiv.) were added to a 2 L three-neck flask, and the reaction mixture was dissolved in dichloromethane (700 mL). Then, TBSCl (44 g, 292 mmol, 1.35 equiv.) was slowly added. After complete consumption of the starting material as detected by TLC (PE / EA = 9:1), the reaction solution was cooled to room temperature, filtered, and extracted twice with dichloromethane (100 mL) and HO (90 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, stirred with silica gel, and purified by normal-phase column chromatography (PE:EA = 10:1) to give a white solid 5B (65 g, 87% yield).

[0234] Step 2: Compound 5B (60 g, 173 mmol, 1.0 equiv.), bis(pinacolato)diboron (44 g, 173 mmol, 1.0 equiv.), Pd(dppf)Cl (6.3 g, 8.6 mmol, 0.05 equiv.), and potassium acetate (51 g, 520 mmol, 3.0 equiv.) were added to a 2 L three-neck flask, and the reaction solution was dissolved in dioxane (1 L). The reaction solution was stirred at 110 °C under a N atmosphere for 12 h. After the reaction was completed as detected by TLC (PE:EA=9:1), the reaction solution was cooled to room temperature, filtered, and extracted twice with ethyl acetate (1 L) and HO (1 L). The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, stirred with silica gel, and purified by normal phase column chromatography (PE:EA=10:1) to give a white solid 5C (60 g, yield: 88%).

[0235] Step 3: Compound 5C (30 g, 76 mmol, 1.0 equiv.), compound 5D (15.2 g, 76 mmol, 1 equiv.), and Pd(dppf)Cl (3 g, 4 mmol, 0.05 equiv.) were added to a 1 L three-neck flask, followed by dioxane (600 mL). CsCO (74 g, 228 mmol, 3.0 equiv.) was dissolved in water (60 mL). The reaction solution was reacted at 110 °C for 2 h under a nitrogen atmosphere. After completion of the reaction as detected by TLC (PE:EA = 9:1), the reaction solution was cooled to room temperature, filtered, and extracted twice with ethyl acetate (1 L) and HO (1 L). The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, stirred with silica gel, and purified by normal phase column chromatography (PE:EA=10:1) to give a white solid 5E (20 g, yield: 68%).

[0236] Step 4: Compound 5E (2.0 g, 5.2 mmol, 1.0 equiv.), compound 1C (1.96 g, 5.2 mmol, 1.0 equiv.), and MgSO4 (1.88 g, 15.6 mmol, 3.0 equiv.) were added to a 250 mL three-neck flask, and acetonitrile (50 mL) was added. The reaction solution was cooled to -20 °C using dry ice and ethanol, and trifluoromethanesulfonic acid (2.34 g, 15.6 mmol, 3.0 equiv.) was slowly added. The resulting mixture was stirred at 0 °C for 2 h. After the reaction was complete as detected by TLC (PE:EA = 1:1), sodium bicarbonate solution was added to adjust the pH to 7-8. The acetonitrile was removed by rotary evaporation, and the reaction solution was extracted and slurried with PE:EA = 3:1 to give crude compound 5F (3.2 g, 98% yield), which was used directly in the next step. MS-ESI: M / z 630.1 [M+H] + .

[0237] Step 5: Compound 5F (3.2 g, 5.08 mmol, 1 eq.) and Fe (2.84 g, 50.8 mmol, 10 eq.) were added to a 250 mL three-neck flask, ethanol (80 mL) was added, and NHCl (2.72 g, 50.8 mmol, 10 eq.) was dissolved in water (80 mL) and then added to the three-neck flask, and the reaction solution was reacted at 60° C. for 2 hours. After the reaction was completed as detected by TLC (DCM:MOH=10:1), ethanol was removed by evaporation under reduced pressure, and the pH was adjusted to 8 by adding aqueous sodium bicarbonate. The reaction solution was extracted twice with 80 mL of dichloromethane and water, and the organic phase was dried over magnesium sulfate, filtered, and concentrated by rotary evaporation to give a yellow solid (2.6 g) as a crude product, 400 mg of which was subjected to reverse phase column chromatography to give compound 5 (43.9 mg, purity: 95% or more). MS-ESI: M / z 600.1 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.38 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 10.1 Hz, 1H), 7.24 (d, J = 7.9 Hz, 2H), 6.96 (s, 1H), 6.90 (s, 1H), 6.76 (s, 1H), 6.16 (dd, J = 10.1, 1.8 Hz, 1H), 5.93 (s, 1H), 5.40 (s, 1H), 4.92 (d, J = 4.8 Hz, 1H), 4.79 (brs, 1H), 4.49 (d, J = 19.4 Hz, 1H), 4.43 (s, 2H), 4.32-4.26 (m, 1H), 4.17 (d, J = 19.5 Hz, 1H), 3.90 (s, 2H), 2.62-2.52 (m, 1H), 2.36-2.26 (m, 1H), 2.18-1.97 (m, 2H), 1.81-1.55 (m, 5H), 1.39 (s, 3H), 1.10-0.94 (m, 2H), 0.86 (s, 3H). compound 6

[0238] [ka]

[0239] Step 1: Compound 6A (10 g, 43.1 mmol) was dissolved in dichloromethane (100 mL), and then DIEA (16.7 g, 129.3 mmol) was added. TBSCl (9.7 g, 64.6 mmol) in dichloromethane (100 mL) was added to the reaction solution. The resulting mixture was stirred overnight for 3 days. After the reaction was complete as detected by TLC (PE / EA = 40 / 1), the reaction solution was directly concentrated under reduced pressure by rotary evaporation and purified by column chromatography with pure petroleum ether to give 6B (12.0 g, 80% yield) as a bright yellow oil.

[0240] Step 2: Compound 6B (1 g, 2.89 mmol), bis(pinacolato)diboron (883 mg, 3.48 mmol), KOAc (853 mg, 8.69 mmol), 1,4-dioxane (10 mL), and Pd(dppf)Cl (212 mg, 0.29 mmol) were added to 1,4-dioxane (10 mL). The reaction solution was purged with nitrogen, heated (110 °C), and stirred for 17 hours overnight. After completion of the reaction as detected by TLC (PE:EA = 40:1), the reaction solution was directly concentrated by rotary evaporation. The residue was directly stirred with silica gel and subjected to column chromatography (petroleum ether:ethyl acetate = 50:1) to give 6C (183 mg, 16% yield) as a bright yellow oil. MS-ESI: M / z 394.3 [M+H] + .

[0241] Step 3: Compound 6C (180 mg, 0.46 mmol), Pd(dppf)Cl (33.5 mg, 0.046 mmol), compound 5D (90.6 mg, 0.46 mg), CsCO (298 mg, 0.92 mmol), 1,4-dioxane (8 mL), and water (2 mL) were added, and the reaction solution was purged with N, heated (110 °C), and stirred overnight (20 h). After completion of the reaction as detected by TLC (PE / EA = 10 / 1), the reaction solution was directly concentrated by rotary evaporation, and the residue was directly stirred with silica gel and subjected to column chromatography (petroleum ether: ethyl acetate = 100:1) to give 6D (95.8 mg, 54% yield) as a bright yellow oily liquid. MS-ESI: M / z 386.1 [M+H] + .

[0242] Step 4: Compound 6D (500 mg, 1.30 mmol), compound 1C (488 mg, 1.30 mmol), MeCN (10 mL), and MgSO (930 mg, 7.73 mmol) were added to a three-neck flask, and the reaction solution was purged with N and cooled to 0 °C. Trifluoromethanesulfonic acid (585 mg, 3.90 mmol) was added via a constant pressure addition funnel, and the resulting mixture was stirred (3 h). After the reaction was complete as detected by TLC (DCM / MeOH = 10 / 1), the reaction solution was directly concentrated by rotary evaporation, and the residue was directly stirred with silica gel and subjected to column chromatography (dichloromethane:MeOH = 50:1) to give white solid 6E (300 mg, yield: 37%); MS-ESI: M / z 630.1 [M+H]. + .

[0243] Step 5: Compound 6E (4 g, 6.35 mmol) and ethanol (100 mL) were added to a recovery flask. Iron powder (2.36 g, 42.1 mmol) and NH4Cl (3.4 g, 63.6 mmol) were dissolved in water (40 mL) and then added to the recovery flask. The reaction solution was purged with N2, heated to 60 °C, and stirred for 2 h. After completion of the reaction as detected by TLC (dichloromethane / CH3OH = 10 / 1), aqueous NaHCO3 was added to adjust the pH to 7-8. The reaction solution was stirred for 10 min, filtered, and the filtrate was concentrated by rotary evaporation. Dichloromethane was added for dissolution, and the resulting mixture was stirred and subjected to column chromatography (dichloromethane:methanol = 30:1) to obtain a white powder solid 6 (3.5 g, yield: 92%). 50 mg of this was collected and subjected to preparative thin-layer chromatography, filtered, and the filtrate was concentrated by rotary evaporation. Acetonitrile and water (purified water) were added, and the resulting mixture was lyophilized to obtain product 6 (38 mg, purity: 90.08%). MS-ESI: M / z 622.3 [M+Na] + . 1H NMR (400 MHz, DMSO-d6) δ 7.35 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 10.2 Hz, 1H), 7.22-7.16 (m, 2H), 6.93 (d, J = 7.6 Hz, 1H), 6.45-6.40 (m, 1H), 6.40-6.34 (m, 1H), 6.16 (dd, J = 10.1, 1.9 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 5.14-5.04 (m, 1H), 4.98-4.83 (m, 3H), 4.78 (d, J = 3.4 Hz, 1H), 4.54-4.45 (m, 1H), 4.34-4.26 (m, 3H), 4.22-4.12 (m, 1H), 3.75 (s, 2H), 2.60-2.53 (m, 1H), 2.36-2.26 (m, 1H), 2.17-1.96 (m, 2H), 1.81-1.60 (m, 5H), 1.39 (s, 3H), 1.09-0.97 (m, 2H), 0.85 (s, 3H). compound 7

[0244] [ka]

[0245] Step 1: Compound 7A (65 g, 280 mmol, 1.0 equiv.) and imidazole (29 g, 420 mmol, 1.5 equiv.) were added to a 2 L three-neck flask. The reaction mixture was dissolved in dichloromethane (700 mL), and then TBSCl (59 g, 392 mmol, 1.4 equiv.) was slowly added. After complete consumption of the starting material as detected by TLC (PE / EA = 9:1), the reaction solution was cooled to room temperature, filtered, and extracted twice with dichloromethane (100 mL) and HO (90 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, stirred with silica gel, and purified by normal-phase column chromatography (PE:EA = 10:1) to give a white solid, 7B (80 g, 82% yield). Step 2: Compound 7B (70 g, 203 mmol, 1.0 equiv.), bis(pinacolato)diboron (51 g, 203 mmol, 1.0 equiv.), Pd(dppf)Cl (7.4 g, 10.1 mmol, 0.05 equiv.), and potassium acetate (60 g, 609 mmol, 3.0 equiv.) were added to a 2 L three-neck flask, and the mixture was dissolved in dioxane (1 L). The reaction solution was stirred at 110 °C for 12 h under a N atmosphere. After the reaction was completed as detected by TLC (PE:EA=10:1), the reaction solution was cooled to room temperature, filtered, and extracted twice with ethyl acetate (1 L) and HO (1 L). The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, stirred with silica gel, and purified by normal phase column chromatography (PE:EA=10:1) to give a white solid 7C (60 g, yield: 75%).

[0246] Step 3: Compound 7C (15 g, 38 mmol, 1.0 equiv.), compound 5D (7.6 g, 38 mmol, 1 equiv.), and Pd(dppf)Cl (1.4 g, 1.9 mmol, 0.05 equiv.) were added to a 1 L three-neck flask, followed by the addition of dioxane (600 mL). CsCO (37 g, 114 mmol, 3.0 equiv.) was dissolved in water (50 mL). The reaction solution was reacted at 110 °C for 2 h under a nitrogen atmosphere. After the reaction was complete as detected by TLC (PE:EA = 10:1), the reaction solution was cooled to room temperature, filtered, and extracted twice with ethyl acetate (1 L) and HO (1 L). The organic phase was dried over anhydrous sodium sulfate, concentrated by rotary evaporation, stirred with silica gel, and purified by normal phase column chromatography (PE:EA=10:1) to give a white solid 7D (10 g, yield: 68%).

[0247] Step 4: Compound 7D (1 g, 2.6 mmol, 1.0 equiv.), compound 1C (0.98 g, 2.6 mmol, 1.0 equiv.), and MgSO (0.94 g, 7.8 mmol, 3.0 equiv.) were added to a 250 mL three-neck flask, and acetonitrile (50 mL) was added. The resulting mixture was cooled to -20 °C using dry ice and ethanol, and trifluoromethanesulfonic acid (1.17 g, 7.8 mmol, 3.0 equiv.) was slowly added. The resulting mixture was stirred at 0 °C for 2 hours. After the reaction was completed as detected by TLC (dichloromethane:MeOH=10:1), sodium bicarbonate solution was added to adjust the pH to 7-8, acetonitrile was removed by rotary evaporation, and the reaction solution was extracted and slurried with PE:EA=3:1 to obtain compound 7E (1.4 g, yield: 86%); MS-ESI: M / z 630.1 [M+H] + .

[0248] Step 5: Compound 7E (200 mg, 0.317 mmol, 1 eq.) and iron powder (177 mg, 3.17 mmol, 10 eq.) were added to a 25 mL three-neck flask, ethanol (6 mL) was added, and NHCl (170 mg, 3.17 mmol, 10 eq.) was dissolved in water (6 mL) and then added to the three-neck flask. The reaction solution was reacted at 60 °C for 2 h. After the reaction was completed as detected by TLC (DCM:MeOH = 10:1), ethanol was removed by evaporation under reduced pressure, and aqueous sodium bicarbonate was added to adjust the pH to 8. The reaction solution was extracted twice with 80 mL of dichloromethane and water. The organic phase was dried over magnesium sulfate, filtered, and concentrated by rotary evaporation to give a yellow solid (200 mg), which was then purified by reverse-phase column chromatography to give compound 7 (42.6 mg, yield: 22%). MS-ESI: M / z 600.8 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 10.1 Hz, 1H), 7.22 (d, J = 7.9 Hz, 2H), 7.20 - 7.14 (m, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 6.16 (dd, J = 10.1, 1.9 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 4.91 (d, J = 4.7 Hz, 1H), 4.79 (s, 1H), 4.54 - 4.45 (m, 3H), 4.31 - 4.26 (m, 1H), 4.17 (d, J = 19.5 Hz, 1H), 3.87 (s, 2H), 2.60 - 2.53 (m, 1H), 2.36 - 2.26 (m, 1H), 2.18 - 1.96 (m, 2H), 1.81 - 1.54 (m, 5H), 1.39 (s, 3H), 1.12 - 0.94 (m, 2H), 0.86 (s, 3H). Compound 8

[0249]

Chem.

[0250] Step 1: Compound 3 (2 g, 3.42 mmol, 1.0 equiv.) and compound 8A (2.52 g, 6.84 mmol, 2.0 equiv.) were added to a reaction flask, and the reaction mixture was dissolved in THF (30 mL). Under a nitrogen atmosphere, TsOH (0.63 g, 3.66 mmol, 1.07 equiv.) was added to an ice-water bath, and the resulting mixture was reacted at 5-10 °C for 30-40 min. After the reaction was completed as detected by TLC (PE / EA = 1 / 2), the reaction solution was added to water, and the product was extracted with EA (100 mL). The organic phase was washed with water (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated. The residue was stirred with silica gel and subjected to column chromatography (PE / EA = 2 / 1) to obtain the product, which was concentrated by rotary evaporation to give a white solid 8B (2.3 g, yield: 75%). MS-ESI: M / z 893.4 [M+H] + .

[0251] Step 2: Compound 8B (2 g, 2.24 mmol) was dissolved in DCM (20 mL), and DEA (6 mL) was added dropwise in an ice-water bath under a nitrogen atmosphere. After the addition was complete, the resulting mixture was allowed to react at room temperature for 2 hours. After the reaction was complete as detected by TLC (PE / EA = 1 / 2), the reaction solution was concentrated by rotary evaporation, extracted three times with DCM, subjected to reverse-phase column chromatography, and lyophilized to give an off-white solid 8C (600 mg, yield: 40%). MS-ESI: M / z 671.3 [M+H] + .

[0252] Step 3: Compound 8C (300 mg, 0.447 mmol, 1.0 equiv) and compound 8D (230 mg, 0.492 mmol, 1.1 equiv) were added to a reaction flask, and the reaction solution was dissolved in DMF (3.5 mL). DIEA (173 mg, 1.341 mmol, 3.0 equiv) was added to the reaction mixture in an ice-water bath under a nitrogen atmosphere. A solution of HATU (221 mg, 0.581 mmol, 1.3 equiv) dissolved in 1 mL of DMF was added dropwise. After the addition was complete, the resulting mixture was stirred at 0-5 °C for 30 min. After the reaction was complete as detected by LC-M, the reaction solution was poured into ice-water, and EA (100 mL) was added to extract the product. The organic phase was washed with water (50 mL × 3), dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to give a yellow solid, 8E (430 mg, 86% yield). MS-ESI: M / z 1119.4 [M+H] + .

[0253] Step 4: Under a nitrogen atmosphere, compound 8E (300 mg, 0.268 mmol, 1 eq.), Pd(PPh3)4 (62 mg, 0.0536 mmol, 0.2 eq.), and N-methylmorpholine (452 ​​mg, 4.47 mmol, 16.7 eq.) were added to a reaction flask, and the reaction mixture was allowed to react at room temperature for 1 hour under a nitrogen atmosphere. After the reaction was completed as detected by TLC, the reaction solution was directly purified by reverse-phase column chromatography to give a white solid 8F (210 mg, yield: 72%). MS-ESI: M / z 1079.3 [M+H] + .

[0254] Step 5: Under a nitrogen atmosphere, compound 8F (200 mg, 0.185 mmol) was dissolved in DCM (2.4 mL), and DEA (0.8 mL) was added dropwise to an ice-water bath. After the addition was complete, the resulting mixture was heated to room temperature for 2 hours. After the reaction was complete as detected by LCMS, the reaction solution was directly extracted and washed three times with PE (60 mL x 3). The product precipitated and adhered to the bottle wall. After dissolving in acetonitrile (3 mL), the reaction solution was purified by reverse-phase column chromatography to give a white solid 8G (62 mg, yield: 39%). MS-ESI: M / z 857.6 [M+H] + .

[0255] Step 6: Compound 8G (60 mg, 0.07 mmol, 1.0 equiv) was dissolved in THF (3 mL), and the reaction solution was dissolved in water (0.6 mL). TEA (414 mg, 4.1 mmol, 58 equiv) was added in an ice-water bath, and the reaction mixture was stirred for 1 min. Bromoacetyl bromide (113 mg, 0.56 mmol, 8.0 equiv) was added, and the resulting mixture was stirred at 0-5 °C for 5 min. After the reaction was complete as detected by LC-MS, the reaction solution was stored with dry ice and ethanol, subjected to preparative chromatography, and lyophilized to give a white solid 8 (11 mg, yield: 16%). MS-ESI: M / z 977.3 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ 8.62 (t, J = 6.6 Hz, 1H), 8.47 (t, J = 5.6 Hz, 1H), 8.24 (t, J = 5.9 Hz, 1H), 8.18 (d, J = 7.7 Hz, 1H), 7.40 - 7.34 (m, 2H), 7.31 (d, J = 10.1 Hz, 1H), 7.27 - 7.20 (m, 3H), 7.16 (brs, 1H), 7.14 - 7.07 (m, 2H), 6.16 (dd, J = 10.1, 1.9 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 4.91 (d, J = 5.1 Hz, 1H), 4.77 (brs, 1H), 4.65 - 4.55 (m, 2H), 4.49 (d, J = 19.4 Hz, aH), 4.39 (s, 2H), 4.33 - 4.25 (m, 2H), 4.17 (d, J = 19.5 Hz, 1H), 3.94 - 3.89 (m, 4H), 3.80 (d, J = 5.6 Hz, 2H), 3.72 (d, J = 5.8 Hz, 2H), 2.57 - 2.52 (m, 2H), 2.30 - 2.24 (m, 2H), 2.18 - 2.06 (m, 1H), 2.06 - 1.89 (m, 2H), 1.83 - 1.59 (m, 6H), 1.39 (s, 3H), 1.12 - 0.97 (m, 2H), 0.85 (s, 3H). Compound 9

[0256]

Chem.

[0257] Note: There seems to be a typo in the original text where "4.49 (d, J = 19.4 Hz, 1H)" is likely a misprint as the "aH" in the translation is assumed to be a mistake. It should probably be "1H". The same goes for "4.17 (d, J = 19.5 Hz, 1H)". Also, "

化

Chem.

[0258] Step 2: Compound 9A (1.3 g, 1.46 mmol) was dissolved in DCM (13 mL), and DEA (2.6 mL) was added dropwise in an ice-water bath under a nitrogen atmosphere. After the addition was complete, the resulting mixture was allowed to react at room temperature for 2 hours. After the reaction was complete as detected by TLC (PE / EA = 1 / 2), the reaction solution was concentrated by rotary evaporation, extracted three times with DCM, subjected to reverse phase column chromatography, and lyophilized to give an off-white solid 9B (350 mg, yield: 36%). MS-ESI: M / z 670.8 [M+H] + .

[0259] Step 3: Compound 9B (350 mg, 0.52 mmol, 1.0 equiv.) and compound 8D (243 mg, 0.52 mmol, 1.0 equiv.) were added to a reaction flask, and the reaction solution was dissolved in DMF (3.5 mL). DIEA (168 mg, 1.3 mmol, 2.5 equiv.) was added to the reaction mixture in an ice-water bath under a nitrogen atmosphere, and a solution of HATU (240 mg, 0.63 mmol, 1.21 equiv.) dissolved in 1 mL of DMF was added dropwise. After the addition was complete, the resulting mixture was stirred at 0-5°C for 30 min. After the reaction was complete as detected by LC-MS, the reaction solution was poured into ice-water, and EA (100 mL) was added to extract the product. The organic phase was washed with water (50 mL x 3), dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to give a foamy yellow solid 9C (600 mg) with negligible excess crude product yield. MS-ESI: M / z 1119.4 [M+H] + .

[0260] Step 4: Under a nitrogen atmosphere, compound 9C (400 mg, 0.35 mmol, 1.0 equiv.), Pd(PPh3)4 (82 mg, 0.071 mmol, 0.2 equiv.), and N-methylmorpholine (353 mg, 3.5 mmol, 10.0 equiv.) were added to a reaction flask, and the reaction mixture was allowed to react at room temperature under a nitrogen atmosphere for 1 hour. After the reaction was complete as detected by LC-MS, the reaction solution was directly purified by reverse-phase column chromatography to give a white solid 9D (150 mg, yield: 39%). MS-ESI: M / z 1079.4 [M+H] + .

[0261] Step 5: Under a nitrogen atmosphere, compound 9D (150 mg, 0.14 mmol) was dissolved in DCM (1.5 mL), and DEA (0.3 mL) was added dropwise in an ice-water bath. After the addition was complete, the resulting mixture was heated to room temperature for 2 hours. After the reaction was complete as detected by LCMS, the reaction solution was directly concentrated by rotary evaporation, extracted three times with DCM, and purified by reverse-phase column chromatography to give a white solid 9E (60 mg, yield: 50%). MS-ESI: M / z 857.4 [M+H] + .

[0262] Step 6: Compound 9E (50 mg, 0.058 mmol, 1.0 equiv) was dissolved in THF (3 mL), 3 drops of water were added for dissolution, and TEA (117 mg, 1.16 mmol, 20.0 equiv) was added in an ice-water bath. The reaction mixture was stirred for 1 min, and bromoacetyl bromide (93 mg, 0.46 mmol, 8.0 equiv) was added. The resulting mixture was stirred at 0-5 °C for 5 min. After completion of the reaction as detected by LC-MS, the reaction solution was stored on dry ice, subjected to preparative chromatography, and lyophilized to give a white solid 9 (11 mg, 19% yield). MS-ESI: M / z 977.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (t, J = 6.6 Hz, 1H), 8.49 (t, J = 5.6 Hz, 1H), 8.26 (t, J = 5.8 Hz, 1H), 8.19 (d, J = 7.5 Hz, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 10.1 Hz, 1H), 7.26-7.14 (m, 6H), 6.16 (dd, J = 10.1, 1.9 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 4.91 (d, J = 4.9 Hz, 1H), 4.79 (brs, 1H), 4.61-4.54 (m, 2H), 4.49 (d, J = 19.4 Hz, 1H), 4.37 (s, 2H), 4.32-4.24 (m, 2H), 4.16 (d, J = 19.4 Hz, 1H), 3.95-3.88 (m, 4H), 3.79 (d, J = 5.5 Hz, 2H), 3.72 (d, J = 5.8 Hz, 2H), 2.58-2.52 (m, 2H), 2.30-2.24 (m, 2H), 2.17-2.05 (m, 1H), 2.05-1.89 (m, 2H), 1.82-1.58 (m, 6H), 1.39 (s, 3H), 1.11-0.96 (m, 2H), 0.85 (s, 3H). compound 10

[0263] [ka]

[0264] Step 1: Compound 5 (1.9 g, 3.21 mmol, 1.0 equiv.), compound 8D (1.5 g, 3.21 mmol, 1.0 equiv.), and HATU (1.6 g, 4.24 mmol, 1.3 equiv.) were added to a 100 mL three-neck flask. The reaction mixture was dissolved in DMF (45 mL). 2,6-lutidine (1.4 g, 12.72 mmol, 4.0 equiv.) was then slowly added. After complete consumption of the starting material as detected by LCMS, the reaction solution was slowly added dropwise to water (300 mL). The product was filtered and dried under oil pump vacuum to give a yellow solid, 10A (3.3 g, 94% yield). MS-ESI: M / z 1048.4 [M+H]. + .

[0265] Step 2: Compound 10A (1.8 g, 1.72 mmol, 1.0 equiv.) and Pd(PPh3)4 (392 mg, 0.34 mmol, 0.2 equiv.) were added to a 50 mL three-neck flask, and the reaction mixture was dissolved in dichloromethane (30 mL). Under a nitrogen atmosphere, N-methylmorpholine (868 mg, 8.6 mmol, 5.0 equiv.) was slowly added, and the resulting mixture was allowed to react for 0.5 h. After the reaction was complete as detected by LCMS, diethylamine (6 mL) was slowly added to the three-neck flask and stirred at room temperature for 1 h. After the reaction was completed as detected by LCMS, DMF (20 mL) was added to the reaction solution, and the resulting mixture was rotary evaporated under reduced pressure at room temperature until 20 mL of solvent remained. Then, the rotary evaporation was stopped, and the residue was subjected to reverse-phase column chromatography (30%-70% aqueous ACN) and lyophilized to give a bright yellow solid 10B (455 mg, yield: 34%).

[0266] Step 3: Compound 10B (150 mg, 0.19 mmol, 1.0 equiv) was dissolved in THF (5 mL), water (0.2 mL) and triethylamine (190 mg, 1.9 mmol, 10 equiv) were added, and the reaction solution was cooled to 0 °C in an ice-water bath. Bromoacetyl bromide (153 mg, 0.76 mmol, 4.0 equiv) was dissolved in 1 mL of THF, and the resulting solution was then slowly added to the above reaction solution and reacted at 0 °C for 15 minutes. After the reaction was completed as detected by LCMS, the reaction solution was subjected to preparative chromatography to obtain a white solid 10 (20 mg, yield: 12%). MS-ESI: M / z 906.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 8.52 (s, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.43 (s, 1H), 7.38 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 9.7 Hz, 2H), 7.23 (d, J = 7.9 Hz, 2H), 6.87 (s, 1H), 6.16 (d, J = 10.3 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 4.91 (d, J = 5.1 Hz, 1H), 4.78 (s, 1H), 4.49 (d, J = 19.2 Hz, 1H), 4.41 (s, 2H), 4.39-4.32 (m, 1H), 4.29 (s, 1H), 4.17 (d, J = 19.5 Hz, 1H), 3.93 (s, 2H), 3.88 (s, 2H), 3.82-3.77 (m, 2H), 2.59-2.53 (m, 1H), 2.35-2.21 (m, 4H), 2.04-1.92 (m, 2H), 1.84-1.63 (m, 6H), 1.39 (s, 3H), 1.09-0.99 (m, 2H), 0.86 (s, 3H). compound 11

[0267] [ka]

[0268] Step 1: Compound 6 (300 mg, 0.5 mmol, 1.0 equiv.), compound 8D (233 mg, 0.5 mmol, 1.0 equiv.), and HATU (228 mg, 0.6 mmol, 1.2 equiv.) were added to a 25 mL three-neck flask. The reaction mixture was dissolved in DMF (5 mL). 2,6-lutidine (150 mg, 1.4 mmol, 2.8 equiv.) was then slowly added. After complete consumption of the starting material as detected by LCMS, the reaction solution was slowly added dropwise to water (100 mL). The product was filtered and dried under reduced pressure on an oil pump to give a yellow solid, 11A (450 mg, 86% yield). MS-ESI: M / z 1048.4 [M+H]. + .

[0269] Step 2: Compound 11A (450 mg, 0.43 mmol, 1.0 equiv.) and Pd(PPh3)4 (92 mg, 0.08 mmol, 0.2 equiv.) were added to a 50 mL three-neck flask, and the reaction mixture was dissolved in THF (10 mL). Under a nitrogen atmosphere, N-methylmorpholine (200 mg, 1.98 mmol, 4.6 equiv.) was slowly added, and the resulting mixture was allowed to react for 0.5 h. After completion of the reaction as detected by LCMS, the reaction solution was subjected to reverse-phase column chromatography (30%-70% aqueous ACN) and lyophilized to give a bright yellow solid 11B (250 mg, 58% yield). MS-ESI: M / z 1008.3 [M+H] + .

[0270] Step 3: Compound 11B (250 mg, 0.25 mmol) was added to a 25 mL three-neck flask, and the reaction mixture was dissolved in DCM (10 mL). Under a nitrogen atmosphere, diethylamine (2 mL) was added, and the resulting mixture was allowed to react for 0.5 h. After the reaction was complete as detected by LCMS, the reaction solution was subjected to reverse-phase column chromatography (20%-50% aqueous ACN) and lyophilized to give a bright yellow solid, 11C (130 mg, yield: 67%). MS-ESI: M / z 786.4 [M+H] + .

[0271] Step 4: Compound 11C (120 mg, 0.153 mmol, 1.0 equiv) was dissolved in THF (5 mL), water (0.2 mL) and triethylamine (155 mg, 1.53 mmol, 10.0 equiv) were added, and the reaction solution was cooled to 0 °C in an ice-water bath. Bromoacetyl bromide (123 mg, 0.61 mmol, 4.0 equiv) was dissolved in 1 mL of THF, and the resulting solution was slowly added to the above reaction solution and reacted at 0 °C for 15 min. After the reaction was completed as detected by LCMS, the reaction solution was subjected to preparative HPLC to give white solid 11 (12.2 mg, yield: 9%). MS-ESI: M / z 906.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.58-8.45 (m, 1H), 8.37 (d, J = 7.6 Hz, 1H), 7.51-7.46 (m, 1H), 7.40-7.34 (m, 2H), 7.31 (d, J = 10.0 Hz, 1H), 7.28-7.19 (m, 3H), 7.01-6.94 (m, 1H), 6.16 (dd, J = 10.0, 1.9 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 4.91 (d, J = 5.0 Hz, 1H), 4.77 (brs, 1H), 4.49 (d, J = 19.5 Hz, 1H), 4.42 (d, J = 3.7 Hz, 1H), 4.39-4.32 (m, 1H), 4.29 (brs, 1H), 4.17 (d, J = 19.5 Hz, 1H), 3.98-3.77 (m, 6H), 2.60-2.52 (m, 2H), 2.34-2.28 (m, 2H), 2.18-1.96 (m, 3H), 1.88-1.55 (m, 6H), 1.39 (s, 3H), 1.13-0.98 (m, 2H), 0.85 (s, 3H). compound 12

[0272] [ka]

[0273] Step 1: TEA (8.94 g, 88.39 mmol, 12.30 mL) was added to a solution of compound 4C (10.0 g, 44.2 mmol) dissolved in DCM (70 mL), and the reaction solution was cooled to 0 °C. MsCl (6.08 g, 53.0 mmol, 4.10 mL) was added, and the resulting mixture was stirred at 25 °C for 12 h. After complete consumption of the starting material as detected by LCMS, the reaction solution was diluted with DCM (50 mL), quenched with water (200 mL), and extracted with DCM (100 mL × 3). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous NaSO, and filtered. The filtrate was concentrated by rotary evaporation to give compound 12A as a yellow oil (8.60 g, crude).

[0274] Step 2: Thioglycolic acid (7.21 g, 63.15 mmol) was added to a solution of compound 12A (8.60 g, 31.58 mmol) dissolved in DMF (51.6 mL), and the reaction solution was stirred at 25 °C for 5 h. After complete consumption of the starting material as detected by TLC (PE / EA = 5:1), water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated saline solution, then with 5% aqueous LiCl, dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated by rotary evaporation to give compound 12B as a brown oil (5.75 g, yield: 57.82%, purity: 90.2%). MS-ESI: M / z 285.2 [M+H] + .

[0275] Step 3: Compound 12B (12.0 g, 42.2 mmol) was dissolved in methanol (240 mL), K2CO3 (8.75 g, 63.3 mmol) was added, and the reaction solution was stirred at 20 °C for 3 h. After complete consumption of the starting material as detected by LCMS, the reaction solution was poured into DCM (240 mL), and 0.5 M HCl (100 mL) was added, followed by liquid separation. The organic phase was dried over anhydrous sodium sulfate and concentrated to give an off-white solid 12C (10.0 g, yield: 97.7%). MS-ESI: M / z 483.2 [M+H]+ .

[0276] Step 4: Compound 12C (9.00 g, 37.1 mmol), compound 1C (13.9 g, 37.1 mmol), and anhydrous magnesium sulfate (13.4 g, 111 mmol) were added to acetonitrile (270 mL). After the addition was complete, TfOH (16.7 g, 111 mmol, 9.84 mL) was added dropwise at 0 °C, and the reaction solution was gradually heated to 20 °C and stirred for 3 h. After complete consumption of the starting material as detected by LCMS, the reaction solution was quenched with saturated aqueous sodium bicarbonate (60 mL) and extracted with ethyl acetate (100 mL × 2). The organic phase was washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, and concentrated to give an off-white solid 12D (15.0 g, yield: 33.6%). MS-ESI: M / z 1199.4 [M+H] + .

[0277] Step 5: Compound 12D (290 mg, crude) was added to DMF (4 mL), followed by the addition of DL-dithiothreitol (200 mg, 1.30 mmol). The reaction solution was allowed to react at room temperature under a nitrogen atmosphere for 16 hours. After the reaction was complete as detected by LCMS, the reaction solution was directly subjected to preparative HPLC to obtain white powdery solid 12 (20 mg, yield: 13.8%). MS-ESI: M / z 601.2 [M+H] + . 1H NMR (400 MHz, DMSO) δ 7.37 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 10.1 Hz, 1H), 7.23 (dd, J = 8.1, 3.3 Hz, 4H), 7.14 (d, J = 8.0 Hz, 2H), 6.16 (dd, J = 10.1, 1.7 Hz, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 4.91 (d, J = 5.0 Hz, 1H), 4.78 (s, 1H), 4.49 (d, J = 19.4 Hz, 1H), 4.29 (s, 1H), 4.17 (d, J = 19.5 Hz, 1H), 3.88 (d, J = 11.3 Hz, 2H), 3.67 (d, J = 7.6 Hz, 2H), 2.77 (t, J = 7.6 Hz, 1H), 2.50-2.55 (m, 1H), 2.45-2.25 (m, 1H), 2.25-2.05 (m, 2H), 1.80 - 1.53 (m, 5H), 1.38 (s, 3H), 1.14 - 0.94 (m, 2H), 0.86 (s, 3H). compound 13

[0278] [ka]

[0279] Step 1: Compound 12 (7.00 g, 11.6 mmol) and compound 8A (4.29 g, 11.6 mmol) were added to DMF (49.0 mL), TfOH (3.50 g, 23.3 mmol, 2.06 mL) was added, and the reaction solution was stirred at 25 °C for 16 hours. After complete consumption of the starting material as detected by LCMS, the reaction solution was quenched with saturated aqueous sodium bicarbonate (20 mL) and extracted with ethyl acetate (100 mL x 3). The organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and concentrated to give compound 13A as a yellow oil (15.0 g, negligible crude yield). MS-ESI: M / z 909.3 [M+H] + .

[0280] Step 2: Compound 13A (15.0 g, 16.5 mmol) was added to acetonitrile (105 mL), and diethylamine (6.03 g, 82.5 mmol, 8.50 mL) was added. The reaction mixture was stirred at 25° C. for 16 hours. After complete consumption of the starting material as detected by LCMS, the reaction mixture was slurried with acetonitrile (20.0 mL), filtered, and the liquid was removed from the resulting filter cake. The crude product was subjected to reverse-phase preparative chromatography (water:acetonitrile = 25% to 55%, 20 min) and lyophilized to give compound 13B (0.900 g, yield: 7.94%) as a white solid. MS-ESI: M / z 687.3 [M+H] + .

[0281] Step 3: Compound 13B (500 mg, 727 μmol) and compound 8D (526 mg, 1.09 mmol) were added to DMF (5 mL), HATU (830 mg, 2.18 mmol) and 2,6-lutidine (56.0 mg, 1.46 mmol, 169 μL) were added, and the reaction solution was stirred at 20 °C for 3 hours. After complete consumption of the starting material as detected by LCMS, the reaction solution was dried under reduced pressure to give the crude product (550 mg), of which 250 mg was subjected to reverse-phase preparative chromatography to give compound 13C as a white solid (100 mg, yield: 26.2%). MS-ESI: M / z 1151.4 [M+H] + .

[0282] Step 4: Compound 13C (50.0 mg, 43.4 μmol) was added to acetonitrile (1 mL), and diethylamine (15.8 mg, 217 μmol, 22.3 μL) was added, and the reaction solution was stirred at 25° C. for 6 hours. After complete consumption of the starting material as detected by LCMS, the reaction solution was concentrated by rotary evaporation and slurried with methyl tert-butyl ether (3.00 mL) to give compound 13D as a yellow solid (40.0 mg, yield: 99.1%). MS-ESI: M / z 929.2 [M+H] + .

[0283] Step 5: EEDQ (31.9 mg, 12 μmol) and bromoacetic acid (11.3 mg, 81.8 μmol) were dissolved in DMF (0.8 mL), and the reaction solution was stirred at room temperature for 1 hour. Compound 13D (40.0 mg, 43.0 μmol) was added, and the resulting mixture was stirred at 25° C. for 2 hours. After complete consumption of the starting material as detected by LCMS, the reaction solution was concentrated by rotary evaporation to give compound 13E as a yellow solid (the crude product was used directly in the next step). MS-ESI: M / z 1049.2 [M+H] + .

[0284] Step 6: Compound 13E (45.0 mg, 42.8 μmol) was dissolved in DCM (1.50 mL), trifluoroacetic acid (770 mg, 6.75 mmol, 500 μL) was added, and the reaction solution was stirred at 25° C. for 1 hour. After complete consumption of the starting material as detected by LCMS, the reaction solution was concentrated by rotary evaporation, and the crude product was then purified by reverse-phase preparative chromatography to give compound 13 as a yellow solid (3.06 mg, yield: 7.18%). MS-ESI: M / z 993.2 [M+H] + . 1HNMR (400 MHz, DMSO-d6) δ 7.83 (br d, 1H, J = 7.6 Hz), 7.63 (br d, 1H, J = 7.2 Hz), 7.4 (m, 2H), 7.26 (br d, 2H, J = 8.0 Hz), 7.16 (br d, 1H, J = 7.6 Hz), 4.92 (br s, 1H), 4.48 (br s, 1H), 4.30 (br s, 2H), 4.1 - 4.3 (m, 2H), 4.02 (br d, 1H, J = 13.6 Hz), 3.92 (br d, 2H, J = 8.4 Hz), 3.81 (br d, 2H, J = 5.2 Hz), 3.74 (br s, 3H), 3.63 (br s, 2H), 2.91 (s, 2H), 2.75 (br s, 1H), 2.3 - 2.4 (m, 2H), 2.27 (br s, 2H), 2.l - 2.1 (m, 1H), 2.02 (br s, 1H), 1.92 (br s, 1H), 1.77 (br s, 3H), 1.3 - 1.5 (m, 8H), 1.25 (s, 2H), 1.18 (br d, 1H, J = 7.2 Hz), 1.02 (br s, 1H), 0.8 - 1.0 (m, 2H). Compound 14 and Compound 14S

[0285]

Chem.

[0286] Step 1: Compound 14A (8 g, 37 mmol), compound 14B (5.8 g, 37 mmol), and K2CO3 (15.3 g, 111 mmol) were added to THF (80 mL), followed by the addition of water (4 mL) and Pd(dppf)Cl2 (2.7 g, 3.7 mmol). Under a nitrogen atmosphere, the external temperature was raised to 80 °C, and the mixture was refluxed for 16 h. After the reaction was completed as detected by TLC (PE:EA = 5:1), the reaction solution was cooled to room temperature, filtered, and the product was extracted by adding water (300 mL) and EA (300 mL). The organic phase was washed with water (100 mL × 3), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, stirred with silica gel, and purified by column chromatography (PE:EA = 10:1) to give 14C (2.86 g, 31% yield). MS-ESI: M / z 248.1 [M+H] + .

[0287] Step 2: Compound 14C (200 mg, 0.81 mmol), compound 14D (304 mg, 0.81 mmol), acetonitrile (6 mL), and MgSO4 (388 mg, 3.24 mmol) were added to a reaction flask. The mixture was cooled to 0 °C under a nitrogen atmosphere. Acetonitrile (1 mL) was dissolved in methanesulfonic acid (233 mg, 2.43 mmol) and added dropwise to the reaction mixture. After the addition, the mixture was stirred at 0 °C to 10 °C for 6 h. After the product content was confirmed to be 70% by LCMS, the reaction solution was poured into saturated aqueous sodium bicarbonate (40 mL), and the product was extracted with EA (40 mL × 2). The organic phase was washed with water (40 mL × 3), dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to give a yellow solid 14E (480 mg, crude yield was negligible). MS-ESI: M / z 606.2 [M+H] + .

[0288] Step 3: Compound 14E (240 mg, 0.40 mmol), iron powder (222 mg, 4.00 mmol), and NHCl (212 mg, 4.00 mmol) were added to a reaction flask, followed by the addition of ethanol (3 mL) and water (0.75 mL). The mixture was heated to 60 °C under a nitrogen atmosphere and reacted for 1 h. After the reaction was completed as detected by LCMS, the reaction solution was cooled to room temperature, filtered, and water (40 mL) and EA (100 mL) were added to extract the product. The organic phase was washed with water (40 mL × 2), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and purified by preparative thin-layer chromatography (PE:EA = 1:2) to give a white solid (40 mg). The solid was purified by reverse-phase preparative chromatography and lyophilized to give compound 14 (15 mg, yield: 6%) and compound 14S (4.4 mg, yield: 2%). Compound 14: MS-ESI: M / z 576.2 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.31 (d, J = 10.1 Hz, 1H), 7.12 - 7.06 (m, 1H), 7.02 (t, J = 8.0 Hz, 1H), 6.73 (d, J = 3.5 Hz, 1H), 6.69 - 6.53 (m, 3H), 6.18 (dd, J = 10.1, 1.9 Hz, 1H), 5.97 (s, 1H), 5.73 (s, 1H), 4.88 (d, J = 4.3 Hz, 1H), 4.80 (s, 1H), 4.48 (d, J = 19.4 Hz, 1H), 4.29 (d, J = 4.2 Hz, 1H), 4.15 (d, J = 19.5 Hz, 1H), 3.98 (s, 2H), 2.60 - 2.54 (m, 1H), 2.37 - 2.28 (m, 1H), 2.18 - 1.94 (m, 2H), 1.79 - 1.54 (m, 5H), 1.39 (s, 3H), 1.02 - 0.91 (m, 1H), 0.88 (dd, J = 11.2, 3.5 Hz, 1H), 0.84 (s, 3H). Compound 14S: MS-ESI: M / z576.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.31 (d, J = 10.1 Hz, 1H), 7.15 - 7.08 (m, 1H), 7.00 (d, J = 3.5 Hz, 1H), 6.75 (d, J = 3.5 Hz, 1H), 6.74 - 6.59 (m, 3H), 6.29 (s, 1H), 6.17 (dd, J = 10.1, 1.9 Hz, 1H), 5.94 (s, 1H), 5.20 (d, J = 7.0 Hz, 1H), 4.78 (s, 1H), 4.36 (d, J = 19.2 Hz, 1H), 4.29 (s, 1H), 4.10 - 3.94 (m, 3H), 2.59 - 2.53 (m, 1H), 2.32 - 2.26 (m, 1H), 2.11 - 1.94 (m, 3H), 1.86 - 1.61 (m, 5H), 1.38 (s, 3H), 1.21 - 1.12 (m, 1H), 1.03 (dd, J = 11.2, 3.4 Hz, 1H), 0.86 (s, 3H). Compound 15

[0289]

change

[0290] Step 1: Compound 15A (5 g, 23.1 mmol), 15B (3.6 g, 23.1 mmol), and K2CO3 (9.6 g, 69.3 mmol) were dissolved in THF (50 mL), and water (5 mL) was added. Under a nitrogen atmosphere, Pd(dppf)Cl2 (3.38 g, 4.6 mmol) was added, the external temperature was raised to 80 °C, and the mixture was refluxed for 16 h. After the reaction was completed as detected by TLC (PE:EA = 5:1), the reaction solution was cooled to room temperature, filtered, and the product was extracted with water (300 mL) and EA (300 mL). The organic phase was washed with water (100 mL × 3), dried over anhydrous sodium sulfate, stirred with silica gel, concentrated, and purified by column chromatography (PE:EA = 15:1) to give a yellow solid 15C (830 mg, yield: 14%). MS-ESI: M / z 248.1 [M+H] + .

[0291] Step 2: Under a nitrogen atmosphere, compound 15C (800 mg, 3.2 mmol), 15D (1.2 g, 3.2 mmol), acetonitrile (20 mL), and MgSO4 (15.5 g, 12.9 mmol) were added to a reaction flask. The mixture was cooled to -20 °C, and a solution of trifluoromethanesulfonic acid (1.46 g, 9.7 mmol) dissolved in acetonitrile (5 mL) was cooled and added dropwise to the reaction mixture. After the addition, the resulting solution was stirred at -20 °C for 1 h. After the reaction was completed as detected by LCMS, the reaction solution was poured into saturated aqueous sodium bicarbonate solution (100 mL), and the product was extracted with EA (100 mL × 2). The organic phase was washed with water (50 mL × 3), dried over anhydrous sodium sulfate, concentrated, stirred with silica gel, and purified by column chromatography to give a yellow solid 15E (1 g, yield: 51%). MS-ESI: M / z 606.3 [M+H] + .

[0292] Step 3: Compound 15E (100 mg, 0.165 mmol), iron powder (93 mg, 1.65 mmol), and NH4Cl (88 mg, 1.65 mmol) were added to a reaction flask, followed by the addition of ethanol (0.8 mL) and water (0.2 mL). Under a nitrogen atmosphere, the mixture was heated to 60 °C and reacted for 1 h. After the reaction was completed as detected by LCMS, the reaction solution was cooled to room temperature, filtered, and the product was extracted with water (40 mL) and EA (100 mL). The organic phase was washed with water (40 mL × 2), purified by preparative thin-layer chromatography (PE:EA = 1:3), and lyophilized to give a yellow solid 15 (46 mg, yield: 48%). MS-ESI: M / z 576.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.36 - 7.28 (m, 1H), 7.04 (d, J = 3.5 Hz, 1H), 6.84 (d, J = 8.3 Hz, 2H), 6.66 (d, J = 3.5 Hz, 1H), 6.45 (d, J = 8.4 Hz, 2H), 6.19 (dd, J = 10.1, 1.9 Hz, 1H), 6.00 (s, 1H), 5.73 (s, 1H), 5.07 (t, J = 5.9 Hz, 1H), 4.93 - 4.85 (m, 3H), 4.82 - 4.76 (m, 1H), 4.48 (dd, J = 19.5, 6.3 Hz, 1H), 4.34 - 4.26 (m, 1H), 4.15 (dd, J = 19.5, 5.6 Hz, 1H), 3.88 (s, 2H), 2.60 - 2.52 (m, 1H), 2.38 - 2.30 (m, 1H), 2.17 - 1.96 (m, 2H), 1.78 - 1.57 (m, 5H), 1.39 (s, 3H), 1.06 - 0.93 (m, 1H), 0.91 - 0.86 (m, 1H), 0.84 (s, 3H). compound 16

[0293] [ka]

[0294] Step 1: Compounds 16A (7.6 g, 35.2 mmol) and 16B (5.5 g, 35.2 mmol) were placed in a 500 mL eggplant-shaped flask and dissolved in THF (60 mL). K2CO3 (14.6 g, 105.6 mmol) and HO (10 mL) were added. Pd(dppf)Cl2 (5.2 g, 7.04 mmol) was added under a nitrogen atmosphere. The resulting solution was refluxed at 80 °C for 2 h. After substantial consumption of the starting material as detected by LCMS, the reaction solution was filtered, and the mother liquor was extracted with EA (100 mL) and HO (70 mL) for liquid separation. The organic phase was dried, concentrated by rotary evaporation, and purified by column chromatography (PE:EA = 3:1) to give yellow solid product 16C (1.2 g, 14% yield). MS-ESI: M / z 248.1 [M+H] + .

[0295] Step 2: Compounds 16C (1.12 g, 4.53 mmol) and 16D (1.79 g, 4.53 mmol) were dissolved in MeCN (10 mL), and MgSO (1.64 g, 13.6 mmol) was added. Under a nitrogen atmosphere, TfOH (1.36 g, 9.07 mmol) was added at 0 °C, and the mixture was reacted for 3 h. After confirming the formation of the product by LCMS, the reaction solution was filtered, directly concentrated by rotary evaporation, stirred with silica gel, and purified (DCM:MeOH = 13:1) to give red-orange-red solid 16E (750 mg, yield: 26%). MS-ESI: M / z 624.3 [M+H] + .

[0296] Step 3: Compound 16E (700 mg, 1.124 mmol) and Fe (629 mg, 11.24 mmol) were dissolved in EtOH (9 mL), and a solution of NH4Cl (596 mg, 11.24 mmol) dissolved in HO (3 mL) was added. The mixture was reacted at 60 °C for 2 h under a nitrogen atmosphere. After the starting material was substantially consumed as detected by LCMS, the reaction solution was filtered and extracted with water (10 mL) and EA (15 mL), and the liquid was separated. The organic phase was concentrated by rotary evaporation to give a yellow solid 16 (600 mg, yield: 90%). MS-ESI: M / z 594.3 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.29 (d, J = 10.1 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H), 7.08 (d, J = 3.5 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.86 - 6.80 (m, 2H), 6.75 (d, J = 3.5 Hz, 1H), 6.24 (dd, J = 10.1, 1.9 Hz, 1H), 6.05 (s, 1H), 5.74 (s, 1H), 5.43 (s, 1H), 4.92 - 4.85 (m, 1H), 4.49 (d, J = 19.4 Hz, 1H), 4.22 - 4.13 (m, 2H), 4.06 (s, 2H), 2.71 - 2.52 (m, 2H), 2.49 - 2.40 (m, 1H), 2.40 - 2.31 (m, 1H), 2.20 - 2.07 (m, 1H), 2.05 - 1.95 (m, 1H), 1.88 - 1.79 (m, 1H), 1.71 - 1.57 (m, 3H), 1.50 (s, 3H), 1.44 - 1.29 (m, 1H), 0.85 (s, 3H). compound 21

[0297] [ka]

[0298] Step 1: Compound 14 (2 g, 3.48 mmol), 21A (1.63 g, 3.48 mmol), and HATU (1.59 g, 4.18 mmol) were added to a 100 mL three-neck flask, DMF (30 mL) was added for dissolution, and 2,6-lutidine (1.12 g, 10.44 mmol) was slowly added. After complete consumption of the starting material as detected by LCMS, the reaction solution was slowly added dropwise to water (300 mL) and filtered to obtain the product, which was dried under reduced pressure in an oil pump to give a white solid, 21B (3.5 g, 98% yield). MS-ESI: M / z 1024.4 [M+H] + .

[0299] Step 2: Compound 19B (1.0 g, 1.0 mmol, 1.0 equiv) and Pd(PPh3)4 (392 mg, 0.339 mmol, 0.35 equiv) were added to a 50 mL three-neck flask, and DCM (30 mL) was added for dissolution. Under a nitrogen atmosphere, NMMP (868 mg, 8.6 mmol, 8.8 equiv) was slowly added. The mixture was reacted for 0.5 h. After completion as detected by LCMS, the mixture was stirred with silica gel, concentrated, and purified by column chromatography (0% to 15% DCM in MeOH) to give a yellow solid 21C (0.88 g, yield: 92%). MS-ESI: M / z 984.4 [M+H] + .

[0300] Step 3: Compound 21C (700 mg, 0.71 mmol) was dissolved in DMF (10 mL) and DEA (2 mL), and the mixture was allowed to react at room temperature for 15 minutes. After the reaction was complete as detected by LCMS, the reaction solution was purified by reverse-phase column chromatography (20%-50% ACN in water) to give a yellow solid 21D (300 mg, yield: 55%). MS-ESI: M / z 762.3 [M+H] + .

[0301] Step 4: Compound 21D (100 mg, 0.13 mmol) was dissolved in THF (5 mL) and water (0.5 mL), and 2,6-lutidine (139 mg, 1.3 mmol) was added at room temperature. Bromoacetyl bromide (130 mg, 0.65 mmol) was dissolved in THF (1 mL), and the resulting solution was slowly added to the reaction solution. The mixture was reacted at room temperature for 15 minutes. After the reaction was completed as detected by LCMS, the reaction solution was purified by preparative HPLC (ACN in 0.1% TFA) to give a white solid 21 (19.4 mg, yield: 17%). MS-ESI: M / z 882.2 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 12.14 (brs, 1H), 9.92 (s, 1H), 8.52 (t, J = 5.6 Hz, 1H), 8.25 (d, J = 7.8 Hz, 1H), 7.51 - 7.42 (m, 2H), 7.31 (d, J = 10.1 Hz, 1H), 7.23 - 7.14 (m, 1H), 7.08 (d, J = 3.5 Hz, 1H), 6.96 - 6.89 (m, 1H), 6.73 (d, J = 3.5 Hz, 1H), 6.18 (dd, J = 10.1, 1.9Hz, 1H), 5.97 (s, 1H), 5.73 (s, 1H), 4.88 (d, J = 4.8 Hz, 1H), 4.79 (s, 1H), 4.49 (d, J = 19.5 Hz, 1H), 4.44 - 4.35 (m, 1H), 4.29 (s, 1H), 4.15 (d, J = 19.5 Hz, 1H), 4.07 (s, 2H), 3.94 (s, 2H), 3.86 - 3.75 (m, 2H), 2.59 - 2.52 (m, 1H), 2.38 - 2.17 (m, 3H), 2.17 - 1.91 (m, 3H), 1.89 - 1.78 (m, 1H), 1.78 - 1.54 (m, 5H), 1.39 (s, 3H), 1.01 - 0.91 (m, 1H), 0.88 (dd, J = 11.2, 3.5 Hz, 1H), 0.84 (s, 3H). compound 22

[0302] [ka]

[0303] Step 1: Compound 15 (350 mg, 0.6 mmol) and compound 22A (289 mg, 0.6 mmol) were added to a 25 mL three-neck flask, and DMF (8 mL) was added. Under a nitrogen atmosphere, 2,6-lutidine (196 mg, 1.8 mmol) was added to an ice-water bath, and the mixture was stirred for 10 minutes. HATU (301 mg, 0.79 mmol) was added dropwise to the DMF solution (1 mL). After the addition, the resulting solution was heated to room temperature and stirred for 1 hour. After the reaction was complete as detected by LCMS, the reaction solution was slowly added dropwise to water (100 mL), precipitating a large amount of solid. The mixture was stirred for 30 minutes and filtered. The solid was lyophilized to give white solid 22B (525 mg, yield: 83%). MS-ESI: M / z 1040.4 [M+H] + .

[0304] Step 2: Compound 22B (200 mg, 0.192 mmol) and DCM (4 mL) were added to a 25 mL recovery flask, and the mixture was stirred under a nitrogen atmosphere until completely dissolved. DEA (0.57 mL) was added dropwise. After the addition, the resulting solution was heated to room temperature and reacted for 4 hours. After the reaction was complete as detected by LCMS, the reaction solution was concentrated to dryness and purified by reverse-phase preparative chromatography to give 22C (70 mg, 44% yield). MS-ESI: M / z 818.4 [M+H] + .

[0305] Step 3: Compound 22C (70 mg, 0.086 mmol) was dissolved in THF (5 mL) and water (0.5 mL). 2,6-lutidine (46 mg, 0.43 mmol) was added in an ice-water bath, and the resulting solution was stirred for 5 minutes. Bromoacetyl bromide (86 mg, 0.43 mmol) was added, and after the addition, the mixture was reacted in an ice-water bath for 10 minutes. After the reaction was completed as detected by LCMS, the reaction solution was added to water, the pH was adjusted to 1-2 with TFA, and the product was extracted with EA (20 mL). The organic phase was washed with water (10 mL × 2), dried over anhydrous sodium sulfate, and concentrated to a small volume. It was used directly in the next step, but the yield of the crude product was negligible. MS-ESI: M / z 938.3 [M+H] + .

[0306] Step 4: Under a nitrogen atmosphere, the organic phase (3 mL) from the previous step was added to TFA (1.5 mL) at room temperature. The resulting solution was stirred at room temperature for 4 hours. After the reaction was completed as detected by LCMS, the reaction solution was directly purified by reverse-phase preparative chromatography and lyophilized to give a white solid 2 (6.5 mg, 9% yield for two steps). MS-ESI: M / z 882.2 [M+H] + . 11H NMR (400 MHz, DMSO-d6) δ 12.14 (brs, 1H), 9.91 (s, 1H), 8.52 (t, J = 5.6 Hz, 1H), 8.27 (d, J = 7.9 Hz, 1H), 7.52 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 10.1 Hz, 1H), 7.15 (d, J = 8.6 Hz, 2H), 7.07 (s, 1H), 6.70 (d, J = 3.5 Hz, 1H), 6.19 (dd, J = 10.1, 1.9 Hz, 1H), 5.99 (s, 1H), 5.72 (s, 1H), 4.88 (d, J = 4.7 Hz, 1H), 4.80 (s, 1H), 4.49 (d, J = 19.5 Hz, 1H), 4.45 - 4.37 (m, 1H), 4.30 (s, 1H), 4.20 - 4.11 (m, 1H), 4.11 - 3.98 (m, 2H), 3.94 (s, 2H), 3.81 (dd, J = 5.7, 1.6 Hz, 2H), 2.60 - 2.53 (m, 1H), 2.38 - 2.24 (m, 3H), 2.17 - 1.93 (m, 3H), 1.89 - 1.77 (m, 1H), 1.77 - 1.70 (m, 2H), 1.70 - 1.57 (m, 3H), 1.39 (s, 3H), 1.05 - 0.92 (m, 1H), 0.91 (dd, J = 11.1, 3.4 Hz, 1H), 0.84 (s, 3H). Compound 23

[0307] [Chemistry]

[0308] Step 1: Compound 16 (540 mg, 0.91 mmol), compound 23A (353 mg, 0.76 mmol), and 2,6-lutidine (121 mg, 1.14 mmol) were added to a three-neck flask (25 mL), DMF (4 mL) was added for dissolution, and HATU (316 mg, 0.833 mmol) was added. The mixture was stirred for 17 hours. After the reaction was complete as detected by LCMS, the reaction solution was slowly added to water (80 mL), causing a large amount of yellow solid to precipitate. The resulting mixture was filtered, and the filter cake was washed with water (80 mL) and lyophilized to give yellow solid 23B (700 mg, 88% yield). MS-ESI: M / z 1042.4 [M+H] + .

[0309] Step 2: Compound 23B (700 mg, 0.67 mmol), N-methylmorpholine (679 mg, 6.7 mmol), and Pd(PPh3)4 (310 mg, 0.27 mmol) were added to a 25 mL three-neck flask, and THF (5 mL) was added for dissolution. The mixture was purged with nitrogen, cooled to 0 °C, and reacted for 2 h. After completion of the reaction as detected by LCMS, the reaction solution was directly stirred with silica gel and purified by normal phase column chromatography (DCM / MeOH = 80 / 20) to give yellow powder 23C (450 mg, yield: 66%). MS-ESI: M / z 1002.3 [M+H] + .

[0310] Step 3: Compound 23C (450 mg) was dissolved in DCM (3 mL), diethylamine (1.5 mL) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete as detected by LCMS, the reaction solution was added dropwise to PE (60 mL), resulting in the precipitation of a large amount of yellow solid. The resulting mixture was allowed to stand for 5 minutes, and then filtered to give a yellow powder (200 mg). This was purified by reverse-phase preparative chromatography to give a white solid 23D (15 mg, yield: 4%). MS-ESI: M / z 780.3 [M+H] + .

[0311] Step 4: Compound 23D (15 mg, 0.019 mmol) was dissolved in THF (0.8 mL) and HO (0.2 mL). The resulting solution was cooled to 5°C in an ice-water bath, and 2,6-lutidine (20.5 mg, 0.19 mmol) and bromoacetyl bromide (15.8 mg, 0.07 mmol) were added. The mixture was stirred for 5 minutes. After the reaction was complete as detected by LCMS, the reaction solution was purified by reverse-phase preparative chromatography to give 23 (6 mg, 34% yield) as a white powder. MS-ESI: M / z 900.2 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.55 - 8.48 (m, 1H), 8.24 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.44 (s, 1H), 7.29 (d, J = 10.1 Hz, 1H), 7.23 - 7.17 (m, 1H), 7.09 - 7.06 (m, 1H), 6.97 - 6.90 (m, 1H), 6.72 (d, J = 3.5 Hz, 1H), 6.24 (dd, J = 10.1, 1.9 Hz, 1H), 6.06 (s, 1H), 5.74 (s, 1H), 5.44 - 5.39 (m, 1H), 4.91 - 4.87 (m, 1H), 4.50 (d, J = 19.4 Hz, 1H), 4.42 - 4.36 (m, 1H), 4.17 (d, J = 19.5 Hz, 3H), 4.07 (s, 2H), 3.94 (s, 2H), 3.83 - 3.78 (m, 2H), 2.40 - 2.09 (m, 2H), 2.05 - 1.75 (m, 3H), 1.69 - 1.57 (m, 3H), 1.49 (s, 3H), 1.45 - 1.28 (m, 1H), 0.84 (s, 3H). compound 24

[0312] [ka]

[0313] Step 1: Compound 24A (4.53 g, 9.38 mmol) was dissolved in pyridine (36.0 mL) and DMF (36.0 mL), EDCI (3.60 g, 18.8 mmol) was added, and compound 15 (4.53 g, 9.38 mmol) was added to the reaction solution at 0 °C. The resulting solution was allowed to warm to room temperature and stirred for 1 h. After complete consumption of the starting material as detected by LCMS, the mixture was poured into water (118 mL). The resulting mixture was diluted with ethyl acetate (118 mL) and extracted with ethyl acetate (100 mL, 50.0 mL). The organic phases were combined, washed with saturated aqueous sodium chloride solution (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid 24B (crude, 6.50 g, negligible crude yield). MS-ESI: M / z 1040.4 [M+H] + .

[0314] Step 2: Compound 24B (6.30 g, 6.06 mmol), tetrazole (4.24 g, 60.6 mmol, 5.37 mL), and compound 24C (9.06 g, 36.3 mmol) were dissolved in DMF (44.0 mL). The mixture was stirred at 20 °C for 2.5 h, and the internal temperature was lowered to 0 °C. HO (3.78 g, 33.3 mmol, 3.20 mL, 30% purity) was added to the reaction mixture at 0 °C, and the resulting solution was allowed to warm to room temperature and stirred for 1 h. After complete consumption of the starting material as detected by LCMS, the mixture was poured into saturated aqueous sodium sulfite solution (44.0 mL). The resulting mixture was diluted with water (88.0 mL) and extracted with ethyl acetate (100 mL, 50.0 mL, 30.0 mL). The organic phases were combined, washed with saturated aqueous sodium chloride (20.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid 24D (crude, 7.46 g, negligible crude yield). MS-ESI: M / z 1232.5 [M+H] + .

[0315] Step 3: Compound 24D (7.16 g, 5.81 mmol) and piperidine (4.20 g, 49.4 mmol, 4.88 mL) were dissolved in acetonitrile (40.0 mL), and the mixture was stirred at 20 °C for 1 h. After complete consumption of the starting material as detected by LCMS, the reaction solution was concentrated under reduced pressure to remove acetonitrile. The residue after concentration was slurried with petroleum ether for 2 h, filtered, and the filter cake was dried under reduced pressure to give yellow solid 24E (6.37 g, yield: 77.7%), which was confirmed by LCMS (LCMS_ET54964-22-p1b1). MS-ESI: M / z 1010.5 [M+H] + .

[0316] Step 4: Bromoacetic acid (1.61 g, 11.6 mmol, 837 μL) and EEDQ (2.87 g, 11.6 mmol) were dissolved in DMF (41.0 mL), and the mixture was stirred at 20° C. for 1 hour. Compound 24E (5.87 g, 5.81 mmol) was added to the reaction solution at 20° C. The resulting solution was reacted at 20° C. for 2.5 hours. After complete consumption of the starting material as detected by LCMS, the reaction solution was diluted with dichloromethane (220 mL), washed twice with 1 N aqueous hydrobromic acid (160 mL×2), once with saturated aqueous sodium bicarbonate (110 mL), once with saturated aqueous sodium chloride (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give yellow oily liquid 24F (crude, 6.57 g, crude yield was negligible). MS-ESI: M / z 1130.3 [M+H] + .

[0317] Step 5: Compound 24F (6.57 g, 5.81 mmol) was dissolved in dichloromethane (50.0 mL), and TFA (25.0 mL) was added to the reaction solution at 20 °C. The resulting solution was stirred for 1 h. After complete consumption of the starting material as detected by LCMS, the reaction solution was concentrated under reduced pressure to give the crude product, which was purified by reverse-phase preparative chromatography [water (NH4HCO3)-acetonitrile] to give a yellow solid 24 (355 mg, yield: 6.36%). MS-ESI: M / z 961.9 [M+H] + . 1 1H NMR (400 MHz, DMSO-d6) δ 11.65 (broad singlet, 1H), 9.93 (singlet, 1H), 8.54 (triplet, J = 5.6 Hz, 1H), 8.32 - 8.24 (multiplet, 1H), 7.52 (doublet, J = 8.5 Hz, 2H), 7.32 (doublet, J = 10.1 Hz, 1H), 7.15 (doublet, J = 8.6 Hz, 2H), 7.06 (doublet, J = 3.6 Hz, 1H), 6.70 (doublet, J = 3.5 Hz, 1H), 6.19 (doublet of doublets, J = 10.1, 1.9 Hz, 1H), 5.99 (singlet, 1H), 5.81 (singlet, 1H), 4.94 - 4.84 (multiplet, 2H), 4.56 (doublet of doublets, J = 18.2, 7.9 Hz, 1H), 4.47 - 4.37 (multiplet, 1H), 4.34 - 4.28 (multiplet, 1H), 4.09 - 3.97 (multiplet, 2H), 3.94 (singlet, 2H), 3.87 - 3.75 (multiplet, 2H), 2.59 - 2.52 (multiplet, 1H), 2.39 - 2.18 (multiplet, 3H), 2.18 - 2.06 (multiplet, 1H), 2.06 - 1.92 (multiplet, 2H), 1.90 - 1.79 (multiplet, 1H), 1.75 (singlet, 2H), 1.71 - 1.54 (multiplet, 3H), 1.39 (singlet, 3H), 0.99 - 0.88 (multiplet, 2H), 0.86 (singlet, 3H). Compound 25

[0318]

Chem.

[0319] Step 1: Compound 24A (6.29 g, 13.0 mmol) was dissolved in pyridine (50.0 mL) and DMF (50.0 mL). EDCI (4.99 g, 26.0 mmol) was added to the reaction solution, and compound 16 (5.00 g, 8.68 mmol) was added to the reaction solution at 0 °C. The resulting solution was warmed to 10-20 °C and stirred for 12 h. After complete consumption of the starting material as detected by LCMS, the reaction solution was poured into water (150 mL), and the resulting mixture was extracted with ethyl acetate (150 mL, 100 mL, 50.0 mL). The organic phases were combined, washed with saturated aqueous sodium chloride (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid 25A (crude, 9.03 g, negligible crude yield). MS-ESI: M / z 1058.4 [M+H] + .

[0320] Step 2: Compound 25A (9.03 g, 8.68 mmol), tetrazole (4.40 g, 62.8 mmol), and compound 24C (13.0 g, 52.1 mmol) were dissolved in DMF (66.0 mL). The mixture was stirred at 20 °C for 2.5 h, and the internal temperature was lowered to 0 °C. HO (3.93 g, 34.7 mmol, 3.33 mL, 30% purity) was added to the reaction mixture at 0 °C, and the resulting solution was allowed to warm to 10-20 °C and stirred for 1 h. After complete consumption of the starting material as detected by LCMS, the mixture was poured into saturated aqueous sodium sulfite (66.0 mL). The resulting mixture was diluted with water (132 mL) and extracted with ethyl acetate (200 mL, 100 mL). The organic phases were combined, washed with saturated sodium chloride solution (50.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow solid 25B (crude, 10.7 g, negligible crude yield). MS-ESI: M / z 1250.5 [M+H] + .

[0321] Step 3: Compound 25B (10.7 g, 8.68 mmol) and piperidine (6.28 g, 73.8 mmol, 7.29 mL) were dissolved in acetonitrile (60.0 mL), and the mixture was stirred at 20° C. for 1 hour. After complete consumption of the starting material as detected by LCMS, the reaction solution was concentrated under reduced pressure to remove acetonitrile. The residue after concentration was slurried with petroleum ether (100 mL) for 2 hours, filtered, and the filter cake was dried under reduced pressure to give a yellow solid 25C (8.77 g, yield: 83.8%). MS-ESI: M / z 1028.4 [M+H] + .

[0322] Step 4: Bromoacetic acid (2.48 g, 17.8 mmol) and EEDQ (4.41 g, 17.8 mmol) were dissolved in DMF (63.0 mL), and the resulting solution was stirred at 20 °C for 1 h. Compound 25C (9.00 g, 8.91 mmol) was added to the reaction solution at 20 °C, and the resulting solution was reacted at 20 °C for 2.5 h. After complete consumption of the starting material as detected by LCMS, the reaction solution was diluted with dichloromethane (220 mL), washed twice with 1 N hydrobromic acid (160 mL × 2), once with saturated aqueous sodium bicarbonate solution (110 mL), once with saturated aqueous sodium chloride solution (50.0 mL), and dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness to give yellow oily liquid 25D (crude, 10.1 g, negligible crude yield). MS-ESI: M / z 1148.4 [M+H] + .

[0323] Step 5: Compound 25D (10.1 g, 8.91 mmol) was dissolved in dichloromethane (80 mL), and TFA (40 mL) was added to the reaction solution at 20 °C. The resulting solution was stirred for 1 hour. After confirming the presence of 54.7% of the product by LCMS, the reaction solution was concentrated under reduced pressure to obtain the crude product, which was purified by reverse-phase preparative chromatography [water (TFA)-acetonitrile] to obtain a yellow solid 25 (500 mg, yield: 8.58%, purity: 93.4%). MS-ESI: M / z 980.0 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 2H), 9.93 (s, 1H), 8.51 (t, J = 5.7 Hz, 1H), 8.24 (d, J = 7.8 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.46 - 7.40 (m, 1H), 7.30 (d, J = 10.2 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 7.07 (d, J = 3.5 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 6.73 (d, J = 3.5 Hz, 1H), 6.25 (dd, J = 10.1, 1.9 Hz, 1H), 6.06 (s, 1H), 5.83 (s, 1H), 5.51 (d, J = 4.5 Hz, 1H), 4.96 - 4.84 (m, 2H), 4.57 (dd, J = 18.2, 8.1 Hz, 1H), 4.44 - 4.35 (m, 1H), 4.23 - 4.16 (m, 1H), 4.07 (s, 2H), 3.94 (s, 2H), 3.88 - 3.73 (m, 2H), 2.70 - 2.54 (m, 2H), 2.40 - 2.32 (m, 1H), 2.32 - 2.22 (m, 2H), 2.22 - 2.09 (m, 1H), 2.06 - 1.91 (m, 2H), 1.90 - 1.76 (m, 3H), 1.71 - 1.57 (m, 3H), 1.49 (s, 4H), 1.44 - 1.29 (m, 1H), 0.87 (s, 3H). 2.2 Preparation of antibody-drug conjugates targeting BDCA2 In this application, an aqueous buffer of 20 mM histidine (pH=5.5) was obtained by adjusting the pH of a 20 mM histidine solution to 5.5 with HOAc. Stock solution 1: 10 mM EDTA buffer, pH=4.7; Stock solution 2: 800 mM aqueous sodium citrate buffer, pH=8.4; Stock solution 3: 500 mM urea solution; Stock solution 4: 10 mM tris(2-carboxyethyl)phosphine solution.

[0324] The protein purity of the ADC in this application was detected by SEC method, and the parameters are shown below.

[0325] [Table 8]

[0326] The DAR value of the ADC in this application can be detected by LC-MS. The experimental procedure was as follows: 8.0 mol / L guanidine hydrochloride (Gdn-HCl, 75.0 μL), 1.0 mol / L Tris-HCl (5.0 μL), and 1 mol / L DTT (2.0 μL) were added to the ADC sample solution (40.0 μg). The solution was diluted to 100.0 μL with ultrapure water, mixed thoroughly, and incubated at 22°C for 30 minutes. The drug-to-antibody ratio (DAR) was then analyzed using LC-MS. The LC parameters are shown below.

[0327] [Table 9]

[0328] The MS parameters are shown below.

[0329] [Table 10]

[0330] The DAR values ​​of the ADCs in this application can also be detected by the HIC method, and the parameters are shown below.

[0331] [Table 11]

[0332] Hu033-03, Hu033-20, and Hu005-04 are humanized anti-BDCA2 antibodies with the antibody subtype IgG1. Immunomodulatory antibody-drug conjugates targeting BDCA2 were constructed using Hu033-03, Hu033-20, and Hu005-04. Conjugation of Hu033-03 antibody-drug conjugate (Hu033-03-ADC)

[0333] [ka]

[0334] To an aqueous buffer solution of antibody Hu033-03 (0.02 M aqueous histidine buffer, pH = 6.0; 1.88 mL, 8.0 mg / mL, 0.101 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.028 mL, 0.283 μmol) at 22 °C. The reaction solution was placed in a water bath shaker, shaken, and reacted at 22 °C for 3 hours, after which the reaction was stopped.

[0335] Compound 9 (1.5 mg, 1.52 μmol) was dissolved in 0.15 mL of DMSO, and the resulting solution was added to the above solution. The mixture was placed in a water bath shaker, shaken, and reacted at 22° C. for 2 hours, after which the reaction was stopped. One-third of the volume of 100 mM aqueous histidine buffer (pH=5.5) was added to the reaction solution, and the mixture was desalted and purified using a Zeba desalting spin column (40K MWCO) (eluent: 0.02 M aqueous histidine buffer, pH 5.5) to obtain the antibody-drug conjugate Hu033-03-ADC (11 mg, 3.27 mg / mL, yield: 73%).

[0336] N a-I was found to be 4.01 according to the detection by HIC. Conjugation of Hu033-20 antibody-drug conjugate (Hu033-20-ADC)

[0337] [ka]

[0338] To an aqueous PBS buffer solution of antibody Hu033-20 (0.05 M aqueous PBS buffer, pH = 7.4; 1.88 mL, 8.0 mg / mL, 0.101 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.030 mL, 0.303 μmol) at 22 °C. The reaction solution was placed in a water bath shaker, shaken, and reacted at 22 °C for 3 hours, after which the reaction was stopped.

[0339] Compound 9 (1.5 mg, 1.52 μmol) was dissolved in 0.15 mL of DMSO, and the resulting solution was added to the above solution. The mixture was placed in a water bath shaker, shaken, and reacted at 22° C. for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Zeba desalting spin column (40K MWCO) (eluent: 0.02 M aqueous histidine buffer, pH 5.5) to obtain the antibody-drug conjugate Hu033-20-ADC (12.5 mg, 4.28 mg / mL, yield: 83%).

[0340] N a-I was found to be 4.46 according to the detection by HIC. Conjugation of Hu005-04 antibody-drug conjugate (Hu005-04-ADC)

[0341] [ka]

[0342] To an aqueous PBS buffer solution of antibody Hu005-04 (0.05 M aqueous PBS buffer, pH = 7.4; 1.88 mL, 8.0 mg / mL, 0.101 μmol) was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.028 mL, 0.283 μmol) at 22 °C. The reaction solution was placed in a water bath shaker, shaken, and reacted at 22 °C for 3 hours, after which the reaction was stopped.

[0343] Compound 9 (1.5 mg, 1.52 μmol) was dissolved in 0.15 mL of DMSO, and the resulting solution was added to the above solution. The mixture was placed in a water bath shaker, shaken, and reacted at 22° C. for 2 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Zeba desalting spin column (40K MWCO) (eluent: 0.02 M aqueous histidine buffer, pH 5.5) to obtain the antibody-drug conjugate Hu005-04-ADC (9 mg, 3.27 mg / mL, yield: 60%).

[0344] N a-I was found to be 4.34 according to the detection by HIC. Control ADC (BIIB059-ADC)

[0345] [ka]

[0346] For the preparation of the control ADC, reference was made to Preparation Example 1.9 in WO 2022171101A1, except that the antibody in this case was BIIB059. Example 3. Inhibition of human peripheral blood mononuclear cell activity by antibody-drug conjugates targeting BDCA2 BDCA2-ADC inhibits CpG-A-induced IFNα and TNFα production from human peripheral blood mononuclear cells the purpose The inhibitory effect of the antibody-drug conjugate molecules of the present application on cytokine production from peripheral blood mononuclear cells was examined. Peripheral blood mononuclear cells produce large amounts of cytokines after stimulation with the TLR9 agonist CpG-A. Peripheral blood mononuclear cells were treated in vitro with different concentrations of BDCA2 antibody-drug conjugates, and after stimulation with 0.5 μM CpG-A for a certain period of time, the IFNα and TNFα produced by the peripheral blood mononuclear cells were quantitatively detected. The in vitro activity of the ligand-drug conjugates was evaluated based on the range of cytokine inhibition. Experimental Process Thaw cryopreserved human peripheral blood mononuclear cells, resuspend in complete culture medium, count, and adjust the cell suspension to 2.5 x 10 cells using culture medium. 6 The cells were adjusted to a concentration of 0.5 μM / mL and cultured overnight. The next day, the suspension was centrifuged to collect the cells, and then the cells were resuspended using fresh culture medium. Test compounds were diluted to 4x the final concentration in cell culture medium, and 50 μL of the compound was added to a 96-well cell plate. 100 μL of the diluted cells (800,000 cells / well) were added to each well of the 96-well plate containing the compound, followed by the addition of CpG-A (50 μL) to a final concentration of 0.5 μM. The cell plate was incubated at 37°C with 5% CO2 for 24 hours. The supernatant was collected, and TNF-α and IFN-α concentration levels in the supernatant were detected using ELISA kits. Nonlinear regression was used to fit sigmoidal curves to the dose-response data, and IC 50 values ​​were calculated.

[0347] TNF-α concentration assays were performed using the Dakewe TNF-α ELISA kit (catalog number 1117202). Serially diluted cytokine standards (100 μL) were added to each standard well, 10-fold diluted test samples (100 μL) were added to each sample well, and 1× Dilution Buffer R (100 μL) was added to each blank control well. Biotinylated antibody working solution (50 μL) was added to each well. After mixing the wells, the plate was covered with plate sealing film and incubated at room temperature for 3 hours. The plate was washed three times: the liquid in each well was removed, and 1× Wash Buffer working solution (300 μL) was added to each well. After allowing to stand for 1 minute, the liquid in each well was discarded. This procedure was repeated three times. Streptavidin-HRP working solution (100 μL) was added to each well. The plate was covered with plate sealing film and incubated at room temperature for 20 minutes. The plate was washed three times, and TMB solution (100 μL) was added to each well. The plate was incubated at room temperature (18-25°C) in the dark for 25 minutes. The reaction was stopped by the rapid addition of 100 μL / well of stop solution. Readings at 450 nm were taken within 10 minutes of stopping the reaction using the M2e instrument.

[0348] IFN-α concentration assays were performed using the Dakewe IFN-α ELISA kit (catalog number 1110012). Serially diluted cytokine standards (100 μL) were added to each standard well, two-fold diluted test samples (100 μL) were added to each sample well, and 1× Dilution Buffer R (100 μL) was added to each blank control well. Biotinylated antibody working solution (50 μL) was added to each well. After mixing the wells, the plate was covered with plate sealing film and incubated at room temperature for 3 hours. The plate was washed three times, the liquid in each well was removed, and 1× Wash Buffer working solution (300 μL) was added to each well. After allowing to stand for 1 minute, the liquid in each well was discarded. This procedure was repeated three times. Streptavidin-HRP working solution (100 μL) was added to each well. The plate was covered with plate sealing film and incubated at room temperature for 20 minutes. The plate was washed three times, and TMB solution (100 μL) was added to each well. The plate was incubated at room temperature (18-25°C) in the dark for 25 minutes. The reaction was stopped by the rapid addition of 100 μL / well of stop solution. Readings at 450 nm were taken within 10 minutes of stopping the reaction using the M2e instrument.

[0349] Analysis was performed using GraphPad Prism software. OD450 values ​​were converted to TNF-α and IFN-α content and compared with the reference therapeutic antibody BIIB059 for cytokine secretion inhibition. Isotype Ib was used as a negative control antibody. Results are shown in Figure 5a and Figure 5b.

[0350] The results show that, compared with the reference therapeutic antibody BIIB059, the BDCA2 antibody-drug conjugate of the present application had a more potent ability to inhibit IFN-α secretion from peripheral blood mononuclear cells and achieved complete inhibition of IFN-α secretion at high concentrations. The antibody-drug conjugate exhibited a dose-dependent inhibitory effect on TNF-α secretion, while BIIB059 had no inhibitory effect on TNF-α secretion. The BDCA2 antibody-drug conjugate of the present application had stronger IFN-α inhibitory ability and a broader inhibitory effect on inflammatory factors, and also had a stronger therapeutic effect on systemic lupus erythematosus or other related autoimmune diseases than the therapeutic antibody BIIB059. BDCA2-ADC inhibits R848-induced IFN-α and TNF-α production from human peripheral blood mononuclear cells the purpose The inhibitory effect of the antibody-drug conjugate molecules of the present application on cytokine production from peripheral blood mononuclear cells was detected. Peripheral blood mononuclear cells produce large amounts of cytokines after stimulation with the TLR7 / 8 agonist R848. Peripheral blood mononuclear cells were treated in vitro with different concentrations of BDCA2 antibody-drug conjugates, and after stimulation with 5 μM R848 for a certain period of time, the IFNα and TNFα produced by the peripheral blood mononuclear cells were quantitatively detected. The in vitro activity of the ligand-drug conjugates was evaluated based on the range of cytokine inhibition. Experimental Process Thaw cryopreserved human peripheral blood mononuclear cells, resuspend in complete culture medium, count, and adjust the cell suspension to 2.5 x 10 cells using culture medium. 6The cells were adjusted to a concentration of 800,000 cells / mL and cultured overnight. The next day, the suspension was centrifuged to collect the cells, and then the cells were resuspended using fresh culture medium. Test compounds were diluted to 4x the final concentration in cell culture medium, and 50 μL of the compound was added to a 96-well cell plate. 100 μL of the diluted cells (800,000 cells / well) were added to each well of the 96-well plate containing the compound, followed by the addition of R848 (50 μL) to a final concentration of 5 μM. The cell plate was incubated at 37°C with 5% CO2 for 24 hours. The supernatant was collected, and TNF-α and IFN-α concentration levels in the supernatant were detected using ELISA kits. Nonlinear regression was used to fit sigmoidal curves to the dose-response data, and IC 50 values ​​were calculated.

[0351] TNF-α concentration assays were performed using the Dakewe TNF-α ELISA kit (catalog number 1117202). Serially diluted cytokine standards (100 μL) were added to each standard well, 10-fold diluted test samples (100 μL) were added to each sample well, and 1x Dilution Buffer R (100 μL) was added to each blank control well. Biotinylated antibody working solution (50 μL) was added to each well. After mixing the wells, the plate was covered with plate sealing film and incubated at room temperature for 3 hours. The plate was washed three times, the liquid in each well was removed, and 1x Wash Buffer working solution (300 μL) was added to each well. After allowing to stand for 1 minute, the liquid in each well was discarded. This procedure was repeated three times. Streptavidin-HRP working solution (100 μL) was added to each well. The plate was covered with plate sealing film and incubated at room temperature for 20 minutes. The plate was washed three times, and TMB solution (100 μL) was added to each well. The plate was incubated at room temperature (18-25°C) in the dark for 25 minutes. The reaction was stopped by the rapid addition of 100 μL / well of stop solution. Readings at 450 nm were taken within 10 minutes of stopping the reaction using the M2e instrument.

[0352] IFN-α concentration assays were performed using the Dakewe IFN-α ELISA kit (catalog number 1110012). Serially diluted cytokine standards (100 μL) were added to each standard well, two-fold diluted test samples (100 μL) were added to each sample well, and 1× Dilution Buffer R (100 μL) was added to each blank control well. Biotinylated antibody working solution (50 μL) was added to each well. After mixing the wells, the plate was covered with plate sealing film and incubated at room temperature for 3 hours. The plate was washed three times, the liquid in each well was removed, and 1× Wash Buffer working solution (300 μL) was added to each well. After allowing to stand for 1 minute, the liquid in each well was discarded. This procedure was repeated three times. Streptavidin-HRP working solution (100 μL) was added to each well. The plate was covered with plate sealing film and incubated at room temperature for 20 minutes. The plate was washed three times, and TMB solution (100 μL) was added to each well. The plate was incubated at room temperature (18-25°C) in the dark for 25 minutes. The reaction was stopped by the rapid addition of 100 μL / well of stop solution. Readings at 450 nm were taken within 10 minutes of stopping the reaction using the M2e instrument.

[0353] The analysis was performed using GraphPad Prism software and OD450 values ​​were converted to TNF-α and IFN-α contents. a) Cytokine secretion inhibition compared to reference antibody BIIB059 The results are shown in Figures 6a and 6b.

[0354] The results show that, compared with the reference antibody BIIB059, the BDCA2 antibody-drug conjugate of the present application had a stronger ability to inhibit IFN-α secretion from peripheral blood mononuclear cells and a higher inhibitory effect on IFN-α secretion at equivalent effective concentrations. BIIB059 had no inhibitory effect on TNF-α secretion. However, the BDCA2 antibody-drug conjugate of the present application exhibited a dose-dependent inhibitory effect on TNF-α secretion. Compared with antibody BIIB059, the BDCA2 antibody-drug conjugate of the present application had a stronger IFN-α / TNF-α inhibitory ability and a broad inhibitory effect on inflammatory factors, and had a strong therapeutic effect on autoimmune diseases. b) Inhibition of cytokine secretion compared to control ADC The results are shown in Figures 6c to 6f.

[0355] The results show that the BDCA2 antibody-drug conjugate Hu033-03-ADC of the present application has a stronger inhibitory effect on IFN-α secretion from peripheral blood mononuclear cells, and this effect is significantly superior to that of the control ADC. The IC50 values ​​of Hu033-03-ADC and the control ADC were 0.2801 and 1.718, respectively. The BDCA2 antibody-drug conjugate Hu033-03-ADC of the present application has a stronger inhibitory effect on TNF-α secretion from peripheral blood mononuclear cells, and this effect is significantly superior to that of the control ADC. The IC50 values ​​of Hu033-03-ADC and the control ADC were 0.2801 and 1.718, respectively. 50 The values ​​were 20.86 and 2040, respectively. The control ADC had no inhibitory effect on TNF-α secretion. Compared to the control ADC, the BDCA2 antibody-drug conjugate of the present application had stronger IFN-α / TNF-α inhibitory ability and a broader inhibitory effect on inflammatory factors, and had a stronger therapeutic effect on autoimmune diseases.

[0356] Example 4. Binding of antibody-drug conjugates targeting BDCA2 to the BDCA2 antigen The inhibitory effects of BDCA2 antibody-drug conjugates on human peripheral blood mononuclear cell function were investigated, and Hu033-03-ADC was selected for subsequent development and validation.

[0357] CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells cultured to logarithmic growth phase were digested with TrypLE trypsin. Cells were harvested and diluted to 2 × 10 cells in FACS buffer (PBS + 2% FBS). 6 The cells were resuspended at a concentration of 100 μL / mL. The resuspended cells were added to a 3799 cell plate at 100 μL per well, centrifuged at 300 g, and the supernatant was discarded. The test sample was diluted 5-fold with FACS buffer (final concentrations of 0.00255 nM to 200 nM) and added to the centrifuged cells. The cells were resuspended and incubated at 4°C for 1 hour. The incubated cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. 100 μL of goat anti-human IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Sigma, A11013; 1:1000) secondary antibody was added. The cells were incubated at 4°C for 1 hour, washed twice with FACS buffer, and resuspended in PBS. The fluorescent signals of the antibodies binding to the cell surface were analyzed using a FACS (BD FACS Canto™ II) instrument, and curve fitting was performed using GraphPad Prism 6 to calculate the EC50 values ​​of the antibodies or antibody-drug conjugates for binding to the BDCA2 antigen. BIIB059 antibody was selected as a positive control, and hIgG1 (ISO) was selected as a negative control antibody.

[0358] The binding activity of the present BDCA2 antibody-drug conjugates against CHOK1-human BDCA2 cells and 293F-cynoBDCA2 cells was superior to that of the positive control antibody BIIB059. The results are shown in Table 6 and Figure 7a / Figure 7b.

[0359] [Table 12]

[0360] Example 5. Internalization assay of antibody-drug conjugates targeting BDCA2 CHOK1-human BDCA2 cells cultured to logarithmic growth phase were digested with TrypLE trypsin. Cells were harvested and diluted to 2 × 10 cells in FACS buffer (PBS + 2% FBS). 6 The cells were resuspended at 100 μL / mL. 100 μL / well of the cells were added to a 3799-cell plate, centrifuged at 300 g, and the supernatant was discarded. The cells were placed in an ice-cooled box and cooled. The test samples were serially diluted with FACS buffer (three concentrations: 100 nM, 10 nM, and 1 nM; five groups were set for each concentration: 0 h; 2 h at 4°C; 2 h at 37°C; 4 h at 4°C; and 4 h at 37°C). After dilution, the test samples were placed in an ice-cooled box and cooled for 15 minutes. After 15 minutes, the test samples were added to the centrifuged cells, resuspended, and incubated at 4°C for 40 minutes. The incubated cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer. The cells in the 0 h group were fixed with paraformaldehyde at room temperature for 10 minutes and washed twice with FACS buffer. The cells in the other two groups were resuspended in 100 μL of FACS buffer and incubated separately at 4°C and 37°C for 1 and 4 hours. The cells in the two groups incubated at 4°C and 37°C were fixed with paraformaldehyde for 10 minutes at room temperature and washed twice with FACS buffer. 100 μL of a secondary antibody, i.e., goat anti-human IgG (H+L) Cross-Absorbed Secondary Antibody-Alexa Fluor 488 (Sigma, A11013; 1:1000), was added separately to the cells in group 3, and the cells were incubated for 1 hour at 4°C. The cells were washed twice with FACS buffer, resuspended in PBS, and analyzed using a FACS instrument. The percentage of surface signal was calculated. The results are shown in Figures 8A-8F. The antibody-drug conjugates exhibited higher cell surface binding at 1 nM and 10 nM concentrations and greater internalization into cells at the same concentrations compared to BIIB059.

[0361] Example 6. Bystander killing efficacy assay of immunomodulatory antibody-drug conjugates targeting BDCA2 HEK293 cells cultured to logarithmic growth phase were digested with TrypLE trypsin. The cells were harvested and diluted to a cell concentration of 2 × 10 cells using DMEM + 10% FBS. 5 30 mL of cells were seeded into a culture flask, and 1.5 mL of transfection reagent was prepared based on the doses in the table.

[0362] [Table 13]

[0363] 1.5 mL of transfection reagent was mixed with 30 mL of cells, and GR reporter gene cells were harvested after 24 hours of culture. The transfected GR reporter gene cells were mixed with HEK293-human BDCA2 cells or HEK293 cells at a 1:1 ratio, and 2 × 10 cells were harvested. 4 The conjugates were added to a 96-well culture plate at 1000 μg / well. Test reagents, a positive control (dexamethasone, Compound 4 in Example 2), and a negative control (isotype IgG-ADC) were added at concentrations ranging from 0.01 to 100 nM according to the experimental design. After 24 hours of incubation, Promega Dual-Glo Luciferase Assay System detection reagents were added according to the manufacturer's instructions, and Firefly and Renilla fluorescent signals were detected using an Enspire reader. The glucocorticoid receptor signal generated by the bystander killing effect of the conjugate was calculated by dividing the Firefly signal by the Renilla signal.

[0364] The results are shown in Figures 9A and 9b. Compared to an isotype control antibody conjugate, the BDCA2 antibody-drug conjugate Hu033-03-ADC specifically activated GR reporter gene cells in the presence of BDCA2 expression, demonstrating bystander killing.

[0365] Example 7. Inhibitory effects of BDCA2 immunomodulatory antibody-drug conjugates on pDC function revealed in cynomolgus monkeys Based on the mechanism of action of BDCA2 targeting and the drug efficacy of ADC, the biological activity of the antibody-drug conjugate was measured by examining the pDC phenotype in cynomolgus monkeys after injection of the BDCA2 antibody-drug conjugate and the IFNa expression level in an in vitro stimulation experiment (EMBO Mol Med (2015) 7:464-476).

[0366] Nine female cynomolgus monkeys were divided into three groups (three cynomolgus monkeys per group). A blank control (vehicle control, Group 1, cynomolgus monkeys 1, 8, and 15), the antibody-drug conjugate Hu033-03-ADC at a dose of 10 mg / kg (Group 2, cynomolgus monkeys 5, 7, and 12), and the antibody BIIB059 at a dose of 10 mg / kg (Group 3, cynomolgus monkeys 3, 4, and 10) were intravenously injected into individual cynomolgus monkeys within each group. For each group, whole blood was collected at days -28, -21, -10, 0, 2, 24, 48, 72, 96, 168, 240, 336, 504, 672, 840, 1008, 1176, 1344, 1512, and 1680 for analysis by flow cytometry and in vitro stimulation experiments. For groups 2 and 3, serum and plasma were collected in addition to whole blood collection, and all antibody serum and ADC concentrations were detected by ELISA.

[0367] The percentage of pDCs in peripheral blood and the expression level of BDCA2 on the surface of pDCs were detected by FACS. Venous blood from cynomolgus monkeys was collected using heparin anticoagulation tubes, and 6 mL of red blood cell lysis buffer was added to 300 μL of blood, and the resulting mixture was allowed to stand at room temperature for 10 minutes. Cells were collected by centrifugation at 400 g for 5 minutes at room temperature. Cells were washed with PBS and resuspended in 100 μL of cell staining buffer (PBS + 1% FBS). 5 μL of Fc Block was added to the cell suspension and allowed to stand at 4°C for 10 minutes. Brilliant Violet421™ anti-human HLA-DR antibody (Biolegend, 307636), APC / Cyanine7 anti-human CD20 antibody (Biolegend, 302314), APC / Cyanine7 anti-human CD14 antibody (Biolegend, 301820), PE mouse anti-human CD123 antibody (BD, 554529), and AF647-coupled hu033-03 antibody were added sequentially to the cell suspension, and the cells were incubated for 40 minutes at 4° C. The cells were washed twice with PBS, resuspended in 200 μL of cell staining buffer, and analyzed by using a flow cytometer.

[0368] The expression of cytokines was detected by whole blood stimulation in vitro. Venous blood from cynomolgus monkeys was collected using heparin anticoagulation tubes, and 150 μL of blood was added to a 96-well plate at a final concentration of 200 μg / mL. 100 μL of CpG-A was added to the wells. The cells were cultured at 37°C for 18 hours, and the supernatant was collected. The TNF-α and IFN-α concentrations in the supernatant were detected using an ELISA kit.

[0369] TNF-α concentration assays were performed using the R&D Primate TNF-α DuoSet ELISA Kit (Cat. No. DY1070). Serially diluted cytokine standards (50 μL) were added to each standard well, test samples (50 μL) were added to each sample well, and PBS (50 μL) was added to each blank control well. Cells were incubated at 37°C for 2 hours. The plate was washed three times, the liquid in each well was removed, and 300 μL of 1x PBST buffer working solution was added to each well. After 1 minute, the liquid in each well was discarded. 100 μL of detection antibody working solution was added to each well, and the cells were incubated at 37°C for 1 hour. The plate was washed three times, the liquid in each well was removed, and 300 μL of 1x PBST working solution was added to each well. After 1 minute, the liquid in each well was discarded. This procedure was repeated three times. 100 μL of streptavidin-HRP working solution was added to each well. The plate was covered with plate sealing film and incubated at 37°C for 0.5 hours. The plate was washed three times, and 100 μL of TMB solution was added to each well. The plate was incubated at room temperature (18-25°C) in the dark for 5 minutes. The reaction was stopped by the rapid addition of 50 μL / well of 1 M hydrochloric acid. Readings at 450 nm were detected within 10 minutes after stopping the reaction using a Molecular Devices Spectra Max Plus 384 instrument.

[0370] IFN-α concentration assays were performed using the Mabtech Human IFN-α (Pan-Specific) ELISA Kit (Cat. No. 3425-1H-20). Serially diluted cytokine standards (50 μL) were added to each standard well, test samples (50 μL) were added to each sample well, and PBS (50 μL) was added to each blank control well. Cells were incubated at 37°C for 2 hours. The plate was washed three times, the liquid in each well was removed, and 300 μL of 1x PBST buffer working solution was added to each well. After 1 minute, the liquid in each well was discarded. 100 μL of detection antibody working solution was added to each well, and the cells were incubated at 37°C for 1 hour. The plate was washed three times, the liquid in each well was removed, and 300 μL of 1x PBST working solution was added to each well. After 1 minute, the liquid in each well was discarded. This procedure was repeated three times. 100 μL of streptavidin-HRP working solution was added to each well. The plate was covered with plate sealing film and incubated at 37°C for 0.5 hours. The plate was washed three times, and 100 μL of TMB solution was added to each well. The plate was incubated at room temperature (18-25°C) in the dark for 5 minutes. The reaction was stopped by the rapid addition of 50 μL / well of 1 M hydrochloric acid. Readings at 450 nm were detected within 10 minutes after stopping the reaction using a Molecular Devices Spectra Max Plus 384 instrument.

[0371] The results are shown in Figures 10 to 13. 10 and 11, no significant change was observed in the percentage of DCs in the blood of the antibody-drug conjugate group or the BIIB059 group compared to the blank control. Additionally, the BDCA2 levels on the surface of pDCs found in the blood of the antibody-drug conjugate group or the BIIB059 group were lower than those of the blank control, and the levels of BDCA2 reduction in the antibody-drug conjugate group and the BIIB059 group were comparable. As can be seen, the antibody-drug conjugate Hu033-03-ADC of the present disclosure has good safety and can significantly reduce BDCA2 levels on the surface of pDCs.

[0372] Evaluation criteria for Figures 12 and 13: Changes in mean IFNα / TNFα expression levels were evaluated after in vitro whole blood stimulation with 200 μg / mL of CpGA on days 0 to 33 after administration. In Figure 12b, in pre-administration animals, the mean plasma IFNα level after in vitro whole blood stimulation was 34.2 pg / mL, and on days 0 to 33 after administration, the mean IFNα expression level was 7.6 pg / mL. In Figure 12c, in pre-administration animals, the mean plasma IFNα expression level after in vitro whole blood stimulation was 34.4 pg / mL, and on days 0 to 33 after administration, the mean IFNα expression level was 12.6 pg / mL. In Figure 13b, in pre-administration animals, the mean plasma IFNα expression level after in vitro whole blood stimulation was 5515 pg / mL, and on days 0 to 33 after administration, the mean IFNα expression level was 4131 pg / mL. In Figure 13c, in animals before treatment, the mean plasma IFNα expression level after in vitro whole blood stimulation was 3977 pg / mL, and from days 0 to 33 after treatment, the mean IFNα expression level was 3706 pg / mL.

[0373] 12 and 13, in vitro whole blood experiments using CpGA revealed that IFN-α expression levels were reduced in both the antibody-drug conjugate and BIIB059 groups compared to the blank control group, with the mean IFN-α expression level in the antibody-drug conjugate group (7.6 pg / mL) being lower than that in the BIIB059 group (12.6 pg / mL). Compared to the blank control group, TNFα expression levels in the BIIB059 group did not change significantly, while TNFα expression levels in the antibody-drug conjugate group were significantly reduced. The antibody-drug conjugate Hu033-03-ADC of the present disclosure exhibited better cytokine inhibitory effects than BIIB059.

[0374] Various modifications and variations in the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the present disclosure. Although the present disclosure has been described in connection with certain preferred embodiments, it should be understood that the present disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications to the modes for carrying out the present disclosure which will be apparent to those skilled in molecular biology, immunology, or related fields are intended to be within the scope of the following claims.

[0375] Sequence Listing (all sequences are amino acid sequences; see previous sections herein for sequence definitions / explanations):

[0376] [Table 14-1]

[0377] [Table 14-2]

[0378] [Table 14-3]

Claims

1. An antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof targeting BDCA2, a linker fragment (L), and a glucocorticoid molecule, wherein the antibody or antigen-binding fragment thereof targeting BDCA2 is (1) HCDR1 having the amino acid sequence shown in SEQ ID NO: 1, HCDR2 having the amino acid sequence shown in SEQ ID NO: 2, HCDR3 having the amino acid sequence shown in SEQ ID NO: 3, LCDR1 having the amino acid sequence shown in SEQ ID NO: 4, LCDR2 having the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 having the amino acid sequence shown in SEQ ID NO: 6, or (2) HCDR1 having the amino acid sequence shown in SEQ ID NO: 7, HCDR2 having the amino acid sequence shown in SEQ ID NO: 8, HCDR3 having the amino acid sequence shown in SEQ ID NO: 9, LCDR1 having the amino acid sequence shown in SEQ ID NO: 10, LCDR2 having the amino acid sequence shown in SEQ ID NO: 11, and LCDR3 having the amino acid sequence shown in SEQ ID NO:

12. The antibody-drug conjugate as described above, comprising:

2. the antibody or antigen-binding fragment thereof (1) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 14, or (2) a heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 15 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 16, or (3) A heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region having the amino acid sequence shown in SEQ ID NO:

18. The antibody-drug conjugate of claim 1, comprising:

3. 2. The antibody-drug conjugate of claim 1, wherein the antibody is selected from a human antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, a monoclonal antibody, and a polyclonal antibody.

4. The antigen-binding fragment may be Fab, Fab', F(ab') ２ 2. The antibody-drug conjugate of claim 1, wherein the antibody-drug conjugate is selected from an Fv, scFv, Fab'-SH, sdAb, VHH, a bispecific antibody, and a linear antibody.

5. 2. The antibody-drug conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises an immunoglobulin constant region, and the immunoglobulin constant region is a human IgG constant region, for example a human IgG1 constant region.

6. The antibody or antigen-binding fragment thereof has the following sequence: (1) a heavy chain having an amino acid sequence set forth in SEQ ID NO: 19 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO: 20 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto; or (2) a heavy chain having an amino acid sequence set forth in SEQ ID NO: 21 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO: 22 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto; or (3) A heavy chain having an amino acid sequence set forth in SEQ ID NO: 23 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO: 24 or having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

2. The antibody-drug conjugate of claim 1, comprising or consisting of:

7. The glucocorticoid molecule comprises a structure represented by formula (I), and tautomers, mesomers, racemates, enantiomers, diastereoisomers, or mixtures thereof, or pharmaceutically acceptable salts or solvates thereof; 【Chemistry 1】 During the ceremony, X is —O—, —S—, and —N(R １ａ )-, where R １ａ But H, C １ ~C ６ Alkyl, and —C １ ~C ６ alkylhydroxy; R １ and R ２ are each independently H, protium, deuterium, tritium, halogen, C １ ~C ６ Alkyl and halogenated C １ ~C ６ alkyl, R ３ But C １ ~C ６ Alkyl, halogenated C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, halogenated C １ ~C ６ Alkoxy, C １ ~C ６ Alkylthio, halogenated C １ ~C ６ Alkylthio, -C １ ~C ６ Alkyl-O-P(=O)(OC １ ~C ６ alkyl) ２ , and -C １ ~C ６ Alkyl-O-P(=O)(OH) ２ is selected from, wherein C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, and C １ ~C ６ each alkylthio is optionally substituted with one or more substituents independently selected from halogen, CN, OH, or SH; Ring B is selected from phenyl or 5- to 6-membered heteroaryl, and Ring B is selected from halogen, OH, CN, NH ２ , C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy and halogenated C １ ~C ６ optionally substituted with one or more substituents selected from alkyl; W is a single bond, —O—, —S—, —C(O)—, —NH—, —C(O)NH—, —NHC(O)—, —C(O)—N(C １ ~C ６ alkyl)-, -N(C １ ~C ６ alkyl)-C(O)-, -C １ ~C ６ Alkylene-, -N(C １ ~C ６ alkyl)-, -C １～６ Alkylene -NH-, -O-C １ ~C ６ Alkylene-, and -C １ ~C ６ alkylene-O-, wherein C １ ~C ６ Alkyl and C １ ~C ６ The alkylene is selected from the group consisting of halogen, C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, halogenated C １ ~C ６ Alkyl, C ３ ~C ６ cycloalkyl, oxo, OH, CN, or NH ２ and optionally substituted with one or more substituents selected from V is a single bond or -(C(R ２ａ ) (R ２ｂ )) ｎ -, where R ２ａ and R ２ｂ are each independently H, halogen, OH, NH ２ , -CN,C １ ~C ６ Alkyl and halogenated C １ ~C ６ alkyl, or R ２ａ and R ２ｂ together with the carbon atom to which they are attached, form a carbonyl or C ３～６ forming a cycloalkyl, where n is selected from 1, 2, 3, 4, 5, or 6; Y １ H, halogen, OH, nitro, NH ２ , —NH(C １ ~C ６ alkyl), -N(C １ ~C ６ alkyl) ２ , C １ ~C ６ Alkyl, -CN, C １ ~C ６ Alkoxy, -C １ ~C ６ Alkylhydroxy and halogenated C １ ~C ６ alkyl, The antibody-drug conjugate of any one of claims 1 to 6, wherein m is selected from 1, 2, or 3.

8. X is —O—, —S—, and —N(R １ａ )-, where R １ａ is H, -CH ３ , and -CH ２ CH ３ The antibody-drug conjugate of claim 7, wherein the antibody-drug conjugate is selected from the group consisting of:

9. The antibody-drug conjugate of claim 8, wherein X is selected from -O- and -NH-.

10. R １ and R ２ are each independently H, F, Cl, and —CH ３ The antibody-drug conjugate of claim 7, wherein the antibody-drug conjugate is selected from the group consisting of:

11. R １ and R ２ The antibody-drug conjugate of claim 10, wherein each of is independently selected from H or F.

12. R ３ But -CH ２ Cl, —CH ２ SH, -CH ２ OH, 【Chemistry 2】 , -OCH ３ , -OCH ２ F, -OCH ２ Cl, —OCH ２ CN, -OCH ２ CH ３ , -SCH ２ F, -SCH ２ Cl, -SCH ２ CF ３ , and -SCH ２ CN. CN.

13. R ３ But -CH ２ OH, -SCH ２ F, and 【Transformation 3】 The antibody-drug conjugate of claim 12, wherein the antibody-drug conjugate is selected from the group consisting of:

14. Ring B is selected from phenyl, pyridinyl, thienyl, pyrrolyl, furanyl, pyrazolyl, imidazolyl, and thiazolyl, and Ring B is selected from halogen, OH, CN, NH ２ , C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy and halogenated C １ ~C ６ 8. The antibody-drug conjugate of claim 7, optionally substituted with one or more substituents selected from alkyl.

15. Ring B is selected from phenyl and thienyl, and Ring B is selected from halogen, OH, CN, NH ２ , -CH ３ , -CH ２ CH ３ , or -OCH ３ 15. The antibody-drug conjugate of claim 14, optionally substituted with one or more substituents selected from:

16. Ring B is 【Chemistry 4】 The antibody-drug conjugate of claim 15, wherein the antibody-drug conjugate is selected from the group consisting of:

17. W is -C １ ~C ６ alkylene-, wherein C １ ~C ６ The alkylene is selected from the group consisting of halogen, C １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, halogenated C １ ~C ３ Alkyl, C ３ ~C ６ cycloalkyl, oxo, OH, CN, or NH ２ 8. The antibody-drug conjugate of claim 7, optionally substituted with one or more substituents selected from:

18. W is, 【Transformation 5】 The antibody-drug conjugate of claim 17, wherein the antibody-drug conjugate is selected from the group consisting of:

19. V is a single bond or -(C(R ２ａ ) (R ２ｂ )) ｎ -, where R ２ａ and R ２ｂ are each independently H, halogen, OH, NH ２ , -CN,C １ ~C ３ Alkyl and halogenated C １ ~C ３ The antibody-drug conjugate of claim 7, wherein n is selected from alkyl, aryl, arylsulfonyl ...

20. V is a single bond, —CH ２ - or -CH ２ CH ２ The antibody-drug conjugate of claim 19, wherein the antibody-drug conjugate is selected from the group consisting of:

21. Y １ H, halogen, OH, NH ２ , C １ ~C ３ Alkyl, -CN, C １ ~C ３ Alkoxy, -C １ ~C ３ Alkylhydroxy and halogenated C １ ~C ３ The antibody-drug conjugate of claim 7, wherein the aryl group is selected from alkyl.

22. Y １ H, halogen, OH, NH ２ , -CH ３ , -CH ２ CH ３ , -CN, -OCH ３ , and -CH ２ 22. The antibody-drug conjugate of claim 21, wherein said conjugate is selected from the group consisting of OH.

23. The structure of the glucocorticoid molecule is Table 1 is selected from the group consisting of During the ceremony, R １ and R ２ is as defined in claim 7, 10 or 11, R ３ is as defined in claim 7, 12 or 13, W is as defined in claim 7, 17 or 18; Y １ The antibody-drug conjugate of any one of claims 7 to 22, wherein is as defined in claim 7, 21, or 22.

24. The structure of the glucocorticoid molecule is Table 2-1 Table 2-2 24. The antibody-drug conjugate of claim 23, selected from the group consisting of:

25. The linker fragment (L) is Tr, L １ , L ２ , and L ３ wherein the antibody-drug conjugate comprises a structure represented by formula (II): 【Transformation 6】 Tr is absent or is any group; L ３ is selected from polypeptide fragments, L ２ is absent or selected from linker fragments, L １ The antibody-drug conjugate of any one of claims 1 to 24, wherein is selected from the coupling units.

26. Tr is not present, or 【Transformation 7】 is selected from L ３ However, glycine-glycine-phenylalanine-glycine (GGGF), alanine-alanine-alanine-glycine (AAAG), glycine-glycine-glycine-glycine (GGGG), valine-alanine-glycine (VAG), valine-citrulline-glycine (VCG), alanine-alanine-glycine (AAG), alanine-alanine-alanine (AAA), valine-alanine (VA), valine-citrulline (VC), alanine-alanine (AA), glutamic acid-alanine valine-lysine-glycine (VKG), glycine-glutamic acid-alanine-glycine (EAGG), glycine-glutamic acid-alanine-glycine (GEAG), glycine-glutamic acid-glycine-glycine (GEGG), glutamic acid-glycine-glycine (EGG), glutamic acid-alanine-glycine (EAG), valine-lysine-glycine (VKG), glycine-glutamic acid-glycine (GEG), glutamic acid-alanine (EA), glutamic acid-glycine (EG), and glycine-glutamic acid (GE); L ２ does not exist, or 【Chemistry 8-1】 【Chemistry 8-2】 wherein q is selected from any integer from 1 to 30 and p is any integer from 1 to 20; (1) L １ is coupled to Ab via a sulfhydryl, L １ has the following structure: 【Chemistry 9】 where R Ｌ１ｃ is selected from hydrogen, optionally substituted alkyl, and optionally substituted aryl; (2) L １ When L is coupled to Ab via an amino group, １ has the following structure: 【Chemistry 10】 is selected from (3) L １ is coupled to Ab via click chemistry, L １ has the following structure: 【Chemistry 11】 The antibody-drug conjugate of claim 25, wherein the antibody-drug conjugate is selected from the group consisting of:

27. Structural unit -Tr-L ３ -L ２ -L １ -but, 【Chemistry 12】 The antibody-drug conjugate of claim 26, selected from

28. Formula (III-A): 【Chemistry 13】 an antibody-drug conjugate of the structure represented by During the ceremony, Ab is as defined in any one of claims 1 to 6, and N ａ－Ｉ is any number from 1 to 10, L is as defined in any one of claims 25, 26, or 27; X is —O—, —S—, and —N(R １ａ )-, where R １ａ But H, C １ ~C ６ Alkyl, and C １ ~C ６ alkylhydroxy; R １ and R ２ are each independently H, protium, deuterium, tritium, halogen, C １ ~C ６ Alkyl and halogenated C １ ~C ６ alkyl, R ３ But halogenated C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, C １ ~C ６ Alkylthio, and —C １ ~C ６ Alkyl-O-P(=O)(OH) ２ is selected from, wherein C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, and C １ ~C ６ each alkylthio is optionally substituted with one or more substituents independently selected from halogen, CN, OH, or SH; Ring B is selected from phenyl or 5- to 6-membered heteroaryl, and Ring B is selected from halogen, OH, CN, NH ２ , C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy and halogenated C １ ~C ６ optionally substituted with one or more substituents selected from alkyl; W is a single bond, —O—, —S—, —C(O)—, —NH—, —C(O)NH—, —NHC(O)—, —C(O)—N(C １ ~C ６ alkyl)-, -N(C １ ~C ６ alkyl)-C(O)-, -C １ ~C ６ Alkylene-, -N(C １ ~C ６ alkyl)-, -C １－６ Alkylene -NH-, -O-C １ ~C ６ Alkylene-, and -C １ ~C ６ alkylene-O-, wherein C １ ~C ６ Alkyl and C １ ~C ６ The alkylene is selected from the group consisting of halogen, C １ ~C ６ Alkyl, C １ ~C ６ Alkoxy, halogenated C １ ~C ６ Alkyl, C ３ ~C ６ cycloalkyl, oxo, OH, CN, or NH ２ and optionally substituted with one or more substituents selected from V is a single bond or -(C(R ２ａ ) (R ２ｂ )) ｎ -, where R ２ａ and R ２ｂ are each independently H, halogen, OH, NH ２ , -CN,C １ ~C ６ Alkyl and halogenated C １ ~C ６ alkyl, or R ２ａ and R ２ｂ together with the carbon atom to which they are attached, form a carbonyl or C ３－６ forming a cycloalkyl, where n is selected from 1, 2, 3, 4, 5, or 6; Y １ H, halogen, OH, NH ２ , C １ ~C ６ Alkyl, -CN, C １ ~C ６ Alkoxy, -C １ ~C ６ Alkylhydroxy and halogenated C １ ~C ６ alkyl, The antibody-drug conjugate of any one of claims 1 to 27, wherein m is selected from 1, 2, or 3.

29. Formula (III-A1) to formula (III-A4): 【Chemistry 14】 an antibody-drug conjugate of the structure represented by During the ceremony, L, 【Chemistry 15】 The antibody-drug conjugate of claim 28, wherein the antibody-drug conjugate is selected from the group consisting of:

30. the below described: 【Chemistry 16-1】 【Chemistry 16-2】 having a structure selected from wherein each Ab is as defined in any one of claims 1 to 6, and N ａ－Ｉ The antibody-drug conjugate of any one of claims 1 to 29, wherein is any number from 1 to 10.

31. the below described: 【Chemistry 17】 having a structure selected from In the formula, N ａ－Ｉ is any number from 1 to 10, and preferably, N ａ－Ｉ is an integer or decimal number between 2 and 8, and preferably, N ａ－Ｉ is an integer or decimal number between 3 and 8, and preferably, N ａ－Ｉ is an integer or decimal number between 3 and 4 or between 4 and 5, and Hu033-03, Hu033-20, and Hu005-04 are anti-BDCA2 antibodies.

32. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 31 and a pharmaceutically acceptable carrier or excipient.

33. Use of the antibody-drug conjugate of any one of claims 1 to 31 or the pharmaceutical composition of claim 32 in the preparation of a medicament for treating and / or preventing a disease or disorder mediated by BDCA2 and plasmacytoid dendritic cells.

34. 33. A method for treating and / or preventing a disease or disorder mediated by BDCA2 and plasmacytoid dendritic cells, comprising administering to a subject in need thereof the antibody-drug conjugate of any one of claims 1 to 31 or the pharmaceutical composition of claim 32.

35. 35. The use according to claim 33, or the method according to claim 34, wherein the disease and / or disorder is selected from systemic lupus erythematosus, discoid lupus erythematosus, lupus nephritis, epidermal lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, systemic sclerosis (scleroderma), psoriasis, type I diabetes, dermatomyositis, and polymyositis.

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