Antibody-hormone drug conjugate and use thereof

Abstract

Disclosed is a class of antibody-hormone drug conjugates having novel structures, or pharmaceutically acceptable salts thereof. Specifically, provided are an antibody-drug conjugate having the general structural formula Ab-(L-D)n or a pharmaceutically acceptable salt thereof, a preparation method therefor, a pharmaceutical composition containing the conjugate, and a use thereof in the treatment of autoimmune diseases.

Description

Antibody-hormone drug conjugates and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to and the benefit of Chinese Patent Application No. 202410738268.9, filed June 7, 2024, the patent application file of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure belongs to the field of biological medicine, and relates to a class of antibody-hormone drug conjugates with novel structure, a preparation method thereof, a pharmaceutical composition containing the conjugate, and the use thereof in treating autoimmune diseases. BACKGROUND

[0004] Glucocorticoids (GCs) are extremely important regulatory molecules in the body, which play an important regulatory role in the development, growth, metabolism, and immune function of the body, and are the most important regulatory hormones of the body's stress response and the most widely used and effective anti-inflammatory and immunosuppressive agents in clinical practice. Glucocorticoids can be used to treat many autoimmune diseases and inflammatory diseases, including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. However, long-term use of glucocorticoids can cause osteoporosis, muscle atrophy, hypertension, and other side effects such as suppression of the body's immune system leading to increased infection. Therefore, the use of hormones in the treatment of autoimmune diseases is limited.

[0005] Th2 cytokines interleukin 4 (IL-4) and IL-13 and heterodimer IL-4 receptor (IL-4R) complex play a key role in the pathogenesis of allergic diseases. IL-4 is a cytokine produced mainly by activated T cells, monocytes, basophils, mast cells, and eosinophils. IL-13 is a cytokine produced by Th2 cells, CD4 cells, natural killer T cells, mast cells, basophils, and eosinophils. IL-13 is a central regulator of IgE synthesis, goblet cell hyperplasia, excessive mucus secretion, airway hyperresponsiveness, fibrosis, and chitinase upregulation. IL-4R is a type I transmembrane protein that can bind interleukin 4 and interleukin 13 to regulate the production of IgE antibodies in B cells.

[0006] IL-4 and its receptors play crucial roles in regulating humoral and adaptive immunity. These roles include stimulating the proliferation of activated B cells and T cells, differentiating CD4+ T cells into type II helper T cells, inducing the conversion of B cell antibody classes to IgE, and upregulating the production of the major histocompatibility complex type II. Studies have shown that IL-4 and its receptors play multiple roles in the immune responses to infectious diseases, autoimmune diseases, and tumors, with significant unmet clinical needs identified in Th2-mediated autoimmune diseases.

[0007] Th2-mediated autoimmune diseases include those affecting the skin, respiratory system, digestive system, and cardiovascular system, such as atopic dermatitis, chronic spontaneous urticaria, asthma, chronic obstructive pulmonary disease, and chronic sinusitis with nasal polyps. Studies have shown that cytokines such as IL-4 and IL-13 are involved in the pathogenesis of these diseases. Summary of the Invention

[0008] This disclosure combines the treatment of biological agents and hormone drugs to provide a novel antibody-hormone drug conjugate for the treatment of autoimmune diseases or inflammatory diseases.

[0009] In one aspect, this disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Ab-(LD). n ;

[0010] in,

[0011] The Ab is an antibody or its antigen-binding fragment;

[0012] L represents the connecting subunit;

[0013] D is the drug unit as shown in the following formula.

[0014] n is 1 to 16.

[0015] In some implementations, the connection subunit L is selected from... Its a-end is covalently connected to Ab, and its b-end is covalently connected to drug unit D, where L 1 The peptide residues are composed of 1 to 8 amino acids, and the peptide residues are further optionally coated with halogens, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic groups, and (C1-C6 alkyl)-(C=O)-(NCH2CH2). j1 -(OCH2CH2) j2 One or more substituents are selected from (OC1-C6 alkyl), wherein j1 and j2 are each independently selected from integers 0 to 20.

[0016] [Corr. to Rule 91 01.07.2025] In some embodiments, the linker unit L is selected from , the a end of which is covalently attached to Ab and the b end of which is covalently attached to the drug unit D, wherein L 1 is a peptide residue consisting of 1 to 8 amino acids, which is further optionally substituted with one or more substituents selected from the group consisting of halogen, CN, =0, C1-C6 alkyl, OH, 0(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl.

[0017] In some embodiments, the linker unit L is selected from , the a end of which is covalently attached to Ab and the b end of which is covalently attached to the drug unit D, wherein L 1 is a peptide residue consisting of 2 or 3 amino acids selected from glycine, alanine or glutamic acid, which is further optionally substituted with (C1-C6 alkyl)-(C=0)-(NCH2CH2) j1 -(OCH2CH2) j2 -(OC1-C6 alkyl), each of j1 and j2 is independently selected from an integer from 1 to 12; preferably, the peptide residue is further optionally substituted with .

[0018] In some embodiments, the linker unit L is selected from , the a end of which is covalently attached to Ab and the b end of which is covalently attached to the drug unit D.

[0019] In some embodiments, the linker unit L is selected from , the a end of which is covalently attached to Ab and the b end of which is covalently attached to the drug unit D.

[0020] [Corr. to Rule 91 01.07.2025] In some embodiments, the antibody-drug conjugate of the general formula Ab-(L-D) n or a pharmaceutically acceptable salt thereof is selected from the following compounds of Formula (I) or Formula (II) or a pharmaceutically acceptable salt thereof:

[0021] wherein Ab and n are as defined above.

[0022] In some embodiments, the Ab can specifically bind one or more antigens selected from the group consisting of: IL-4R, TNFa, AXL, BAFFR, BCMA, BCR, BDCA2, BDCA4, BTLA, BTNL2 BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD163, CD168, CD 177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR’s, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNAR, IFNAR1, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL6R, IL5R, IL7R, IL9R, Integrins, LAG3, LIFR, MAG / Siglec-4 (sialic acid-binding immunoglobulin-like lectin-4), MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, OX40 (CD134), PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1 (sialic acid-binding immunoglobulin-like lectin-1), Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4, IL2R-y, TL1A, and VTCN1.

[0023] In some embodiments, the Ab is an antibody or antigen-binding fragment thereof that specifically binds TNFa, IL-4R, or TL1A.

[0024] In some embodiments, the Ab is an antibody or antigen-binding fragment thereof that specifically binds IL-4R.

[0025] In some embodiments, the Ab is an antibody or antigen-binding fragment thereof that specifically binds IL-4R, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and / or a light chain variable region (VL);

[0026] the heavy chain variable region comprises CDR1, CDR2 and CDR3 selected from the VH as set forth in any one of SEQ ID NO. 1, 3, 5 and 7;

[0027] the light chain variable region comprises CDR1, CDR2 and CDR3 selected from the VL as set forth in any one of SEQ ID NO. 2, 4, 6 and 8.

[0028] Preferably, the heavy chain variable region comprises CDR1, CDR2 and CDR3 as set forth in:

[0029] (1) SEQ ID NO. 11, 12 and 13, respectively;

[0030] (2) SEQ ID NO. 17, 18 and 19, respectively;

[0031] (3) SEQ ID NO. 23, 24 and 25, respectively;

[0032] (4) SEQ ID NO. 29, 30 and 31, respectively; or

[0033] (5) a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or having at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid insertion, deletion and / or substitution, preferably a substitution to a conservative amino acid, to the sequence as set forth in any one of (1) to (4) above, respectively.

[0034] Preferably, the light chain variable region comprises CDR1, CDR2 and CDR3 as set forth in:

[0035] (1) SEQ ID NO. 14, 15 and 16, respectively;

[0036] (2) SEQ ID NO. 20, 21 and 22, respectively;

[0037] (3) SEQ ID NO. 26, 27 and 28, respectively;

[0038] (4) SEQ ID NO. 32, 33 and 34, respectively; or

[0039] (5) a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of any one of (1) to (4) above, respectively, or having at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to IL-4R comprises a heavy chain variable region comprising an amino acid sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs. 1, 3, 5, and 7; and / or a light chain variable region comprising an amino acid sequence having at least 80% identity to the sequence set forth in any one of SEQ ID NOs. 2, 4, 6, and 8.

[0041] Preferably, (1) the heavy chain variable region comprises the sequence set forth in SEQ ID NO. 1, and the light chain variable region comprises the sequence set forth in SEQ ID NO. 2;

[0042] (2) the heavy chain variable region comprises the sequence set forth in SEQ ID NO. 3, and the light chain variable region comprises the sequence set forth in SEQ ID NO. 4;

[0043] (3) the heavy chain variable region comprises the sequence set forth in SEQ ID NO. 5, and the light chain variable region comprises the sequence set forth in SEQ ID NO. 6;

[0044] (4) the heavy chain variable region comprises the sequence set forth in SEQ ID NO. 7, and the light chain variable region comprises the sequence set forth in SEQ ID NO. 8;

[0045] (5) the heavy chain variable region and / or the light chain variable region comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence of any one of (1) to (4), respectively; or, a sequence having at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations compared to the sequence of any one of (1) to (4), respectively; the mutations can be selected from insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds IL-4R further comprises a heavy chain constant region and / or a light chain constant region;

[0047] Preferably, the heavy chain constant region is selected from an IgG, such as IgGl, IgG2, IgG3, or IgG4, which can be selected from a human IgG, such as human IgG4; alternatively, the heavy chain constant region can be selected from an Fc region, a CH3 region, or a complete heavy chain constant region, which can be a human Fc region; alternatively, the heavy chain constant region has the sequence set forth in SEQ ID NO. 9 or 39; the light chain constant region is selected from a kappa chain or a lambda chain, preferably a kappa chain; alternatively, the light chain constant region has the sequence set forth in SEQ ID NO. 10.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds IL-4R is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds IL-4R is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific molecule (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a whole antibody, a fragment of a whole antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a Fab, a Fab’, a Fab’-SH, a F(ab’)2, a Fd, a Fv, a scFv, or a diabody.

[0050] Preferably, the multispecific molecule further comprises an antibody or antigen-binding fragment that specifically binds an antigen other than IL-4R or binds a different epitope of IL-4R than any of the above antibodies or antigen-binding fragments;

[0051] Preferably, the antigen other than IL-4R is selected from the group consisting of: (1) a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA); (2) an immune checkpoint; (3) a target that recruits and / or activates an immune cell.

[0052] In some embodiments, the Ab is an antibody or antigen-binding fragment thereof that specifically binds TNFα, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and / or a light chain variable region (VL);

[0053] the heavy chain variable region comprising CDR1, CDR2, and CDR3 of a VH set forth in SEQ ID NO. 40;

[0054] Alternatively, the heavy chain variable region comprises CDRs that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or have at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions, to the CDR1, CDR2, and CDR3 of the VH as set forth in SEQ ID NO. 40.

[0055] In some embodiments, the light chain variable region comprises CDR1, CDR2, and CDR3 of the VL as set forth in SEQ ID NO. 41;

[0056] Alternatively, the light chain variable region comprises CDRs that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or have at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions, to the CDR1, CDR2, and CDR3 of the VL as set forth in SEQ ID NO. 41.

[0057] In some embodiments, the antibody or antigen binding fragment thereof that specifically binds TNFα comprises: a heavy chain variable region comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence as set forth in SEQ ID NO. 40; or, a sequence that differs from the sequence as set forth in SEQ ID NO. 40 by at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations; said mutations can be selected from insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions; or / and a light chain variable region comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence as set forth in SEQ ID NO. 41; or, a sequence that differs from the sequence as set forth in SEQ ID NO. 41 by at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations; said mutations can be selected from insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions.

[0058] Preferably, the heavy chain variable region comprises a sequence as set forth in SEQ ID NO. 40, and the light chain variable region comprises a sequence as set forth in SEQ ID NO. 41;

[0059] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds TNFa further comprises a heavy chain constant region and / or a light chain constant region;

[0060] Preferably, the heavy chain constant region is selected from an IgG, such as IgGl, IgG2, IgG3, or IgG4, which can be selected from a human IgG, such as human IgG4; alternatively, the heavy chain constant region can be selected from an Fc region, a CH3 region, or a complete heavy chain constant region, which can be a human Fc region; alternatively, the heavy chain constant region has a sequence as set forth in SEQ ID NO. 42; the light chain constant region is selected from a kappa chain or a lambda chain, preferably a kappa chain; alternatively, the light chain constant region has a sequence as set forth in SEQ ID NO. 10.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds TNFa is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds TNFa is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific molecule (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a whole antibody, a fragment of a whole antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a Fab, a Fab’, a Fab’-SH, a F(ab’)2, a Fd, a Fv, a scFv, or a diabody.

[0063] Preferably, the multispecific molecule further comprises an antibody or antigen-binding fragment that specifically binds an antigen other than TNFa or binds a different epitope of TNFa than any of the above antibodies or antigen-binding fragments;

[0064] Preferably, the antigen other than TNFa is selected from the group consisting of: (1) a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA); (2) an immune checkpoint; (3) a target that recruits and / or activates an immune cell.

[0065] In some embodiments, the Ab is an antibody or antigen-binding fragment that specifically binds TL1A, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and / or a light chain variable region (VL);

[0066] the heavy chain variable region comprising CDR1, CDR2, and CDR3 of a VH as set forth in SEQ ID NO. 44;

[0067] Alternatively, the heavy chain variable region comprises CDRs that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or have at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions, to the CDR1, CDR2, and CDR3 of the VH as set forth in SEQ ID NO. 44.

[0068] In some embodiments, the light chain variable region comprises CDR1, CDR2, and CDR3 of the VL as set forth in SEQ ID NO. 45;

[0069] Alternatively, the light chain variable region comprises CDRs that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or have at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions, to the CDR1, CDR2, and CDR3 of the VL as set forth in SEQ ID NO. 45.

[0070] In some embodiments, the antibody or antigen binding fragment thereof that specifically binds to TL1A comprises: a heavy chain variable region comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence as set forth in SEQ ID NO. 44; or, a sequence that differs from the sequence as set forth in SEQ ID NO. 44 by at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations; said mutations can be selected from insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions; or / and a light chain variable region comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence as set forth in SEQ ID NO. 45; or, a sequence that differs from the sequence as set forth in SEQ ID NO. 45 by at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations; said mutations can be selected from insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions.

[0071] Preferably, the heavy chain variable region comprises a sequence as set forth in SEQ ID NO. 44, and the light chain variable region comprises a sequence as set forth in SEQ ID NO. 45;

[0072] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to TL1A comprises further comprises a heavy chain constant region and / or a light chain constant region;

[0073] Preferably, the heavy chain constant region is selected from an IgG, such as IgGl, IgG2, IgG3, or IgG4, which can be selected from a human IgG, such as human IgG4; alternatively, the heavy chain constant region can be selected from an Fc region, a CH3 region, or a complete heavy chain constant region, which can be a human Fc region; alternatively, the heavy chain constant region has a sequence as set forth in SEQ ID NO. 46; the light chain constant region is selected from a kappa chain or a lambda chain, preferably a kappa chain; alternatively, the light chain constant region has a sequence as set forth in SEQ ID NO. 10.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to TL1A comprises is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.

[0075] In some embodiments, the antibody or antigen-binding fragment thereof that specifically binds to TL1A comprises is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific molecule (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a whole antibody, a fragment of a whole antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a Fab, a Fab’, a Fab’-SH, a F(ab’)2, a Fd, a Fv, a scFv, or a diabody.

[0076] Preferably, the multispecific molecule further comprises an antibody or antigen-binding fragment that specifically binds to an antigen other than TL1A or binds to a different epitope of TL1A than any of the aforementioned antibodies or antigen-binding fragments;

[0077] Preferably, the antigen other than TL1A is selected from the group consisting of: (1) a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA); (2) an immune checkpoint; (3) a target that recruits and / or activates an immune cell.

[0078] In some embodiments, the foregoing general formula is Ab-(L-D) nan antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n is selected from 1-16, for example n is selected from 1-10, for example n is selected from 1-8, for example n is selected from 2-8, for example n is selected from 2-6, for example n is selected from 2-4, for example n is selected from 4-8, for example n is selected from 6-8.

[0079] In some embodiments, n is selected from 2-8, for example n is 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

[0080] In a second aspect, the present disclosure also provides a drug-linker compound or a pharmaceutically acceptable salt thereof, having the general structure of X-L-D, wherein:

[0081] X is selected from halogen, OS(O)2CH3, or OS(O)2CF3;

[0082] [Corresponding to Rule 91 correction 01.07.2025] L is selected from wherein the a end is covalently attached to X and the b end is covalently attached to the drug unit D, wherein L 1 is a peptide residue consisting of 1 to 8 amino acids, which is further optionally substituted with one or more substituents selected from the group consisting of halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclyl, and (C1-C6 alkyl)-(C=O)-(NCH2CH2) j1 -(OCH2CH2) j2 -(OC1-C6 alkyl), j1 and j2 are each independently selected from an integer from 0 to 20;

[0083] D is a drug unit having the following formula

[0084] In some embodiments, L is selected from wherein the a end is covalently attached to X and the b end is covalently attached to the drug unit D, wherein L 1is a peptide residue consisting of 1 to 8 amino acids, said peptide residue being further optionally substituted with one or more substituents selected from the group consisting of halogen, CN, =0, C1-C6 alkyl, OH, 0(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4- to 7-membered heterocyclyl.

[0085] [Corrected according to Rule 91 01.07.2025] In some embodiments, L is selected from wherein the a end is covalently attached to X and the b end is covalently attached to the drug unit D, wherein L 1 is a peptide residue consisting of 2 or 3 amino acids selected from glycine, alanine or glutamic acid, said peptide residue being further optionally substituted with (C1-C6 alkyl)-(C=0)-(NCH2CH2) j1 -(OCH2CH2) j2 -(OC1-C6 alkyl), said j1 and j2 are each independently selected from an integer from 1 to 12; preferably, said peptide residue is further optionally substituted with .

[0086] In some embodiments, L is selected from wherein the a end is covalently attached to X and the b end is covalently attached to the drug unit D.

[0087] In some embodiments, L is selected from wherein the a end is covalently attached to X and the b end is covalently attached to the drug unit D.

[0088] In some embodiments, X is selected from Br or I.

[0089] In some embodiments, X is selected from Br.

[0090] In some embodiments, the drug-linker compound of the present disclosure of the general formula X-L-D or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0091] In another aspect, the present disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate of the aforementioned general formula Ab-(L-D) n of the present disclosure or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0092] In another aspect, the present disclosure provides a method of treating an inflammatory disease or an autoimmune disease in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment, a therapeutically effective amount of an antibody-drug conjugate of the aforementioned general formula Ab-(L-D) n of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0093] In another aspect, the present disclosure provides use of an antibody-drug conjugate of the general formula: Ab-(L-D) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the manufacture of a medicament for treating an inflammatory disease or an autoimmune disease.

[0094] In another aspect, the present disclosure provides use of an antibody-drug conjugate of the general formula: Ab-(L-D) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in treating an inflammatory disease or an autoimmune disease.

[0095] In another aspect, the present disclosure provides an antibody-drug conjugate of the general formula: Ab-(L-D) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for treating an inflammatory disease or an autoimmune disease.

[0096] In some embodiments, the inflammatory disease or autoimmune disease can be selected from rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, atopic dermatitis, urticaria, myocarditis, encephalitis, uveitis, chronic obstructive pulmonary disease, vasculitis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, microscopic colitis, collagenous colitis, indeterminate colitis, necrotizing enterocolitis, transmural colitis, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma.

[0097] In another aspect, the present disclosure provides a method for preparing an antibody-drug conjugate of the general formula: Ab-(L-D) n or a pharmaceutically acceptable salt thereof, comprising the step of coupling a drug-linker compound of the general formula: X-L-D with the antibody.

[0098] In another aspect, the present disclosure provides a method for preparing an antibody-drug conjugate of the general formula: Ab-(L-D) n or a pharmaceutically acceptable salt thereof, comprising the step of linking a drug unit D of the present disclosure with the antibody Ab; optionally, through the linker unit L.

[0099] The antibody-drug conjugate provided by the present disclosure has significant anti-inflammatory activity and / or excellent plasma stability and / or reduced toxic side effects and / or significant bystander effect.

[0100] Definitions and explanations

[0101] Unless otherwise indicated, the terms used in the present disclosure have the following meanings, the definitions of the groups and terms recited in the present disclosure, including the definitions as examples, exemplary definitions, preferred definitions, the definitions recited in the tables, the definitions of the specific compounds in the examples, etc., can be combined and integrated with each other arbitrarily. A specific term should not be considered as indefinite or unclear without a special definition, but should be understood according to the ordinary meaning in the art. When a trade name appears herein, it is intended to refer to its corresponding product or active ingredient thereof.

[0102] “Antibody drug conjugate (ADC)” refers to a monoclonal antibody or antibody fragment connected with a small molecule compound having biological activity through a stable linker unit.

[0103] The term “linker” or “linker unit” refers to a fragment of chemical structure or a chemical bond which is connected with an antibody or antibody fragment at one end and with a drug at the other end.

[0104] The term “drug” refers to a small molecule compound having biological activity in a living organism. In some embodiments of the present disclosure, the drug is a glucocorticoid receptor agonist having anti-inflammatory function.

[0105] The term “DAR” or “drug antibody ratio” refers to the average number of small molecule glucocorticoid receptor agonist drugs connected with each antibody molecule. In an antibody-drug conjugate of the general formula Ab-(L-D) n , the DAR is defined by the variable “n”, which can be an integer or a decimal number.

[0106] The term "antibody" is used in the broadest sense, and refers to a polypeptide or combination of polypeptides that contain sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region to specifically bind to an antigen. "Antibody" herein encompasses various formats and various structures, as long as they exhibit the desired antigen binding activity. "Antibody" herein includes surrogate protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and all-synthetic scaffolds containing, for example, biocompatible polymers. See, e.g., Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds can also include non-antibody-derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to, tenascin, fibronectin, peptide aptamers, and the like.

[0107] "Antibody" herein includes a typical "four-chain antibody" which belongs to an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC); the heavy chain refers to a polypeptide chain consisting of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, a heavy chain constant region CH3 domain in the direction of N-terminal to C-terminal; and, when the full-length antibody is of IgE isotype, optionally further comprising a heavy chain constant region CH4 domain; the light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction of N-terminal to C-terminal; the heavy chain and the heavy chain are connected by a disulfide bond, and the heavy chain and the light chain are connected by a disulfide bond, forming a "Y" shape structure. Due to the difference in the amino acid composition and the arrangement order of the immunoglobulin heavy chain constant region, the antigenicity is also different. Accordingly, "immunoglobulin" herein can be divided into five categories, or called isotypes of immunoglobulin, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain, respectively. The same category of Ig can be divided into different subcategories according to the difference in the amino acid composition of the hinge region and the number and position of the heavy chain disulfide bond, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4, IgA can be divided into IgA1 and IgA2. The light chain is divided into κ chain or λ chain through the constant region. Each of the five categories of Ig can have κ chain or λ chain.

[0108] The term "antibody" herein also includes antibodies that do not comprise light chains, such as heavy-chain antibodies (HCAbs) produced by Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe, and Vicugna pacos, and immunoglobulin new antigen receptor (IgNAR) found in cartilaginous fishes such as sharks.

[0109] The term "antibody" herein can be derived from any animal, including but not limited to human and non-human animals, which can be selected from primates, mammals, rodents, and vertebrates, such as a camelid, a llama, a guanaco, a vicuna, a sheep, a rabbit, a mouse, a rat, or a cartilaginous fish (e.g., a shark).

[0110] The term "antibody" herein includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, an intact antibody, a fragment of an intact antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0111] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variants that can arise during production of the antibody, such variants are typically present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. The modifier "monoclonal" is not to be construed as requiring production of the antibody or antigen binding molecule by any particular method. For example, the monoclonal antibodies can be prepared by a variety of techniques, including but not limited to the hybridoma technique, recombinant DNA methods, phage display techniques, and use of transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

[0112] The term "natural antibody" refers to an antibody manufactured and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by genetic engineering, antibody engineering, and the like. Exemplarily, the "engineered antibody" includes a humanized antibody, a small molecule antibody (e.g., scFv, etc.), a bispecific antibody, and the like.

[0113] The term "monospecific" refers to having one or more binding sites, wherein each binding site binds the same epitope of the same antigen.

[0114] The term "multispecific antibody" refers to an antibody having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," "quadrispecific," and the like refer to the number of different epitopes that the antibody / antigen binding molecule can bind.

[0115] The term "valency" denotes the presence of a specified number of binding sites in an antibody / antigen binding molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen binding molecule.

[0116] "Full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure.

[0117] "Antigen binding fragment" and "antibody fragment" are used herein interchangeably herein to refer to a portion of an intact antibody that specifically binds an antigen, but which does not comprise a full-length antibody. "Antigen binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, and scFv.

[0118] Papain digestion of a whole antibody produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. The Fab fragment also contains, in addition to the VL and VH domains, the constant domains of the light and heavy chains, CL and CH1, respectively. Thus, the term "Fab fragment" herein refers to a light chain fragment containing the VL and CL domains of the light chain, and a heavy chain fragment containing the VH and CH1 domains of the heavy chain. Fab' fragments differ from Fab fragments by the presence of one or more additional residues at the carboxy terminus of the CH1 domain of the heavy chain, including the presence of one or more cysteines from the hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains carry a free thiol group. Pepsin treatment yields an F(ab')2 fragment that has two antigen binding sites and a part of the Fc region.

[0119] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Roseburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond between the VH and VL of the scFv can also be present, forming a disulfide-bonded Fv (dsFv).

[0120] The term "diabody" has VH and VL domains on a single polypeptide chain, but uses a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and creating two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).

[0121] The term "chimeric antibody" refers to an antibody in which a portion of the light chain or / and the heavy chain is derived from one antibody (which can be derived from a particular species or belong to a particular antibody class or subclass), and the other portion of the light chain or / and the heavy chain is derived from another antibody (which can be derived from the same or a different species or belong to the same or a different antibody class or subclass), but retains the binding activity of the target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 6855 (1984)). For example, the term "chimeric antibody" can include an antibody (e.g., a human murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are from a first antibody (e.g., a murine antibody), while the heavy and light chain constant regions of the antibody are from a second antibody (e.g., a human antibody).

[0122] The term "humanized antibody" refers to a non-human-derived antibody that has been genetically engineered to have an amino acid sequence that is modified to increase homology to the sequence of a human-derived antibody. Typically, a humanized antibody has all or a portion of the CDR regions from a non-human-derived antibody (donor antibody) and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) from a human-derived immunoglobulin (acceptor antibody). A humanized antibody typically retains or partially retains the desired properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to enhance immune cell activity, ability to enhance immune response, etc.

[0123] The term "fully human antibody" refers to an antibody having variable regions in which both the FRs and the CDRs are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region also is derived from a human germline immunoglobulin sequence. A fully human antibody herein can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro, or by somatic mutation in vivo). However, a "fully human antibody" herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.

[0124] The term "variable region" refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen, "variable region of the heavy chain" and "VH" or "HCVR" are used interchangeably, and "variable region of the light chain" and "VL" or "LCVR" are used interchangeably. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain can be sufficient to confer antigen-binding specificity. The term "complementarity determining region" and "CDR" are used interchangeably herein to refer to the hypervariable region of a heavy chain variable region (VH) or light chain variable region (VL), which is also known as a hypervariable loop (HVR) because it forms loops that are believed to be involved in binding to an antigen, where the heavy chain variable region CDRs can be abbreviated as HCDRs and the light chain variable region CDRs can be abbreviated as LCDRs. The term "framework region" or "FR region" is interchangeable and refers to those amino acid residues in a variable region of a heavy chain or light chain that are outside the CDRs. Typically, a canonical antibody variable region is composed of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0125] The "CDRs" herein can be annotated and defined in ways known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tools websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). The CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0126] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system set forth by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0127] The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that is not directly involved in binding of an antibody to an antigen, but exhibits effector functions such as interaction with Fc receptors, which has a more conserved amino acid sequence relative to the variable domains of the antibody. The "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. The "heavy chain constant region" includes both "full length heavy chain constant region" and "heavy chain constant region fragment", the former having substantially similar structure as the native antibody constant region, while the latter includes only "a portion of the full length heavy chain constant region". Exemplarily, a typical "full length antibody heavy chain constant region" consists of CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it further includes CH4 domain; when the antibody is heavy chain antibody, it does not include CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" can be selected from CH1, Fc or CH3 domain.

[0128] The term "light chain constant region" herein refers to the carboxy-terminal portion of an antibody light chain that is not directly involved in binding of an antibody to an antigen, which can be selected from constant kappa domain or constant lambda domain.

[0129] The term "Fc" herein refers to the carboxy-terminal portion of an intact antibody that results from papain cleavage, which typically comprises the CH3 and CH2 domains of an antibody. The Fc region includes, e.g., native sequence Fc region, recombinant Fc region, and variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary slightly, the Fc region of a human IgG heavy chain typically extends from the Cys226 position or from Pro230 to the carboxy-terminus thereof. The C-terminal lysine (residue 447 according to Kabat numbering system) of the Fc region can be removed, e.g., during production or purification of the antibody, or by recombinant engineering of nucleic acid encoding the antibody heavy chain, and therefore, the Fc region can include or exclude Lys447.

[0130] The term "conservative amino acid" herein refers generally to amino acids that are of the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Illustratively, the amino acids within each of the following groups are of conservative amino acid residues to each other, substitution of an amino acid residue within a group is a substitution of a conservative amino acid:

[0131] Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

[0132] 1) Alanine (A), Serine (S), Threonine (T);

[0133] 2) Aspartic acid (D), Glutamic acid (E);

[0134] 3) Asparagine (N), Glutamine (Q);

[0135] 4) Arginine (R), Lysine (K), Histidine (H);

[0136] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0137] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0138] The term "identity" herein can be calculated by determining the percentage of "identical" amino acid residues or nucleotides between two amino acid sequences or two nucleic acid sequences, by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-identical sequences can be disregarded for comparison purposes). Subsequently, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

[0139] The percentage of identity between two sequences varies as a function of the number of identical positions, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each gap.

[0140] Sequence comparisons and percent identity calculations between two sequences can be accomplished using a mathematical algorithm. For instance, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. By way of further example, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the set that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a shift - frame gap penalty of 5.

[0141] As used herein indicates a point of attachment.

[0142] [Corr. to Rule 91 01.07.2025] The graphical representation of racemic or enantiomerically pure compounds herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, a wedge and a dashed wedge indicates the absolute configuration of a stereocenter, with a solid and a dashed bond indicates the relative configuration of a stereocenter (e.g., the cis or trans configuration of an alicyclic compound).

[0143] The compounds of the present disclosure can have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, or asymmetric double bonds, and thus the compounds of the present disclosure can exist in particular geometric or stereoisomeric forms. The particular geometric or stereoisomeric forms can be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)-isomers, (L)-isomers, as well as racemic mixtures or other mixtures thereof, such as those that contain an enantiomeric or diastereomeric excess, all of which are within the scope of the compounds of the present disclosure. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms can be present in a substituent group, and all such isomers and mixtures thereof are included within the definition of the compounds of the present disclosure. The compounds of the present disclosure containing an asymmetric atom can be isolated in optically active form or as racemic mixtures, and the optically active forms can be obtained by separation from the racemic mixtures or by using chiral starting materials or chiral reagents in the synthetic sequence.

[0144] The term "substituted" means that any one or more hydrogen atoms on the particular atom is replaced with a substituent group, provided that the valence of the particular atom is not exceeded, and that the substituted compound is stable. When the substituent is oxo (i.e., =0), it means that two hydrogen atoms are replaced by the oxo group. The oxo group cannot be on an aromatic group.

[0145] The term "optionally" or "optional" means that the subsequently described event or circumstance can or can not occur, and this description includes instances where the event or circumstance occurs and instances where it does not. For example, an ethyl group "optionally" substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), mono-substituted (CH2CH2F, CH2CH2C1, etc.), poly-substituted (CHFCH2F, CH2CHF2, CHFCH2C1, CH2CHC12, etc.), or fully substituted (CF2CF3, CF2CC13, CC12CC13, etc.). One skilled in the art will appreciate that for any given group containing one or more substituents, no substitution or substitution pattern is introduced that is not spatially possible and / or synthetically feasible.

[0146] When any variable (e.g., R a , R b ) occurs more than one time in a compound, its definition in each occurrence is independent of the other. For example, if a group is substituted with 2 R b groups, then each R b is selected independently.

[0147] C m -C n" is intended to mean that the group can have an integer number of carbon atoms in the given range. For example, "Ci-C6alkyl" is intended to mean that the alkyl group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. 10 " is intended to mean that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0148] The term "alkyl" refers to a hydrocarbon group of formula C n H 2n+1 The term "Ci-C6alkyl" can be understood to mean a straight or branched chain saturated hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. Specific examples of said alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like. The term "Ci-C3alkyl" can be understood to mean a straight or branched chain saturated alkyl group having 1 to 3 carbon atoms. Said "Ci-C6alkyl" can further comprise "Ci-C3alkyl".

[0149] The term "cycloalkyl" refers to a carbocyclic group that is fully saturated and exists as a monocyclic, fused ring, bridged ring, or spiro ring, among others. Unless otherwise indicated, the carbocyclic ring is typically a 3- to 10-membered ring. The term "C3-C6cycloalkyl" can be understood to mean a saturated monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms, specific examples including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, among others.

[0150] The term "heterocyclyl" refers to a monocyclic, bicyclic, spiro, or bridged ring radical which is completely saturated or partially saturated (i.e., is not a heteroaromatic radical which is aromatic overall), which contains from 1 to 5 heteroatoms or heteroatom groups (i.e., groups of atoms containing a heteroatom) in its ring atom count, including but not limited to a nitrogen atom (N), an oxygen atom (O), a sulfur atom (S), a phosphorus atom (P), a boron atom (B), -S(=0)2-, -S(=0)-, -P(=0)2-, -P(=0)-, -NH-, -S(=0)(=NH)-, -C(=0)NH-, or -NHC(=0)NH-, and the like, in its ring atoms. The term "4-7 membered heterocyclyl" refers to a heterocyclyl radical having a ring atom count of 4, 5, 6, or 7, and which contains from 1 to 3 heteroatoms or heteroatom groups independently selected from those described above in its ring atom count. Specific examples of 4-membered heterocyclyl groups include, but are not limited to, azetidinyl or oxetanyl; specific examples of 5-membered heterocyclyl groups include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazolyl, or 2,5-dihydro-lH-pyrrolyl; specific examples of 6-membered heterocyclyl groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[l,3,4]thiadiazinyl; and specific examples of 7-membered heterocyclyl groups include, but are not limited to, diazepinyl.

[0151] The term "halo" or "halogen" refers to fluoro, chloro, bromo, or iodo.

[0152] The term "treatment" refers to a surgical or therapeutic treatment whose purpose is to prevent, slow down (reduce), or halt the progress of an undesirable physiological change or pathological condition, such as cancer, autoimmune disease, and viral infection, in a subject. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. A subject in need of treatment includes a subject who has a condition or disease, a subject who is predisposed to having a condition or disease, or a subject who is intended to prevent a condition or disease. When referring to the terms slow down, reduce, diminish, ameliorate, palliate, and the like, the meaning also includes elimination, disappearance, nonoccurrence, and the like.

[0153] The term "effective amount" means the amount of a therapeutic agent, administered alone or in combination with another therapeutic agent, which prevents, slows or halts the progression of a disease condition or the disease. An "effective amount" also means the amount of a compound which alleviates a symptom, e.g., treats, cures, prevents or slows the progression of, or increases the speed of treatment, cure, prevention or slowing of, the relevant medical condition. When the active ingredient is administered individually to an individual, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to combined amounts of the active ingredients that will achieve the therapeutic result, regardless of whether administration is combined, sequential or simultaneous.

[0154] The term "subject" refers to an organism that receives treatment for a particular disease or condition as described herein. Examples of subjects and patients include mammals such as humans, primates (e.g., monkeys) or non-primate mammals that receive treatment for a disease or condition.

[0155] The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" will vary depending on the compound, the disease state and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art considering the knowledge in the art and the disclosure.

[0156] The term "pharmaceutically acceptable" pertains to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0157] The term "pharmaceutically acceptable salt" refers to salts of a compound that are pharmaceutically acceptable, including salts of inorganic acids or organic acids, and salts of inorganic bases or organic bases.

[0158] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure, or salts thereof, with pharmaceutically acceptable excipients. The goal of a pharmaceutical composition is to facilitate administration of a compound of the present disclosure to an organism.

[0159] The term "pharmaceutically acceptable excipient" refers to an excipient that is not biologically or otherwise undesirable, i.e., the excipient can be administered to an organism without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The presence of an excipient in a composition does not imply that the excipient is intended to be part of the dosage form. Suitable excipients are well known to those skilled in the art.

[0160] The words "comprise" or "comprising" and variations thereof such as "comprises" or "comprising", when used in this document, can be understood to encompass the terms "consisting of" or "consisting of" and variations thereof, e.g., "consists" or "consisting of".

[0161] The present disclosure also includes isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I, and 36 Cl, and the like.

[0162] Certain isotopically-labeled compounds of the present disclosure (for example, with 3 H and 14 C) can be used in compound and / or substrate tissue distribution analysis. Tritiated (i.e., with 3 H), and carbon-14 (i.e., with 14 C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically-labeled compounds of the present disclosure can generally be prepared by substituting a readily available isotopically-labeled reagent for a non-isotopically labeled reagent in a procedure similar to those disclosed in the schemes and / or examples below.

[0163] The pharmaceutical compositions of the present disclosure can be adapted for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms. For example, the pharmaceutical compositions of the present disclosure can be in the form of sterile injectable aqueous solutions. Other solvents or solvents systems can be acceptable for use in the pharmaceutical compositions of the present disclosure at the time of use, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0164] In all methods of administration of the compounds described herein, the daily dose administered is from 0.001 mg / kg to 600 mg / kg of body weight, preferably from 0.05 mg / kg to 200 mg / kg of body weight, more preferably from 0.1 mg / kg to 100 mg / kg of body weight, in single or divided doses.

[0165] The compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by a combination of the specific embodiments set forth below with other chemical synthetic methods well known to those skilled in the art, and equivalents thereof as appreciated by those skilled in the art, preferred embodiments including but not limited to the examples of the present disclosure.

[0166] The chemical reactions of the specific embodiments of the present disclosure are performed in solvents appropriate to the reagents and materials employed and suitable for the chemical changes being effected. In some instances, the solvent can be changed after a given reaction to facilitate the further reactions and / or to facilitate purification. In some instances, it can be desired to remove protecting groups by solvolysis. The synthesis of compounds of the present disclosure can require protection of intermediates as appreciated by those skilled in the art. The protection and deprotection of functional groups can be affected according to established procedures well known to those skilled in the art.

[0167] An important consideration in the synthetic route planning in the art is the selection of appropriate protecting groups for reactive functional groups (e.g., amino, carboxyl in the present disclosure), for example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited by the present disclosure are incorporated herein in their entirety. BRIEF DESCRIPTION OF DRAWINGS

[0168] Figure 1 is a graph showing the results of preparing human IL4Rα-293T cells overexpressing in Test Example 1 of the present disclosure.

[0169] Figure 2 is a graph showing the results of detecting ear thickness in an atopic dermatitis mouse model in Test Example 4 of the present disclosure.

[0170] Figure 3 is a graph showing the results of detecting serum IgE in an atopic dermatitis mouse model in Test Example 4 of the present disclosure.

[0171] Figure 4 is a graph showing the results of detecting aspartate aminotransferase (AST) in SD rat models of hormone compounds 3 and 4 in Test Example 7 of the present disclosure.

[0172] Figure 5 is a graph showing the results of detecting alanine aminotransferase (ALT) in SD rat models of hormone compounds 3 and 4 in Test Example 7 of the present disclosure.

[0173] Figure 6 is a graph showing the results of detecting aspartate aminotransferase (AST) in SD rat models of hormone compounds 1 and 2 in Test Example 7 of the present disclosure.

[0174] Figure 7 is a graph showing the results of detecting alanine aminotransferase (ALT) in SD rat models of hormone compounds 1 and 2 in Test Example 7 of the present disclosure.

[0175] Figure 8 shows the detection results of the activity of the anti-IL-4R antibody-drug conjugate disclosed in the GRE reporter gene assay.

[0176] Figure 9 shows the detection results of the activity of the anti-TNFα antibody-drug conjugate disclosed in the GRE reporter gene assay.

[0177] Figure 10 shows the detection results of the activity of the anti-TL1A antibody-drug conjugate disclosed in the GRE reporter gene assay.

[0178] Figure 11 shows the detection results of the bystander effect of the anti-IL-4R antibody-drug conjugate disclosed in this invention.

[0179] Figure 12 shows the detection results of the bystander effect of the anti-TNFα antibody-drug conjugate disclosed in this invention.

[0180] Figure 13 shows the detection results of the bystander effect of the anti-TL1A antibody-drug conjugate disclosed in this invention. Detailed Implementation

[0181] The invention is described in detail below with reference to embodiments, but this does not imply any adverse limitation of the present disclosure. The present disclosure has been described in detail, including specific embodiments thereof. Various changes to the specific embodiments of the present disclosure will be apparent to those skilled in the art without departing from the spirit and scope of the present disclosure. All reagents used in this disclosure are commercially available and can be used without further purification.

[0182] Unless otherwise stated, the proportions expressed for mixed solvents are volume-based.

[0183] Unless otherwise stated, % refers to wt%.

[0184] Compounds are processed manually or Software naming conventions are used; commercially available compounds use supplier catalog names.

[0185] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are measured in units of 10⁻¹⁰. -6 (ppm). The solvents used for NMR determination were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., with tetramethylsilane (TMS) as the internal standard; "IC 50 "Hardest effective concentration (HIC)" refers to the concentration at which half of the maximum inhibitory effect is achieved. 50 "Refers to the concentration that produces half of the maximum effect concentration, i.e., the concentration that causes 50% of the maximum effect."

[0186] Example 1: Synthesis of ((S)-4-(2-(2-bromoacetamido)acetamido)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (Drug-Linker 1)

[0187] First step: Synthesis of tert-butyl (S)-4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoate (Intermediate 2)

[0188] (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5- yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b- decahydro-lH-naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxepin-4(2H)-one (Hormone Compound 1, 10.5 g, 17.16 mmol), (2S)-2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl]amino]-5-oxo- pentanoic acid tert-butyl ester (8.28 g, 17.16 mmol), 2,6-dimethylpyridine (5.52 g, 51.49 mmol, 6.00 mL) and 2-(7-azabenzotriazol-l-yl)-l,l,l',l'-tetramethyluronium hexafluoro-phosphate (HATU, 9.71 g, 25.75 mmol) were dissolved in N,N-dimethylformamide (80 mL) and the reaction was stirred at 25 °C for 1 hour. After the reaction was completed, the reaction was diluted with 500 mL of ethyl acetate, washed with water (300 mL x 3), washed with brine (300 mL x 1), dried over anhydrous sodium sulfate, concentrated, and the residue was separated and purified by column chromatography (silica, eluent: dichloromethane:methanol = 10: 1) to obtain Intermediate 2 (11.5 g, 62%).

[0189] MS m / z (ESI): 1076.4 [M+H];

[0190] 1H NMR (400 MHz, DMSO-d6) δ 9.35 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.71 (d, J = 7.4 Hz, 2H), 7.62 - 7.51 (m, 1H), 7.49 - 7.26 (m, 8H), 7.23 - 7.14 (m, 2H), 6.83 (s, 1H), 6.21 - 6.12 (m, 1H), 5.97 - 5.89 (m, 1H), 5.38 (s, 1H), 5.04 (dt, J = 28.3, 6.0 Hz, 1H), 4.91 (d, J = 5.2 Hz, 1H), 4.77 (d, J = 3.3 Hz, 1H), 4.58 - 4.44 (m, 4H), 4.34 - 4.11 (m, 5H), 3.80 (s, 2H), 3.72 - 3.58 (m, 2H), 3.19 - 3.07 (m, 3H), 2.64 - 2.51 (m, 1H), 2.38 - 2.19 (m, 3H), 2.19 - 2.08 (m, 1H), 2.02 (s, 1H), 1.97 - 1.87 (m, 1H), 1.84 - 1.58 (m, 6H), 1.38 (d, J = 7.6 Hz, 11H), 1.12 - 0.98 (m, 2H), 0.87 (d, J = 10.4 Hz, 3H).

[0191] Step 2: Synthesis of tert-butyl (S)-4-(2-aminoacetamido)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoate (Intermediate 3)

[0192] Piperidine (147.16 mg, 1.73 mmol) was added dropwise to a solution of Intermediate 2 (620 mg, 576.08 μmol) in acetonitrile (8 mL), and the reaction was stirred at 25 °C for 2 h. After the reaction was completed, the reaction was diluted with ethyl acetate (50 mL), washed with water (30 mL x 1), and the organic phase was separated, washed with saturated brine (20 mL), dried, filtered, and concentrated. The residue was separated and purified by normal phase column chromatography (silica, eluent: dichloromethane:methanol = 10:1) to give Intermediate 3 (320 mg, 65%).

[0193] MS m / z (ESI): 854.40 [M+H];

[0194] 1 H NMR (400 MHz, DMSO-d6) δ 9.44 (s, 1H), 8.11 (s, 1H), 7.46 (s, 1H), 7.41 - 7.34 (m, 2H), 7.31 (d, J = 10.1 Hz, 1H), 7.22 (d, J = 7.8 Hz, 2H), 6.85 (s, 1H), 6.21 - 6.14 (m, 1H), 5.96 - 5.89 (m, 1H), 5.39 (s, 1H), 5.09 (t, J = 5.9 Hz, 1H), 4.92 (d, J = 5.4 Hz, 1H), 4.79 (d, J = 3.4 Hz, 1H), 4.61 - 4.45 (m, 3H), 4.29 (q, J = 3.4 Hz, 1H), 4.23 - 4.16 (m, 1H), 4.16 - 4.06 (m, 2H), 3.81 (s, 2H), 3.19 - 3.11 (m, 5H), 2.56 (dd, J = 13.6, 5.5 Hz, 1H), 2.37 - 2.19 (m, 3H), 2.12 (qd, J = 11.1, 3.9 Hz, 1H), 2.07 - 1.91 (m, 2H), 1.84 - 1.66 (m, 4H), 1.63 (tt, J = 10.4, 5.8 Hz, 1H), 1.39 (d, J = 6.1 Hz, 9H), 1.05 (ddd, J = 24.5, 12.0, 4.2 Hz, 2H), 0.86 (s, 3H).

[0195] Step 3: Synthesis of tert-butyl (S)-4-(2-(2-bromoacetamido)acetamido)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2,3-dihydrobenofuran-7-yl)amino)-5-oxopentanoate (Intermediate 4)

[0196] Intermediate 3 (360 mg, 443.37 µmol) and bromoacetic acid (123.21 mg, 886.75 µmol) were dissolved in N,N-dimethylformamide (3 mL), 2-ethoxy-1,2-dihydro-1- quinolinecarboxylic acid ethyl ester (EEDQ, 175.43 mg, 709.40 µmol) was added, and the reaction solution was stirred at 25 °C for 30 min. After the reaction was completed, the reaction solution was diluted with ethyl acetate (30 mL), washed with water (10 mL x 1), and separated into organic and aqueous phases. The organic phase was washed with saturated brine (10 mL), dried, filtered, and concentrated. The residue was separated and purified by column chromatography (filler C18 (40-60 µm), eluent: water / acetonitrile = 3 / 1) to obtain intermediate 4 (280 mg, 68%).

[0197] MS m / z (ESI): 974.30 [M+H];

[0198] 1 H NMR (400 MHz, DMSO-d6) δ 9.36 (s, 1H), 8.49 (t, J = 5.7 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 7.44 (d, J = 1.6 Hz, 1H), 7.36 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 10.1 Hz, 1H), 7.21 (d, J = 7.9 Hz, 2H), 6.85 (d, J = 1.6 Hz, 1H), 6.17 (dd, J = 10.1, 1.9 Hz, 1H), 5.93 (t, J = 1.6 Hz, 1H), 5.39 (s, 1H), 5.10 (t, J = 6.0 Hz, 1H), 4.92 (d, J = 5.4 Hz, 1H), 4.79 (d, J = 3.2 Hz, 1H), 4.57 - 4.47 (m, 4H), 4.29 (p, J = 3.2 Hz, 1H), 4.18 (dd, J = 19.4, 5.3 Hz, 1H), 3.94 (s, 1H), 3.85 - 3.75 (m, 4H), 3.15 (t, J = 8.7 Hz, 2H), 2.56 (dd, J = 13.4, 5.4 Hz, 1H), 2.35 - 2.18 (m, 4H), 2.12 (tt, J = 10.9, 5.4 Hz, 1H), 2.02 (d, J = 6.8 Hz, 1H), 1.98 - 1.91 (m, 1H), 1.81 - 1.58 (m, 6H), 1.39 (d, J = 4.0 Hz, 9H), 1.05 (ddd, J = 23.3, 11.7, 4.2 Hz, 2H), 0.86 (s, 3H).

[0199] Step 4: Synthesis of (S)-4-(2-(2-bromoacetamido)acetamido)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (Drug-Linker 1)

[0200] Intermediate 4 (261.27 mg, 267.98 μmol) was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (1 mL), and the reaction solution was stirred at 25 °C for 30 min. After the reaction was completed, the reaction solution was concentrated to remove the solvent. The residue was separated and purified by reverse column chromatography (filler C18 (40-60 μm), eluent: water / acetonitrile = 4 / 1) to obtain Drug-Linker 1 (123 mg, 50%).

[0201] MS m / z (ESI): 918.30 [M+H];

[0202] 1 H NMR (400 MHz, DMSO-d6) δ 12.14 (s, 1H), 9.36 (s, 1H), 8.52-8.46 (m, 1H), 8.19 (d, J = 7.9 Hz, 1H), 7.41 (s, 1H), 7.36 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 10.1 Hz, 1H), 7.21 (d, J = 7.8 Hz, 2H), 6.84 (s, 1H), 6.19-6.13 (m, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 5.12-5.06 (m, 1H), 4.91 (d, J = 5.0 Hz, 1H), 4.78 (d, J = 3.2 Hz, 1H), 4.61-4.42 (m, 4H), 4.29 (s, 1H), 4.20-4.14 (m, 1H), 3.93 (s, 2H), 3.86-3.73 (m, 4H), 3.17-3.11 (m, 2H), 2.56-2.52 (m, 1H), 2.39-2.23 (m, 3H), 2.11 (d, J = 10.6 Hz, 1H), 2.06-1.90 (m, 2H), 1.84-1.60 (m, 6H), 1.39 (s, 3H), 1.13-0.98 (m, 2H), 0.86 (s, 3H).

[0203] Example 2: (S)-2-(2-bromoacetamido)-N1 - ((S)-1-((S)-1-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxo-10-yl)benzyl)-2,3- dihydrobenzofuran-7-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-N 5 Synthesis of (2,5,8,11,14,17,20,23,26-nonoxyl)octacosanamide (Drug-Linker 2)

[0204] Synthesis of ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert- butoxy)-5-oxopentanoyl)-L-alanyl-L-alanine

[0205] (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (2-1, 5.00 g, 11.7 mmol) and N-hydroxysuccinimide (OHSu, 1.50 g, 12.9 mmol) were dissolved in tetrahydrofuran (30 mL), N,N’-dicyclohexylcarbodiimide (DCC, 2.60 g, 12.9 mmol) was added and reacted at room temperature for 2 h. The reaction solution was then filtered, L-alanyl-L-alanine (2.10 g, 12.8 mmol) and sodium bicarbonate (1.50 g, 17.5 mmol) were added to the filtrate and water (5 mL) was added and reacted for 2 h. LC-MS detection showed that the reaction was complete, the reaction solution was added with dilute hydrochloric acid (2M, 8.7 mL), then diluted with ethyl acetate (10 mL), the aqueous phase was extracted with ethyl acetate (20 mL*3), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure until a large amount of solid was precipitated, then filtered, and the filter cake was dried to obtain the compound (2-2, 2.8 g).

[0206] LC-MS (ESI): m / z = 568.2 [M+H] +

[0207] Step 2: Synthesis of tert-butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 5-((S)-1-((S)-1-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydronaphtho[2',1':4,5]indeno[1,2-d][1,3]dioxepin-10-yl)benzyl)-2,3- dihydrobenzofuran-7-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)- 5-oxopentanoic acid

[0208] ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)- L-alanyl-L-alanine (2-2, 500 mg, 881 μmol) was dissolved in N,N-dimethylformamide (5 mL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (365 mg, 969 μmol) and 2,6-dimethylpyridine (103 mg, 969 μmol) were added, and the reaction was carried out at room temperature for 30 min. (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5-yl)methyl)phenyl)- 7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b- decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxepin-4(2H)-one (hormone compound 1, 538.8 mg, 0.881 mmol) was added and the reaction was continued for 1 h. LC-MS detection showed that the starting material was completely reacted, and the reaction solution was concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain compound (2-3, 703 mg).

[0209] LC-MS (ESI): m / z = 1161.5 [M+H] +

[0210] Step 3: Synthesis of (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1- ((S)-1-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydronaphtho[2',1':4,5]indeno[1,2-d][1,3]dioxepin-10-yl)benzyl)-2,3- dihydrobenofuran-7-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)- 5-oxopentanoic acid

[0211] tert-Butyl (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1-((S)-1-((5-(4- ((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a- dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydronaphtho[2',1':4,5] indeno[1,2-d][1,3]dioxepin-10-yl)benzyl)-2,3-dihydrobenofuran-7-yl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-5-oxopentanoate (2-3, 300 mg, 258 pmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (2 mL) was added and reacted at room temperature for 2 h. LC-MS detection showed that the raw material was completely reacted, and the reaction solution was concentrated to dryness under reduced pressure to obtain the crude product 2-4, which was directly subjected to the next step without further purification.

[0212] LC-MS (ESI): m / z = 1105.5 [M+H] +

[0213] Step 4: Synthesis of (9H-fluoren-9-yl)methyl ((33S,36S,39S)-40-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydronaphtho[2',l':4,5]indeno[l,2-d][l,3]dioxon-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-36,39-30,34,37,40-tetraoxa-2,5,8,11,14,17,20,23,nonoxa-29,35,38-triazinetriyl)carbamate

[0214] To a solution of crude (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-l-((S)-l-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydronaphtho[2',l':4,5]indeno[l,2-d][l,3]dioxon-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-l-oxopropan-2-yl)amino)-l-oxopropan-2-yl)amino)-5-oxopentanoic acid (2-4) in N,N-dimethylformamide (5 mL) was added O-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (110 mg, 293 μmol) and 2,6-dimethylpyridine (65 mg, 611 μmol). After the mixture was stirred at room temperature for 30 min, amino nonaethylene glycol monomethyl ether (104 mg, 244 μmol) was added and the reaction was continued for 1 h. LC-MS detection showed that the starting material was completely consumed. The reaction mixture was concentrated to dryness under reduced pressure and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give the product 2-5 (185 mg).

[0215] LC-MS (ESI): m / z = 758.1 [M / 2 + H] +

[0216] Step 5: Synthesis of (S)-2-amino-N 1- ((S)-1-((S)-1-(5-(4-(((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxyn-10-yl)benzyl)-2,3-dihydrobenzofuran-7- yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-N 5 Synthesis of (2,5,8,11,14,17,20,23,26-nonaoxyoctacosan-28-yl)pentanediamide (S-014-3)

[0217] (9H-fluoren-9-yl)methyl ((33S,36S,39S)-40-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxyn-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-36,39-30,34,37,40-tetraoxo-2,5,8,11,14,17,20,23,nonaoxy-29,35,38-triazinetetra carbon-33-yl)carbamate (2-5, 185 mg, 122 μmol) was dissolved in acetonitrile (5 mL), piperidine (52 mg, 611 μmol) was added and the reaction was continued at room temperature for 2 h. LC-MS detection showed that the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain the product 2-6 (85 mg).

[0218] LC-MS (ESI): m / z = 1292.6 [M+H] +

[0219] Step 6: (S)-2-(2-bromoacetamido)-N 1- ((S)-1-((S)-1-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxo-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)- 1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-N 5 Synthesis of (2,5,8,11,14,17,20,23,26-nonoxaoctacosyl)pentanediamide

[0220] (S)-2-amino-N 1 - ((S)-1-((S)-1-(5-(4-(((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxo-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)- 1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-N 5 (2,5,8,11,14,17,20,23,26-nonaoctacosyl)pentanediamide (2-6,75 mg, 58 μmol) was dissolved in N,N-dimethylformamide (1 mL), bromoacetic acid-N-succinimidyl ester (14 mg, 58 μmol) was added and the reaction was continued at room temperature for 1 h. LC-MS indicated the reaction was complete and the reaction solution was directly purified by reverse phase C-18 column chromatography (column: Agela C18, 20 g, 40-60 um, 30-60% acetonitrile in water, 10 min) to give drug-linker 2 (21 mg).

[0221] LC-MS (ESI): m / z = 1412.5, 1414.5 [M+H] +

[0222] 1 ​H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 8.54 - 8.32 (m, 1H), 8.24 - 8.07 (m, 2H), 7.95 - 7.81 (m, 1H), 7.52 (s, 1H), 7.40 - 7.27 (m, 3H), 7.23 - 7.18 (m, 2H), 6.81 (s, 1H), 6.19 - 6.13 (m, 1H), 5.93 (s, 1H), 5.39 (s, 1H), 5.14 - 5.07 (m, 1H), 4.91 (d, J = 5.0 Hz, 1H), 4.79 (d, J = 3.3 Hz, 1H), 4.57 - 4.42 (m, 4H), 4.36 - 4.23 (m, 3H), 4.22 - 4.12 (m, 1H), 3.91 (s, 1H), 3.81 (s, 2H), 3.49 (d, J = 3.0 Hz, 31H), 3.44 - 3.40 (m, 2H), 3.38 (d, J = 6.1 Hz, 2H), 3.23 (s, 3H), 3.20 - 3.10 (m, 4H), 2.31 (d, J = 13.0 Hz, 1H), 2.17 - 2.07 (m, 3H), 2.06 - 1.94 (m, 2H), 1.92 - 1.81 (m, 1H), 1.79 - 1.59 (m, 6H), 1.39 (s, 3H), 1.26 (d, J = 6.9 Hz, 3H), 1.21 (s, 3H), 1.09 - 0.99 (m, 2H), 0.85 (s, 3H).

[0223] Example 3: Synthesis of Drug-Linker 3

[0224] Drug-Linker 3 was prepared according to the method of WO2023186072A1 Example 15,

[0225] Example 4: Synthesis of (S)-2-(2-bromoacetylamino)-N5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxoheptatriacontyl-N1-((S)-1-(4-(3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxo-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)1-oxopropan-2-yl)pentanediamide (Drug-Linker 4)

[0226] Step 1: Synthesis of tert-butyl (4-(3-formylbenzyl)phenyl)carbamate (4-2)

[0227] Tert-butyl (4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)carbamate (4-1, 2.0 g, 6.27 mmol), 3-bromomethylbenzaldehyde (1.31 g, 6.58 mmol), [l,l-bis(diphenylphosphino)ferrocene]dichloropalladium (0.23 g, 0.31 mmol) and potassium carbonate (2.6 g, 18.80 mmol) were dissolved in mixed solvents (dioxane / water = 10 / 1, 22 mL) and stirred at 80 °C for 16 hours under nitrogen atmosphere. The reaction was monitored by LCMS. The reaction was filtered, the filtrate was diluted with ethyl acetate (100 mL) and washed with saturated aqueous sodium chloride solution (50 mL), dried, filtered and concentrated. The residue was separated and purified by column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give the title compound (1.8 g).

[0228] MS m / z (ESI): 256.1 [M+H-tBu] +

[0229] Step 2: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-(4- aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-l,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',l':4,5]indeno[l,2-d][l,3]dioxin-4-one (4-3)

[0230] (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5- yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12, 12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxepin-4-one (2.18 g, 5.78 mmol) was dissolved in acetonitrile (40 mL), magnesium sulfate (2.09 g, 17.34 mmol) was added, stirred at 25 °C for 1 hour, then tert-butyl (4-(3-formylbenzyl)phenyl)carbamate (4-2, 1.8 g, 5.78 mmol) was added, stirred at 25 °C for 15 minutes. Trifluoromethanesulfonic acid (2.6 g, 17.34 mmol) was added dropwise at 0 °C, stirred at 0 °C for 10 minutes. The reaction was detected by LCMS to be completed. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (100 mL) at 0 °C, the organic phase was extracted with ethyl acetate (100 mL x 3), washed with saturated aqueous sodium chloride solution (100 mL), dried, filtered and concentrated, and the residue was purified by silica gel column (dichloromethane / methanol = 20 / 1) to obtain the title compound (2.8 g).

[0231] MS m / z (ESI): 570.2 [M+H] +

[0232] Step 3: Synthesis of tert-butyl (S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5- ((S)-1-((S)-1-((4-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H- naphtho[2',1]:4,5]indeno[1,2-d][1,3]dioxepin-10-yl)benzyl)phenyl)amino)-1-oxopropan-2- yl)amino)-1-oxopropan-2-yl)amino)-5-oxopentanoate (4-4)

[0233] ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5- oxopentanoyl)-L-alanyl-L-alanine (876.82 mg, 1.54 mmol) was dissolved in N,N- dimethylformamide (10 mL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (365 mg, 969 μmol) and 2,6-dimethylpyridine (300.95 mg, 2.81 mmol) were added and reacted for 30 min in an ice bath, then (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(3-(4- aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxin-4-one (4-3, 0.8 g, 1.4 mmol) was added and the reaction was continued for 2 h. The reaction was detected by LC-MS. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (100 mL), the organic phase was extracted with ethyl acetate (100 mL x 3), washed with saturated aqueous sodium chloride solution (100 mL), dried, filtered and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 20 / 1) to obtain the title compound (1.57 g).

[0234] Step 4: Synthesis of (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1- ((S)-1-((4-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)- 1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-5-oxopentanoic acid (4-5)

[0235] (S)-4-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1-((S)-1-((4-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indono[1,2-d][1,3]dioxo-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-5-oxopentanoic acid tert-butyl ester (4-4, 1.49 g, 1.33 mmol) was dissolved in dichloromethane (15 mL), trifluoroacetic acid (5 mL) was added and reacted at room temperature for 2 h. LC-MS detection showed that the starting material was completely reacted, and the reaction solution was concentrated to dryness under reduced pressure to obtain the crude title compound, which was directly subjected to the next step.

[0236] MS m / z (ESI): 1063.4 [M+H] +

[0237] Step 5: Synthesis of (9H-fluoren-9-yl)methyl ((42S,45S,48S)-49-((4-(3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indono[1,2-d][1,3]dioxo-10-yl(benzyl)phenyl)amino)-45,48-dimethyl-39,43,46,49-tetraoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxo-38,44,47-triazacyclotetradecane-42-yl)carbamate (4-6)

[0238] (S)-2-amino-N5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptan-37-yl)-N1-((S)-1- ((S)-1-((4-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)pentanediamide (4-7) was synthesized according to the following procedure: To a solution of the above (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((S)-1-((S)-1-((4-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-5-oxopentanoic acid (4-5, 1.32 g, 1.24 mmol) in N,N-dimethylformamide (20 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (562.07 mg, 1.49 mmol) and 2,6-dimethylpyridine (665.17 mg, 6.21 mmol) at 0 °C for 30 min, then 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptan-37-ylamine (694.87 mg, 1.24 mmol) was added and the reaction was continued for 2 h. LC-MS detection showed that the starting material was completely reacted. The reaction solution was quenched by adding saturated aqueous sodium bicarbonate solution (200 mL), and the organic phase was extracted with ethyl acetate (100 mL x 3), washed with saturated aqueous sodium chloride solution (100 mL), dried, filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give the title compound (750 mg).

[0239] LC-MS (ESI): m / z = 803.2 [M / 2+H] +

[0240] Step 6: Synthesis of (S)-2-amino-N5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptan-37-yl)-N1-((S)-1-((S)-1-((4-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)pentanediamide (4-7)

[0241] (9H-fluoren-9-yl)methyl ((42S,45S,48S)-49-((4-(3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl(benzyl)phenyl)amino)-45,48-dimethyl-39,43,46,49-tetraoxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxo-38,44,47-triazacyclotetraoxane-42-yl)aminocarbamate (4-6, 100 mg, 62.3 μmol) was dissolved in acetonitrile (2 mL), piperidine (26.53 mg, 311.55 μmol) was added and the reaction was continued at room temperature for 2 h. LC-MS detection showed that the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 5 / 1) to give the title compound (47 mg).

[0242] LC-MS (ESI): m / z = 691.9 [M / 2+H] +

[0243] Step 7: Synthesis of (S)-2-(2-bromoacetylamino)-N5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxoheptadecyl)-N1-((S)-1-(4-(3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)phenyl)amino)-1-oxoprop-2-yl)amino)1-oxopropan-2-yl)pentanediamide (Drug-Linker 4)

[0244] (S)-2-amino-N5-(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptan-37-yl)-N1-((S)-1- ((S)-1-((4-((3-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro- 1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolo[4,5-c]pyrrol-10-yl)benzyl)amino)-1- oxopropan-2-yl)amino)-1-oxopropan-2-yl)pentanediamide (4-7, 47 mg, 33.9 μmol) was dissolved in N,N-dimethylformamide (2 mL), 2,5-dioxopyrrolidin-1-yl 2-bromoacetate (12.0 mg, 50.99 μmol) was added and the reaction was continued at room temperature for 2 h. LC-MS indicated the reaction was complete, the reaction solution was directly purified by reverse phase C-18 column (column: Agela C18, 20 g, 40-60 um, mobile phase: A: water, B: acetonitrile; B%: 5%-95%, 10 min) to give the title compound (30 mg). LC-MS (ESI): m / z = 1502.6, 1504.6 [M+H]

[0245] LC-MS (ESI): m / z = 1502.6, 1504.6 [M+H] +

[0246] 1H NMR (400 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.17 (d, J = 7.2 Hz, 1H), 8.10 (d, J = 7.2 Hz, 1H), 7.91 - 7.82 (m, 1H), 7.51 - 7.45 (m, 2H), 7.36 - 7.22 (m, 5H), 7.09 (d, J = 8.5 Hz, 2H), 6.21 - 6.15 (m, 1H), 6.01 - 5.94 (m, 1H), 5.41 (s, 1H), 5.10 - 5.02 (m, 1H), 4.91 (d, J = 4.8 Hz, 1H), 4.79 (d, J = 3.3 Hz, 1H), 4.56 - 4.45 (m, 1H), 4.43 - 4.22 (m, 4H), 4.22 - 4.13 (m, 1H), 3.91 (s, 2H), 3.86 (s, 2H), 3.55 - 3.45 (m, 45H), 3.44 - 3.40 (m, 2H), 3.39 - 3.35 (m, 2H), 3.23 (s, 3H), 3.20 - 3.13 (m, 2H), 2.39 - 2.34 (m, 1H), 2.17 - 2.07 (m, 2H), 1.93 - 1.81 (m, 1H), 1.80 - 1.56 (m, 6H), 1.40 (s, 3H), 1.32 - 1.28 (m, 3H), 1.24 - 1.20 (m, 3H), 1.02 - 0.93 (m, 2H), 0.86 (s, 3H).

[0247] Example 5: Synthesis of N-((S)-5-(2-bromoacetylamino)-6-((S)-1-((S)-1-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3-dioxole-10-yl)benzyl)-2-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6-oxohexyl)-2,5,8,11,14,17,20,23,26,29,32,35,38-trioxadotetradocosahexaenoic acid-41-amide (Drug-Linker 5)

[0248] Step 1: Synthesis of ((4-bromo-2-nitrobenzyl)oxy)(tert-butyl)dimethylsilane (5-2)

[0249] (4-bromo-2-nitrophenyl)methanol (5-1, 5.0 g, 21.55 mmol) was dissolved in dichloromethane (100 mL), under nitrogen atmosphere, triethylamine (4.1 g, 32.3 mmol) and tert-butyldimethylsilyl chloride (3.9 g, 25.8 mmol) were added at 0 °C, and the reaction was allowed to warm to room temperature and stirred for 16 h. The reaction was checked by LCMS. The reaction mixture was diluted with dichloromethane (100 mL), washed with water (100 mL) and saturated aqueous sodium chloride solution (100 mL), dried, filtered and concentrated. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1) to give the title compound (6.9 g).

[0250] Step 2: Synthesis of tert-butyldimethyl((2-nitro-4-(4,4,5,5-tetramethyl-1,3,2- dioxaborolan-2-yl)benzyl)oxy)silane (5-3)

[0251] ((4-bromo-2-nitrobenzyl)oxy)(tert-butyldimethyl)silane (5-2, 1.0 g, 2.89 mmol), pinacol diborane (0.87 g, 3.47 mmol), potassium acetate (0.85 g, 8.66 mmol), [1,1'- bis(diphenylphosphino)ferrocene]palladium dichloride (0.1 g, 0.14 mmol) were dissolved in dioxane (10 mL), and the reaction was allowed to warm to 80 °C for 4 h under nitrogen atmosphere. The reaction was checked by LC-MS. The reaction mixture was diluted with ethyl acetate (100 mL), filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether / ethyl acetate = 5 / 1) to give the title compound (1.02 g).

[0252] Step 3: Synthesis of 4-(4-((tert-butyldimethylsilyl)oxy)methyl)-3-nitrobenzaldehyde (5-4)

[0253] tert-butyldimethyl((2-nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzyl)oxy)silane (5-3, 1.02 g, 2.59 mmol), 4-bromomethylbenzaldehyde (0.54 g, 2.72 mmol), [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride (0.09 g, 0.13 mmol) and cesium carbonate (2.53 g, 7.78 mmol) were dissolved in a mixed solvent (dioxane / water = 10 / 1, 11 mL), and the reaction was allowed to warm to 100 °C and stirred for 4 h under nitrogen atmosphere. The reaction was checked by LCMS. The reaction mixture was filtered, diluted with ethyl acetate (100 mL), washed with saturated aqueous sodium chloride solution (50 mL), dried, filtered and concentrated. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1) to give the title compound (0.79 g).

[0254] Step 4: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2- hydroxyacetyl)-10-(4-(hydroxymethyl)-3-nitrobenzyl)phenyl)-6a,8a-dimethyl- 1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2- d][1,3]dioxepin-4-one (5-5)

[0255] (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5- yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b, 11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxepin-4-one (0.77 g, 2.05 mmol) was dissolved in acetonitrile (20 mL), magnesium sulfate (0.73 g, 6.15 mmol) was added, stirred at 25 °C for 1 h, then 4-(4-((tert-butyldimethylsilyl)oxy)methyl)-3-nitrobenzaldehyde (5-4, 0.79 g, 2.05 mmol) was added, stirred at 25 °C for 15 min. Trifluoromethanesulfonic acid (0.92 g, 6.15 mmol) was added dropwise at 0 °C, stirred at 0 °C for 10 min. The reaction was detected by LCMS to be completed. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (200 mL) at 0 °C, the organic phase was extracted with ethyl acetate (100 mL x 3), washed with saturated aqueous sodium chloride solution (100 mL), dried, filtered and concentrated. The residue was purified by silica gel column (dichloromethane / methanol = 20 / 1) to give the title compound (0.78 g).

[0256] MS m / z (ESI): 629.9 [M+H] +

[0257] Step 5: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-amino-4- (hydroxymethyl)benzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl- 1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2- d][1,3]dioxepin-4-one (5-6)

[0258] (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-10-(4- (hydroxymethyl)-3-nitrobenzyl)phenyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b- dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxin-4-one (5-5, 0.78 g, 1.24 mmol) was dissolved in ethanol (20 mL) and water (10 mL), ammonium chloride (0.66 g, 12.39 mmol) and iron powder (0.69 g, 12.39 mmol) were added, and the mixture was stirred at 60 °C for 2 h. LCMS showed the reaction was completed. The reaction mixture was diluted with ethyl acetate (200 mL), filtered, the filtrate was washed with saturated sodium chloride aqueous solution (100 mL), dried, filtered and concentrated. The residue was purified by silica gel column (dichloromethane / methanol = 20 / 1) to give the title compound (0.53 g).

[0259] MS m / z (ESI): 581.9 [M+H-H20] +

[0260] Step 6: Synthesis of 2,5-dioxopyrrolidin-1-yl N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine (5-8)

[0261] N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysine (5-7, 5.0 g, 10.67 mmol) and N-hydroxysuccinimide (1.35 g, 11.74 mmol) were dissolved in tetrahydrofuran (25 mL), N,N'-dicyclohexylcarbodiimide (2.42 g, 11.74 mmol) was added and the mixture was reacted at room temperature for 2 h. LCMS showed the reaction was completed. The reaction mixture was filtered, and the filtrate was used directly in the next step.

[0262] LC-MS (ESI): m / z = 465.9 [M+H-Boc] +

[0263] Step 7: Synthesis of N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysyl-L-alanyl-L-alanine (5-9)

[0264] To the above filtrate was added L-alanine-L-alanine (1.78 g, 11.14 mmol) and sodium bicarbonate (1.34 g, 15.91 mmol) and water (5 mL) was added and the reaction was continued for 2 h. LC-MS showed the reaction was complete. The reaction mixture was diluted with dilute hydrochloric acid (2 M, 8.0 mL) and then diluted with ethyl acetate (100 mL). The aqueous phase was extracted with ethyl acetate (100 mL x 3) and the combined organic phase was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The residue was filtered and dried to give the title compound (5.0 g).

[0265] LC-MS (ESI): m / z = 511.0 [M+H-Boc] +

[0266] Step 8: Synthesis of benzyl N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert- butoxycarbonyl)-L-lysyl-L-alanyl-L-alaninate (5-10)

[0267] N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysyl-L- alanyl-L-alanine (5-9, 0.5 g, 0.81 mmol) was dissolved in N,N-dimethylformamide (5 mL), cesium carbonate (0.32 g, 0.98 mmol) and benzyl bromide (0.14 g, 0.85 mmol) were added at 0 °C and the reaction was continued at room temperature for 2 h. LC-MS showed the reaction was complete. The reaction mixture was diluted with ethyl acetate (100 mL), filtered, the filtrate was washed with saturated aqueous sodium chloride solution (50 mL), dried, filtered and concentrated. The residue was purified by silica gel column (dichloromethane / methanol = 20 / 1) to give the title compound (362 mg).

[0268] LC-MS (ESI): m / z = 601.2 [M+H-Boc] +

[0269] Step 9: Synthesis of benzyl ((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysyl-L- alanyl-L-alaninate (5-11)

[0270] Benzyl N2-((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L- lysyl-L-alanyl-L-alaninate (5-10, 0.31 g, 0.44 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (0.5 mL) was added and the reaction was continued at room temperature for 2 h. LC-MS showed the starting material was consumed completely. The reaction mixture was concentrated under reduced pressure to dryness to give the title compound as a crude product which was used directly in the next step.

[0271] MS m / z (ESI): 601.2 [M+H] +

[0272] Step 10: Synthesis of benzyl ((S)-47-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 41-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38-trioxatetraconta-42- azoniasecoctan-48-oyl)-L-alanyl-L-alaninate (5-12)

[0273] The above benzyl(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysyl-L-alanyl-L- alaninate (5-11, 0.26 g, 0.44 mmol) crude was dissolved in N,N-dimethylformamide (5 mL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (251.53 mg, 0.66 mmol) and 2,6-dimethylpyridine (238.14 mg, 2.22 mmol) were added and reacted at 0 °C for 30 min, then 2,5,8,11,14,17,20,23,26,29,32,35,38-trioxatetracontanoic acid-41-oleic acid (295.3 mg, 0.46 mmol) was added and the reaction was continued for 2 h. LC-MS detection showed that the raw material was completely reacted. The reaction solution was quenched by adding saturated aqueous sodium bicarbonate solution (100 mL), the organic phase was extracted with ethyl acetate (50 mL x 3), washed with saturated aqueous sodium chloride solution (50 mL), dried, filtered and concentrated, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to give the title compound (345 mg).

[0274] LC-MS (ESI): m / z = 608.3 [M / 2+H] +

[0275] Step 11: Synthesis of ((S)-47-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)- 41-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38-trioxatetracontan-48-oyl)-L-alanyl-L- alaninate (5-13)

[0276] Benzyl ((S)-47-(((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-oxo- 2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxy-42-azaoctacosyl-48-acyl)-L- alaninyl-L-alaninate (5-12, 0.34 g, 0.28 mmol) was dissolved in methanol (5 mL) and reacted under hydrogen atmosphere (1 atm) at room temperature for 1 h. LC-MS detection showed the starting material was completely reacted. The reaction solution was filtered and the filtrate was concentrated to dryness under reduced pressure to give the crude title compound which was used directly in the next reaction.

[0277] LC-MS (ESI): m / z = 1125.6 [M+H] +

[0278] Step 12: Synthesis of (9H-fluoren-9-yl)methyl ((47S,50S,53S)-54-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo- 2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2- d][1,3]dioxol-10-yl(benzyl)-2-(hydroxymethyl)phenyl)amino)-50,53-dimethyl-41,48,51,54- tetraoxo-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxy-42,49,52-triazacyclopentan-47- yl)carbamate (5-14)

[0279] ((S)-47-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-41-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxy-42-azaoctacosan-48-oic acid-L-alaninyl-L-alanine (5-13, 305 mg, 0.27 mmol) was dissolved in N,N-dimethylformamide (5 mL), O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (132 mg, 352 μmol) and 2,6-dimethylpyridine (58.09 mg, 0.54 mmol) were added and reacted for 30 min in an ice bath, then (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3-amino-4- (hydroxymethyl)benzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxin-4-one (5-6, 0.17 g, 0.28 mmol) was added and the reaction was continued for 2 h. The reaction was detected by LC-MS. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (100 mL), and the organic phase was extracted with ethyl acetate (100 mL x 3), washed with saturated aqueous sodium chloride solution (100 mL), dried, filtered and concentrated. The residue was purified by silica gel column chromatography (dichloromethane / methanol = 10 / 1) to obtain the title compound (371 mg).

[0280] LC-MS (ESI): m / z = 854.1 [M / 2+H] +

[0281] Step 13: Synthesis of N-((S)-5-amino-6-((S)-1-((S)-1-((5-((4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxin-10-yl)benzyl)-2- (hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6- oxohexyl)-2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxypentanoyl)-L-alaninyl-L-alanine (5-15)

[0282] (9H-fluoren-9-yl)methyl ((47S,50S,53S)-54-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl(benzyl)-2-(hydroxymethyl)phenyl)amino)-50,53-dimethyl-41,48,51,54-tetraoxo-2,5,8,11,14,17,20,23,26,29,32,35,38-trioxadec-47-yl)aminocarbamate (5-14, 60 mg, 35.1 μmol) was dissolved in acetonitrile (2 mL), piperidine (14.96 mg, 175.75 μmol) was added and the reaction was continued at room temperature for 1 h. LC-MS detection showed that the reaction was complete, the reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol = 5 / 1) to give the title compound (34 mg).

[0283] LC-MS (ESI): m / z = 743.2 [M / 2+H] +

[0284] Step 14: Synthesis of N-((S)-5-(2-bromoacetamido)-6-((S)-1-((S)-1-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-10-yl)benzyl)-2-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6-oxohexyl)-2,5,8,11,14,17,20,23,26,29,32,35,38-trioxadecahexaenoic acid-41-amide (Drug-Linker 5)

[0285] N-((S)-5-amino-6-((S)-1-((S)-1-((5-((4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecano-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxane-10-acyl)benzyl 2-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)amino)-6-oxohexyl)-2,5,8,11,14,17,20,23,26,29,32,35,38-tetraoxopentanetetracarbon-41-amide (5-15,34 mg, 22.9 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 2,5-dioxopyrrolidone-1-yl-2-bromoacetate (8.1 mg, 34.35 μmol) was added. The reaction was continued at room temperature for 2 h. LC-MS analysis showed that the reaction was complete. The reaction solution was directly purified by reverse-phase C-18 column chromatography (col μmn: Agela C18, 20 g, 40-60 μm). (Mobile phase: A: water, B: acetonitrile; B%: 5%-95%, 10 min) yielded the title compound (19 mg).

[0286] LC-MS(ESI):m / z=802.6,803.5[M / 2+H] +

[0287] 1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.44 - 8.36 (m, 1H), 8.20 - 8.10 (m, 2H), 7.82 - 7.71 (m, 1H), 7.52 - 7.46 (m, 1H), 7.40 - 7.35 (m, 2H), 7.33 - 7.29 (m, 1H), 7.27 - 7.24 (m, 1H), 7.24 - 7.20 (m, 2H), 7.02 - 6.91 (m, 1H), 6.21 - 6.10 (m, 1H), 5.98 - 5.86 (m, 1H), 5.39 (s, 1H), 5.30 - 5.24 (m, 1H), 5.10 - 5.04 (m, 1H), 4.91 (d, 1H), 4.77 (d, J = 3.3 Hz, 1H), 4.54 - 4.12 (m, 9H), 3.95 - 3.85 (m, 4H), 3.60 - 3.54 (m, 2H), 3.52 - 3.48 (m, 48H), 3.47 - 3.44 (m, 4H), 3.44 - 3.40 (m, 2H), 3.03 - 2.94 (m, 2H), 2.30 - 2.27 (m, 2H), 2.15 - 2.06 (m, 1H), 1.79 - 1.68 (m, 4H), 1.67 - 1.60 (m, 2H), 1.55 - 1.43 (m, 2H), 1.39 (s, 3H), 1.32 - 1.25 (m, 8H), 1.10 - 0.98 (m, 2H), 0.85 (s, 3H).

[0288] Synthesis of control molecule (hormone compound 3)

[0289] Synthesis of 2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3- aminobenzyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl phosphate (hormone compound 3)

[0290] Synthesis route

[0291] First step: synthesis of tert-butyl (3-(4-formylbenzyl)phenyl)carbamate

[0292] Intermediate 1 (33.92 g, 106.33 mmol), 4-bromomethylbenzaldehyde (22.29 g, 112.01 mmol), [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (6.83 g, 9.33 mmol) and potassium carbonate (38.70 g, 280.02 mmol) were dissolved in anhydrous tetrahydrofuran (250 mL) under nitrogen atmosphere, stirred at 70 °C for 16 hours under nitrogen atmosphere. The reaction was checked by LCMS. The reaction solution was filtered, the filtrate was diluted with ethyl acetate (500 mL), then washed with saturated aqueous sodium chloride solution (150 mL), dried, filtered and concentrated. The residue was separated and purified by column chromatography (0-10% ethyl acetate / petroleum ether) to give compound 2 (10.50 g).

[0293] MS m / z (ESI): 256.10 [M-tBu];

[0294] 1H NMR (400 MHz, DMSO-d6) δ 10.01 - 9.90 (m, 1H), 9.35 - 9.23 (m, 1H), 7.95 - 7.78 (m, 2H), 7.53 - 7.33 (m, 3H), 7.33 - 7.12 (m, 2H), 6.92 - 6.78 (m, 1H), 3.99 (s, 2H), 1.47 (s, 9H).

[0295] Second Step: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3- aminobenzyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-4H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxepin-4-one

[0296] (8S,9S,10R,11S,13S,14S,16R,17S)-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one (21.76 g, 57.81 mmol) was dissolved in acetonitrile (100 mL), magnesium sulfate (86.98 g, 722.60 mmol) was added, stirred at 25 °C for 1 hour, then intermediate 2 (15.00 g, 48.17 mmol) was added, stirred at 25 °C for 15 minutes. Trifluoromethanesulfonic acid (108.45 g, 722.60 mmol, 57.78 mL) was added dropwise at 0 °C, stirred at 0 °C for 10 minutes. The reaction was detected by LCMS to be completed. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (300 mL) at 0 °C, the organic phase was extracted with ethyl acetate (500 mL x 2), washed with saturated aqueous sodium chloride solution (300 mL), dried, filtered and concentrated. The residue was purified by silica gel column (eluent, dichloromethane / methanol = 10 / 1) first, then the residue was purified by reverse phase column (eluent, water / acetonitrile = 2 / 1) to obtain compound 3 (14.40 g).

[0297] MS m / z (ESI): 570.20 [M+H];

[0298] 1H NMR (400MHz, DMSO-d6) δ 7.39-7.26 (m, 3H), 7.24-7.10 (m, 2H), 6.93-6.83 (m, 1H), 6.39-6.31 (m, 3H), 6.20-6.10 (m, 1H), 5.92 (s, 1H), 5.43-5.35 (m, 1H), 5.09-5.01 (m, 1H), 4.96-4.85 (m, 3H), 4.79-4.71 (m, 1H), 4.55-4.43 (m, 1H), 4.32-4.25 (m, 1H), 4.21-4.11 (m, 1H), 3.73 (s, 2H), 2.57-2.51 (m, 1H), 2.31-2.21 (m, 1H), 2.15-2.07 (m, 1H), 1.83-1.54 (m, 5H), 1.43-1.32 (m, 3H), 1.13-0.94 (m, 2H), 0.89-0.82 (m, 3H).

[0299] Step 3: Synthesis of tert-butyl (3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxan-10-yl)benzyl)phenyl)carbamate

[0300] Intermediate 3 (7.2 g, 12.64 mmol) and di-tert-butyl dicarbonate (3.59 g, 16.43 mmol) were dissolved in ethanol (35 mL) and tetrahydrofuran (35 mL), stirred at 25 °C for 16 hours. The reaction was detected by LCMS to be completed. The reaction was diluted with ethyl acetate (250 mL), washed with saturated aqueous ammonium chloride solution (100 mL x 2) and saturated aqueous sodium chloride solution (100 mL), dried, filtered and concentrated, and the residue was separated and purified by normal phase column chromatography (petroleum ether / ethyl acetate = 1 / 3) to obtain intermediate 4 (6.40 g).

[0301] MS m / z (ESI): 614.30 [M-tBu];

[0302] Step 4: Synthesis of tert-butyl (3-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxan-10-yl)benzyl)phenyl)carbamate

[0303] Intermediate 4 (6.2 g, 9.26 mmol) and 1H-tetrazole (6.48 g, 92.56 mmol) were dissolved in N,N-dimethylformamide (60 mL), di-tert-butyl-N,N diethylphosphoramide (27.69 g, 111.08 mmol) was added dropwise at 0 °C, after dropwise addition, 25 °C was stirred for 30 minutes. TLC monitoring reaction was complete. Hydrogen peroxide (11.06 g, 113.85 mmol, 35% mass fraction) was added at 0 °C, after dropwise addition, 25 °C was stirred for 1 hour. LCMS detection reaction was complete. Sodium sulfite aqueous solution (100 mL) was added at 0 °C to quench the reaction, extracted with ethyl acetate (200 mL x 3), the organic phase was washed with saturated brine (100 mL), dried, filtered and concentrated, the residue was separated and purified by normal phase column chromatography (silica, petroleum ether / ethyl acetate = 1 / 4) to give intermediate 5 (5.20 g).

[0304] MS m / z (ESI): 650.3 [M-2tBu-Boc];

[0305] Fifth step: synthesis of 2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(3- aminobenzyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl phosphonic acid

[0306] Intermediate 5 (4.60 g, 5.34 mmol) was dissolved in dichloromethane (46 mL), trifluoroacetic acid (15 mL) was added, 25 °C was stirred for 30 minutes. LCMS detection reaction was complete. The reaction was concentrated under reduced pressure at low temperature (water bath below 20 °C). The residue was dissolved in dichloromethane (30 mL) and concentrated under reduced pressure at low temperature, repeated 2 times. The residue was purified by reverse phase chromatography (column: Agela C18, 20 g, 40-60 um, mobile phase: A: water, B: acetonitrile; B%: 0%-30%, 30 min) to give the product (913 mg). mobile phase: A: water, B: acetonitrile; B%: 0%-30%, 30 min) to give the product (913 mg).

[0307] MS m / z (ESI): 650.20 [M+H];

[0308] 1H NMR (400 MHz, DMSO-d6) δ 7.29 - 7.23 (m, 3H), 7.21 - 7.08 (m, 1H), 7.05 (br, s, 2H), 6.87 (t, J = 7.7 Hz, 1H), 6.38 - 6.31 (m, 2H), 6.30 (d, J = 7.5 Hz, 1H), 6.13 (dd, J = 10.1, 1.8 Hz, 1H), 5.91 (d, J = 1.5 Hz, 1H), 5.44 (s, 1H), 5.16 (s, 1H), 4.90 (d, J = 4.7 Hz, 1H), 4.82 (dd, J = 18.4, 6.5 Hz, 1H), 4.67 (dd, J = 18.5, 6.5 Hz, 1H), 4.21 (s, 1H), 3.67 (s, 2H), 2.31 - 2.26 (m, 1H), 2.09 - 2.01 (m, 1H), 1.99 - 1.93 (m, 2H), 1.79 - 1.66 (m, 2H), 1.63 - 1.57 (m, 2H), 1.57 (s, 2H), 1.37 (s, 3H), 0.93 - 0.88 (m, 1H), 0.86 (s, 3H), 0.81 - 0.73 (m, 1H).

[0309] Example 6: Preparation of Antibodies

[0310] Example 6-1: Preparation of Anti-Human IL-4R Antibodies

[0311] The antibody variable region sequences for preparing the anti-human IL-4R antibody-drug conjugate are derived from patent document WO2025026332A1, the entire contents of which are incorporated by reference into the disclosure of the present application. The VH, VL and constant region amino acid sequences of the anti-human IL-4R antibody are shown in Table 1-1, and the CDR amino acid sequences of the anti-human IL-4R antibody are shown in Table 1-2.

[0312] According to the above sequence, the heavy chain (HC) and light chain (LC) plasmids are constructed (the nucleic acid sequences encoding the antibody VH and VL are recombined to the pTT5 expression vector (purchased from Yobio) with signal peptide and heavy chain constant region / light chain constant region sequence to obtain the recombinant plasmid expressing VH-CH1-Fc / VL-CL). The antibody corresponding heavy chain, light chain plasmid and transfection reagent PEI (Polysciences, item number: 24765-1) are added to the OPTI-MEM (Gibco, item number: 11058021) and mixed, then placed for 15 min, added to Expi293 cells (Thermofisher, item number: A14527), placed in 5% CO2, 120 rpm, 37℃ shaking bed culture. The second day after transfection, OPM-293ProFeed (Shanghai Oupu Mai, item number: F081918-001) and glucose (Sigma, item number: G7528) are added. The sixth day after transfection, the cell supernatant is collected, purified with Protein A (GE, item number: 28985254), and the eluted sample is dialyzed to PBS buffer, pH 7.4, to obtain the anti-human IL-4R antibody.

[0313] Table 1-1: Amino acid sequence of anti-human IL-4R antibody

[0314] Table 1-2 CDR amino acid sequence of anti-human IL-4R antibody (Kabat)

[0315] The M252Y / S254T / T256E mutation is introduced into the Mab-2 heavy chain constant region, and the nucleic acid sequences encoding the antibody VH and VL are recombined to the pTT5 expression vector (purchased from Yobio) with signal peptide and mutant heavy chain constant region / light chain constant region sequence, and the preparation method is the same as above, to obtain the antibody Mab-5, and the sequence is shown in Table 1-3.

[0316] Table 1-3 Amino acid sequence of anti-human IL-4R antibody containing YTE mutation

[0317] Example 6-2: Preparation of anti-TNFa antibody Adalimumab

[0318] The VH, VL and constant region amino acid sequences of the anti-human TNF-α antibody are shown in Table 2.

[0319] According to the above sequences, heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding the antibody VH and VL were recombined to the pTT5 expression vector (purchased from Yobio) with a signal peptide and heavy chain constant region / light chain constant region sequence to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL). The antibody corresponding heavy chain, light chain plasmid and transfection reagent PEI (Polysciences, item number: 24765-1) were mixed in OPTI-MEM (Gibco, item number: 11058021) and allowed to stand for 15 min, then added to Expi293 cells (Thermofisher, item number: A14527) and placed in a 5% CO2, 120 rpm, 37°C shaker. The next day after transfection, OPM-293ProFeed (Shanghai Oupu Mai, item number: F081918-001) and glucose (Sigma, item number: G7528) were added. On the sixth day after transfection, the cell supernatant was collected, purified with Protein A (GE, item number: 28985254), and the eluted sample was dialyzed into PBS buffer, pH 7.4, to obtain the anti-human TNF-a antibody.

[0320] Table 2: Amino acid sequences of anti-human TNF-a antibodies

[0321] Example 6-3: Preparation of anti-TL1A antibody RVT-3101

[0322] The VH, VL and constant region amino acid sequences of the anti-human TL1A antibody are shown in Table 3.

[0323] According to the above sequences, heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding the antibody VH and VL were recombined to the pTT5 expression vector (purchased from Yobio) with a signal peptide and heavy chain constant region / light chain constant region sequence to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL). The antibody corresponding heavy chain, light chain plasmid and transfection reagent PEI (Polysciences, item number: 24765-1) were mixed in OPTI-MEM (Gibco, item number: 11058021) and allowed to stand for 15 min, then added to Expi293 cells (Thermofisher, item number: A14527) and placed in a 5% CO2, 120 rpm, 37°C shaker. The next day after transfection, OPM-293ProFeed (Shanghai Oupu Mai, item number: F081918-001) and glucose (Sigma, item number: G7528) were added. On the sixth day after transfection, the cell supernatant was collected, purified with Protein A (GE, item number: 28985254), and the eluted sample was dialyzed into PBS buffer, pH 7.4, to obtain the anti-human TNF-a antibody.

[0324] Table 3: Amino acid sequences of anti-human TL1A antibodies

[0325] Example 7: Preparation of antibody-drug conjugates

[0326] Coupling: TCEP (tris(2-carboxyethyl)phosphine hydrochloride) was added to an antibody solution (1x PBS, pH 7.4) at 2-10 mg / ml, and incubated at 37 °C for 2 hours. Then 12-15 molar equivalents of drug-linker compound (e.g., drug-linker 1 to drug-linker 5 prepared in Examples 1-5) was added, and mixed well at 37 °C for 2 hours. After the reaction, the antibody-drug conjugate was obtained by ultrafiltration with 20 mM Histidine, 0.85% NaCl, pH 6.0 buffer for 3 times to remove the residual unreacted free small molecules. Representative structures of antibody-drug conjugates are as follows:

[0327] wherein Ab is Adalimumab, RVT-3101, Dupilumab, Mab-2, Mab-3, Mab-4, or Mab-5.

[0328] SEC-HPLC and LC-MS methods were used to analyze the purity and DAR values of ADC samples.

[0329] ADC SEC purity analysis: SEC-HPLC method was used to analyze the test protein samples, to characterize the molecular size homogeneity of the recombinant proteins, and to determine the purity of the recombinant proteins. The HPLC used in this method was Agilent 1260, and the chromatographic column was TSKgel G3000SWXL (from Tosoh Bioscience). The mobile phase was a mixture of 200 mM pH = 7.0 phosphate buffer / isopropanol (v / v 9:1), the detection temperature was 25 °C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading amount was 50 μg, and the analysis time was 30 minutes.

[0330] Measurement of ADC DAR value by mass spectrometry: The DAR value of ADC molecules was determined by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) method. First, the ADC molecules to be tested were treated with dithiothreitol (DTT) and incubated at 37°C for 1 hour to reduce the light chain and heavy chain, and then analyzed by Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 1 μg of protein was injected into a Waters ACQUITY Protein BEH molecular exclusion chromatography column, the mobile phase was 0.1% formic acid, 0.05% TFA, 25% acetonitrile in water, the flow rate was 0.2 mL / min, the analysis time was 30 minutes, the mass spectrometer was Thermo Q Exactive Plus, and the main parameters of the mass spectrometer were 3.8 kV of spray voltage, 300°C of capillary heating temperature, 35 arb of sheath gas flow rate, 900-4500 of parent ion scanning range, etc. Finally, the mass spectrometry data analysis software Biopharma Finder4.1 was applied to calculate the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component by Respect algorithm deconvolution processing, so as to calculate the DAR value of the ADC sample to be tested.

[0331] According to the above general preparation method, the antibody-drug conjugate or its pharmaceutically acceptable salt shown in Table 4 below was prepared.

[0332] Table 4 Preparation of antibody-drug conjugate, its DAR value and SEC purity

[0333] * The amino acid sequence of Anti-FITC-hIgG4 in the control ADC is as follows:

[0334] Test Example 1, flow cytometry experiment (FACS) for detecting the binding activity of anti-IL-4R antibody-drug conjugate to 293T cells overexpressing human IL-4R

[0335] Preparation of 293T cells overexpressing human IL4Rα:

[0336] The nucleotide sequence encoding the full-length amino acid sequence of hIL4R alpha (NCBI: NP_000409.1) was cloned into pLVX vector (purchased from Clontech) and plasmid was prepared. The 293T cell line (purchased from Chinese Academy of Sciences) was transfected with the plasmid to prepare lentivirus (PEI MAX, purchased from polyscience, item number: 24765-1). After harvesting the virus, the 293T cells were infected again, and the positive monoclonal cells were sorted into 96-well plates using IL4Ra antibody (dupilumab, self-made, VH, VL sequences as SEQ ID NO: 1-2) and donkey anti-human IgG h+l antibody (Jackson, item number: 109605088) on a flow cytometer FACSAria II (purchased from BD Biosciences) and cultured at 37°C, 5% (v / v) CO2. After about 2 weeks, some monoclonal wells were selected for amplification. The amplified clones were screened by flow cytometry. The results are shown in Figure 1.

[0337] Full-length hIL4R alpha (NCBI: NP_000409.1): used to construct human IL4R alpha overexpression cell line 293T-hIL4R

[0338] The 293T-hIL4R cells prepared above were expanded in a T-75 cell culture flask (purchased from Corning, item number: 430720). The cells were collected by centrifugation at 1000 rpm for 5 minutes at room temperature, washed twice with PBS buffer (purchased from Hyclone, item number: SH30256.01), resuspended in PBS and counted. 1x10 5 The anti-human IL-4R antibody-drug conjugate was diluted with PBS containing 1% (w / w) BSA (purchased from Sangon, item number: A500023-0100) at a concentration of 100 nM starting with a 5-fold gradient dilution, and 50 μL was added to each well of the cell mixture. After incubation at 4°C for 1 hour, 200 μL of PBS buffer was added to each well, and centrifugation was performed at 1500 rpm for 5 minutes. The washing step was repeated twice. After discarding the supernatant, 100 μL of diluted fluorescently labeled secondary antibody (purchased from Jackson Immuno Research, item number: 109-605-098) was added to each well, and incubation was performed at 4°C for 1 hour. The cells were washed twice with PBS by centrifugation, and 100 μL of PBS was added to each well to suspend the cells. The results were detected and analyzed using a FACS instrument. As shown in Table 5, the ability of the test molecules to bind to the IL-4R on the surface of 293T-hIL4R cells was comparable to that of their respective monoclonal antibodies. The data in Table 5 are shown in the following table: maxThe maximum mean fluorescence intensity (MFI) of the measured cell population.

[0339] Table 5. Binding activity of antibody-drug conjugates to 293T-hIL4R cells.

[0340] Test Example 2: K562-GRE reporter gene assay to determine the activity of anti-IL-4R antibody-drug conjugate.

[0341] K562 cells were fed at a rate of 5 × 10⁻⁶ 5 Cells / well were seeded into 6-well culture dishes (Costar, catalog number 3516) containing 2 mL of complete culture medium (RPM1640, 10% FBS, penicillin-streptomycin) and incubated at 37°C and 5% CO2 for 24 hours. The next day, 3 μg of pNL2.2[NLucP / MMTV / Hygro-NANO] (Promega) and 3 μl of PLUS reagent (Invitrogen, catalog number 11514-015) were diluted in 150 μL of Opti-MEM (Gibco, catalog number 11058021) and incubated at room temperature for 5 minutes. The pNL2.2[NLucP / MMTV / Hygro-NANO] vector contains MMTV LTR (mouse mammary tumor virus long terminal repeat), which responds to activation of several nuclear receptors (such as glucocorticoid receptor and androgen receptor) and drives the transcription of the luciferase reporter gene NanoLuc. After incubation, the diluted DNA solution was mixed with Lipofectamine LTX solution (Invitrogen, catalog number 15338-100) at a 1:1 ratio (6 μl Lipofectamine LTX + 144 μl Opti-MEM) and pre-incubated at room temperature for 15 minutes to form the DNA-Lipofectamine LTX complex. Following incubation, 300 μl of the DNA-Lipofectamine complex was added directly to the cell wells. K562 cells were transfected at 37°C and 5% CO2 for 24 hours. After transfection, the cells were washed with 3 mL of PBS and selectively grown for two weeks in complete growth medium containing 125 μg / mL hygromycin B (Invitrogen, catalog number 10687010) to obtain K562-GRE cells.

[0342] The nucleotide sequence encoding the human IL-4R amino acid sequence (SEQ ID NO.37) was cloned into the pLVX-IRES-Puro lentiviral vector, and viral particles were prepared in HEK293T cells. The aforementioned constructed K562-GRE cells were then used at 5 × 10⁻⁶ cells per cell line. 5K562-GRE cells were seeded at 1 x 105cells / well into 6-well plates (Costar, 3516) containing 1.5 mL of complete growth medium and incubated at 37 °C, 5% CO2for 24 hours. On the next day, 1.5 mL of human IL-4R lentivirus particle solution prepared in advance was added to the cell culture plate, and 8 g / mL of polybrene (Santa Cruz, sc-134220) was added to the final concentration for lentivirus infection. K562-GRE cells were infected at 37 °C, 5% CO2for 24 hours. After incubation, the cells were washed with 3 mL of PBS, and selected with complete growth medium containing 125 g / mL of hygromycin B (Invitrogen, 10687-010) and 1 g / mL of puro (Gibco, A1113802) for two weeks to generate K562-human IL-4R-GRE-Luc reporter cells.

[0343] K562-human IL4R-GRE-Luc cells were collected and resuspended in assay medium (RPM1640 + 1% carbon absorbed fetal bovine serum + 1% sodium pyruvate + 1% non-essential amino acids + 1% penicillin / streptomycin). Cells were seeded at 5 x 105cells / well (50 μl) in 96-well plates (Costar, 3917). The anti-IL-4R antibody-drug conjugates to be tested were diluted in 6 points with 5-fold gradient starting from 40 nM in the above assay medium. The diluted drugs were added to the 96-well cell plates at 50 μl per well, mixed well, and incubated at 37 °C, 5% (v / v) CO2incubator for 24 hours. After incubation, the 96-well plates were placed at room temperature for 5 min, and 100 μl / well of Nano-Glo luciferase detection reagent (Promega, N1110) was added to the plates. After shaking at room temperature for 10 min, the fluorescence signal value of the plates was detected using a PE Envision instrument-US Lumi module. The induction curve of the drug was plotted with the drug concentration as the abscissa and the fluorescence signal value RLU as the ordinate, and the EC50and maximum luminescence intensity (Emax) of the induction curve were calculated by four-parameter fitting (GraphPad Prism 9). 4 50 max ), and the results are shown in Table 6.

[0344] Table 6 Activity of anti-IL-4R antibody-drug conjugates in GRE reporter gene assay

[0345] Test Example 3 Inhibition of CD23 expression on B cells in a human primary T, B cell co-culture system by anti-IL-4R antibody-drug conjugates

[0346] ​​PBMC cryo-cells (Stemexpress, Cat: PBMNC050C) were taken out from liquid nitrogen tank, put into 37°C water bath, gently shaken to dissolve the cells. The cell suspension was transferred into a centrifuge tube containing 8 ml preheated 1640 medium (Gibco, Cat: 72400047) and centrifuged for 8 min (centrifugal condition: 300g, 25°C), and the supernatant was discarded. 8 ml Easysep TM buffer buffer (Stemcell, Cat: 20144) was added to resuspend the cells, and the cells were counted by a cell counter (Beckman Coulter, Vi-Cell). After centrifugation for 8 min (centrifugal condition: 300g, 25°C), the supernatant was discarded. According to the counting result, the cells were resuspended with Easysep TM buffer buffer, and the density was adjusted to 5x10 7 6 cells / mL. Human primary T cells and B cells were sorted by human T cell sorting kit (Stemcell, Cat: 17951) and human B cell sorting kit (Stemcell, Cat: 17954) respectively, and the cells were counted and ready for use.

[0347] The following reagents were prepared: 1) human primary T cells were adjusted to 1x10 6 6 cells / mL, and human primary B cells were adjusted to 0.5x10 6Cell / mL; 2) Prepare the anti-IL-4R antibody-drug conjugate working solution (4 times final concentration) with 1640 complete medium, so that the final concentration of the drug is 500 nM, 100 nM and 20 nM; 3) Prepare the Staphylococcus aureus enterotoxin A + human recombinant interleukin-4 + anti-human CD3 / CD28 magnetic bead mixed working solution (4 times final concentration), add human T cell activator anti-CD3 / CD28 magnetic beads (6250 magnetic beads / 50 μL / well, Gibco, Cat: 11131D), human recombinant interleukin-4 (final concentration of 10 ng / mL, Peprotech, Cat: 200-04) and Staphylococcus aureus enterotoxin A (final concentration of 1 ng / mL, Toxin technology, Cat: AT101) in 1640 complete medium, mix well and wait for use. In the 96-well-U-shaped plate (Corning, Cat: 3799), add human primary T cells (50000 cells / 50 μL / well), human primary B cells (25000 cells / 50 μL / well) and anti-IL-4R antibody-drug conjugate (50 μL / well) in turn, mix gently, and incubate at room temperature for 10 min. Then add the Staphylococcus aureus enterotoxin A + human recombinant interleukin-4 + anti-human CD3 / CD28 magnetic bead mixed working solution, and incubate at 37°C, 5% CO2 for 3 days. After the incubation time is over, take out the plate, mix the cells, centrifuge for 5 min (centrifugation conditions: 350g, 25°C), and then aspirate the supernatant, and detect the secretion level of IL-13 by ELISA method; the cell pellet is detected for the expression of CD23 on the living B cells by FACS method.

[0348] The cell precipitate of the above human primary T, B cell co-culture was resuspended with 200 μL / well PBS, centrifuged for 5 min (centrifugation condition: 350 g, 25°C) and the supernatant was discarded. After incubation with a fixable near-IR dead live cell staining kit dye (Thermofisher, Cat: L10119) at room temperature for 10 min, 100 μL / well of staining buffer (2% FBS + PBS) was added, centrifuged for 5 min (centrifugation condition: 350 g, 25°C) and the supernatant was discarded. A human Fc receptor blocker (Biolegend, Cat: 422302) was added and incubated at 4°C for 10 min, followed by the addition of 2 μL / well of PE anti-human CD19 antibody (Biolegend, Cat: 302208) and 2 μL / well of APC anti-human CD23 antibody (Biolegend, Cat: 338514), and incubation at 4°C in the dark for 30 min for cell surface staining. After staining, 100 μL / well of staining buffer was added to the cells, centrifuged for 5 min (centrifugation condition: 350 g, 25°C) and the supernatant was discarded, and the cells were resuspended with 120 μL / well of staining buffer. The proportion of CD23-positive cells in live CD19-positive cells was detected using a flow cytometer (BD, Canto II), and the data were analyzed using Flowjo V10 software. The results, as shown in Table 7, indicated that the higher the proportion of CD23-positive cells, the higher the expression level of CD23 on B cells, and the weaker the inhibitory activity of the anti-IL-4R antibody-drug conjugate.

[0349] Table 7 Percentage of CD23-positive B cells in T-B co-culture system

[0350] Test Example 4 Pharmacological and pharmacodynamic effects of anti-IL-4R antibody-drug conjugate on atopic dermatitis mouse model

[0351] Disease model construction: 6-7 week-old C57BL / 6-hIL-4 / h-IL-4RA double humanized mice (provided by Biorbyt). On day 0, the back and right ear of the normal control group of mice were each uniformly coated with 25 μL of a mixture of acetone and olive oil (4:1); the back and right ear of the remaining groups of mice were each uniformly coated with 25 μL of 0.8% oxazolone (OXA) sensitization (solvent: mixture of acetone and olive oil 4:1). From day 7 to day 25, the back and right ear of the normal control group of mice were each uniformly coated with 25 μL of a mixture of acetone and olive oil (4:1) for challenge; the back and right ear of the model group of mice were each uniformly coated with 25 μL of 0.4% OXA solution (solvent as above) for challenge.

[0352] Drug intervention design: drug administration started from the 6th day after the first OXA sensitization, and the normal control mice were grouped separately; the model group of mice were divided into 7 groups according to ear thickness, with 8 mice in each group. The mice in each group were administered intraperitoneally, twice a week.

[0353] The in vivo efficacy experiment was ended on day 26, and serum was taken for subsequent detection. During the entire in vivo experiment, ear thickness measurements were taken for each group of mice before each OXA induction and at the end of the experiment. As shown in Figure 2, both Dupilumab and anti-IL-4R antibody-drug conjugates (ADC-1-1, ADC-1-2, ADC-2-1, ADC-2-2) can significantly inhibit mouse ear thickening compared with the vehicle control group of mice, and the inhibition effect of anti-IL-4R antibody-drug conjugates of the same dose is stronger than that of Dupilumab; at the same time, as shown in Figure 3, Dupilumab and anti-IL-4R antibody-drug conjugates can inhibit the production of IgE in mice.

[0354] Test Example 5-1, Plasma stability test 1 of anti-IL-4R antibody-drug conjugates

[0355] The ADC was diluted (the final concentration of ADC was 100 μg / ml) in human plasma (Aobio, PB021-C), monkey plasma (Sinnovida, SND-X0107) and rat plasma (Sinnovida, SND-X0101) respectively, and incubated in a 37°C incubator. The day of incubation was marked as day 0, and then samples were taken out on day 7, day 14, day 21 and day 28 respectively for detection of free small molecules.

[0356] 20 μL of sample was taken, 300 μL of internal standard working solution (acetonitrile) was added, vortexed for 5 minutes, centrifuged for 5 minutes (14000 rpm), and 4 μL of supernatant was injected into LC-MS / MS (AB Sciex Triple Quad 5500+) for analysis. The results are shown in Table 8, which show that the ADC is relatively stable in human, monkey and rat plasma, and the concentration of free small molecules is lower than the lower limit of quantification (0.8 ng / ml).

[0357] Table 8 Plasma stability of anti-IL4R antibody-drug conjugates

[0358] BLOQ- lower than the lower limit of quantification (1 ng / ml) * 80%

[0359] Test Example 5-2, Plasma stability test 2 of anti-IL-4R antibody-drug conjugates

[0360] The ADC was incubated in wild type mouse plasma (Sino Biological, SND-X0102), mCeslc knockout mouse plasma (Bio-Connect) at a final concentration of 1000 pg / ml, or in human plasma (Aobio, FTS-P-50) at a final concentration of 500 pg / ml. The human plasma was incubated with Protein A affinity chromatography media (Nanojo, 17013-090100) at a volume ratio of 1:1 at 4°C overnight, and the plasma was collected after centrifugation to remove Protein A. The incubation was carried out in a 37°C incubator. The day of sample collection was labeled as day 0, and samples were collected at day 3 for free small molecule detection, and at day 7 for ADC purity determination.

[0361] Take 20 pL sample, add 300 pL internal standard working solution (acetonitrile), vortex for 5 minutes, centrifuge for 5 minutes (14000 rpm), and take 4 pL supernatant for LC-MS / MS (AB Sciex Triple Quad 5500+) analysis, or use SEC-HPLC method to analyze the purity of the ADC molecule to be tested. The results are shown in Table 9, which show that the ADC of the application is stable after plasma incubation.

[0362] Table 9 Plasma stability of anti-IL4R antibody-drug conjugate

[0363] BLOQ - below the lower limit of quantification (1 ng / ml) * 80%

[0364] Test Example 6, cell incubation experiment of anti-IL-4R antibody-drug conjugate

[0365] K562-human IL-4R alpha-GRE-Luc cells (5 x 10 5 / well) were seeded in a 24-well plate, and after the addition of 2000 nM ADC, the cells were incubated in a 37°C cell incubator. The sample taken at the time when the cells were mixed with the ADC-2-1 was labeled as 0h, and then samples were taken at 24h, 48h and 72h, and acetonitrile was added to mix and homogenize. Take 40 pL of the above sample, add 200 pL internal standard working solution (acetonitrile), vortex for 5 minutes, centrifuge for 5 minutes (14000 rpm), mix 60 pL supernatant with 40 pL 0.1% formic acid, and then take 4 pL supernatant for LC-MS / MS (API 6500+) analysis.

[0366] Test Example 7, toxicological evaluation experiment of drug loading

[0367] SD rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., 6-8 weeks, 200-300 g) were repeatedly administered intravenously for 14 consecutive days, 3 female and 3 male rats in each group. The doses of hormone compound 3 and hormone compound 4 were 0.3, 1 or 1 mg / kg / day. After the last administration, serum was collected for serum biochemical analysis, as shown in Figures 4 and 5 below. Compared with hormone compound 3, the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of female and male animals in each dose group of hormone compound 4 were slightly elevated.

[0368] SD rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., 6-8 weeks, 200-300 g) were repeatedly administered intravenously for 14 consecutive days, 3 female and 3 male rats in each group. The doses of hormone compound 1 and hormone compound 2 were 0.3 or 1 mg / kg / day and 1 mg / kg / day, respectively. After the last administration, serum was collected for serum biochemical analysis, as shown in Figures 6 and 7 below. Compared with hormone compound 1, the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) of female and male animals in the dose group of hormone compound 2 were significantly elevated.

[0369] Test Example 8, Rat Pharmacokinetic Test

[0370] I. Test Materials

[0371] SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0372] PEG200 (polyethylene glycol 200) and HP-β-CD (hydroxypropyl-β-cyclodextrin) were purchased from Sigma, methanol, acetonitrile were purchased from Honeywell (USA), formic acid was purchased from ACS (USA), tolbutamide was purchased from Shanghai Zhenzhun Biological Technology Co., Ltd. K2EDTA anticoagulation blood collection tubes were purchased from Jiangsu Xinkang Medical Instrument Co., Ltd.

[0373] II. Test Methods

[0374] 1. Animal Test

[0375] For each test compound, 3 male SD rats (200-300 g, 6-8 weeks) were selected and administered by tail vein injection. The solvent of hormone compound 3 (a prodrug of hormone compound 2) was 50% PEG200 + 50% (10% HP-β-CD), the solvent of hormone compound 4 (a prodrug of hormone compound 1) was 20% PEG200 + 80% water, and the solvent of hormone compound 1 and hormone compound 2 was 10% PEG200 + 90% (10% HP-β-CD). Before the test, all animals were normally fed and watered. During the test, the rats in the hormone compound 3 and hormone compound 4 groups were intravenously bled at 0.083, 0.167, 0.333, 0.5, 1, 2, 4, 8, and 24 hours before and after administration, and the rats in the hormone compound 1 and hormone compound 2 groups were intravenously bled at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours before and after administration. The collected whole blood samples were placed in K2EDTA anticoagulant tubes, centrifuged for 5 min (4000 rpm, 4°C), and the plasma was taken for testing.

[0376] 2. Sample processing and biological analysis

[0377] 10 μL of rat plasma sample was taken, 100 μL of acetonitrile solvent (containing internal standard, 500 ng / mL tolbutamide) was added to precipitate the protein, vortexed for 5 min, then centrifuged for 10 min (4000 rpm, 4°C), 50 μL of supernatant was mixed with an equal volume of water, vortexed and mixed, and then quantitatively detected by LC-MS / MS system (AB Sciex Triple Quad 6500+). The standard curve of male SD rat plasma and the quality control sample were determined when determining the sample concentration. For the 20x diluted sample, 2 μL of sample was added to 38 μL of blank plasma, vortexed for 1 min, then 400 μL of acetonitrile solvent (containing internal standard, 500 ng / mL tolbutamide) was added to precipitate the protein, and the remaining processing steps were the same as above.

[0378] 3. Data processing

[0379] The Phoenix WinNonlin 8.0 software (Certara, USA) was used to calculate the pharmacokinetic parameters by non-compartment model statistical moment method.

[0380] Exposure ratio = (plasma exposure of drug 母体药物 * molecular weight of drug 前药 ) / (plasma exposure of drug 前药 * molecular weight of drug 母体药物 )

[0381] 4. Test results

[0382] The results are shown in Table 10, which indicates that the plasma exposure of the corresponding hormone compound 1 (parent drug) is very low in SD rats after administration of hormone compound 4 (prodrug), and the exposure ratio of parent drug / prodrug is only 0.09. The plasma exposure of the corresponding hormone compound 2 (parent drug) is higher in SD rats after administration of hormone compound 3 (prodrug), and the exposure ratio of parent drug / prodrug reaches 0.74.

[0383] Table 10 Rat PK parameters of hormone compounds of the disclosure

[0384] Test Example 9, Stability test of anti-IL-4R antibody-drug conjugate

[0385] The ADC solution stability test was performed in 20 mM histidine / HCl, 0.85% NaCl, 0.02% Tween-80. 50 mg / mL or 100 mg / mL ADC was incubated in the above solution at 37 degrees for 1 week for sampling and testing. SEC-HPLC and LC-MS methods were used to analyze the purity and DAR value of ADC samples. The specific experimental method is described in Example 3. The results are shown in Table 11, which shows that the ADCs of the present application exhibit good solution stability.

[0386] Table 11 Solution stability of anti-IL4R antibody-drug conjugate

[0387] Test Example 10, K562-GRE reporter assay for activity of anti-IL-4R antibody-drug conjugate

[0388] K562-human IL4R-GRE-Luc cells constructed in Test Example 2 were collected and resuspended in assay medium (RPM1640 + 10% charcoal-stripped fetal bovine serum + 1% sodium pyruvate + 1% non-essential amino acids + 1% penicillin / streptomycin). Cells were plated at 5 x 10 450 μl of anti-IL-4R antibody-drug conjugate (RLU) was seeded per well in a 96-well plate (purchased from Cabernet Biotechnology, catalog number 210102961). The anti-IL-4R antibody-drug conjugate and the control ADC were serially diluted 4-fold at 8 wells, starting from 200 nM, using the aforementioned assay medium. 50 μl of the diluted drug was added to each well of the 96-well cell culture plate, mixed well, and incubated at 37°C in a 5% (v / v) CO2 incubator for 24 hours. After incubation, the 96-well plate was equilibrated at room temperature for 5 min, and 50 μl of Nano-Light luciferase assay reagent (purchased from Meilun, catalog number MA0521) was added to each well. After shaking at room temperature for 10 min, the fluorescence signal value in the wells was detected using a PE Envision instrument-US Lumi module. An activation curve was plotted with drug concentration on the x-axis and RLU fluorescence signal value on the y-axis. Four-parameter fitting (GraphPad Prism 10) was used to calculate the EC50 of the activation curve. 50 and maximum luminous intensity E max The results are shown in Table 12 and Figure 8.

[0389] Table 12 Activity of anti-IL-4R antibody-drug conjugate in GRE reporter gene assay

[0390] Test Example 11: K562-GRE reporter gene assay to detect the activity of anti-TNFα antibody-drug conjugate.

[0391] The nucleotide sequence encoding the human TNFα amino acid sequence (NCBI sequence number: NP_000585.2, missing amino acids 77-88) was cloned into the pLVX-IRES-Puro lentiviral vector, and viral particles were prepared in HEK293T cells. To generate the human transmembrane K562-humanTNFα-GRE reporter gene cell line, the aforementioned K562-GRE was used at a rate of 5 × 10⁻⁶ cells / year. 5Cells were seeded at 1 cell / well into 6-well plates (purchased from Costar, Cat# 3516) with 1.5 mL complete growth medium and incubated at 37°C, 5% CO2 for 24 hours. On the next day, 1.5 mL of human TNFα lentivirus particle solution prepared in advance was added to the cell plates, and 8 ug / mL polybrene (purchased from Santa Cruz, Cat# sc-134220) was added to the final concentration for lentivirus infection. The parental K562-GRE cells were infected at 37°C, 5% CO2 for 24 hours. After incubation, the cells were washed with 3 mL of PBS, and selected with complete growth medium containing 125 ug / mL hygromycin B (purchased from Invitrogen, Cat# 10687-010) and 2 ug / mL puromycin (purchased from Gibco, Cat# A1113802) for two weeks. K562-humanTNFα-GRE-Luc cells were generated.

[0392] Full length hTNFα (NCBI: NP_000585.2): for construction of human TNFα overexpression cell line K562-humanTNFα-GRE-Luc

[0393] K562-humanTNFα-GRE-Luc cells were collected and resuspended in assay medium (RPM1640 + 10% charcoal-stripped fetal bovine serum + 1% sodium pyruvate + 1% non-essential amino acids + 1% penicillin / streptomycin). Cells were seeded at 5 x 10 4 cells / well (50 μl) in 96-well plates (purchased from Corning, Cat# 210102961). The test anti-TNFα antibody-drug conjugates and control ADCs were diluted at 8 points with 4-fold ratio gradient starting from 200 nM in the assay medium described above. The diluted drugs were added to the 96-well cell plates at 50 μl per well, and after mixing, the plates were incubated at 37°C, 5% (v / v) CO2 incubator for 24 hours. After incubation, the 96-well plates were equilibrated at room temperature for 5 min, and 50 μl / well of Nano-Light luciferase detection reagent (purchased from Meilun, Cat# MA0521) was added to the plates. After shaking at room temperature for 10 min, the plates were detected for fluorescence signal values using a PE Envision instrument-US Lumi module. The activation curves of the drugs were plotted with drug concentration as the abscissa and the fluorescence signal values RLU as the ordinate, and the EC50 and maximum luminescence intensity Emax of the activation curves were calculated by four-parameter fitting (GraphPad Prism 10). 50 max The results are shown in Table 13 and Figure 9.

[0394] ​Table 13 Activity of anti-TNFα antibody-drug conjugate in GRE reporter gene assay

[0395] Test Example 12: HEK293-GR reporter gene assay to detect the activity of anti-TL1A antibody-drug conjugate.

[0396] GR-GAL4 Luciferase Reporter HEK293 was purchased from Nanjing Kebai Biotechnology (Catalog No.: CBPM0010, hereinafter referred to as HEK293-GR). Upon receiving the cells, they were immediately expanded to T75 culture flasks (purchased from Corning, Catalog No. 430641).

[0397] HEK293-GR at 3×10 6 Cells were seeded into T25 culture flasks (Corning, catalog number 430639) containing 5 mL of complete medium (DMEM + 10% carbon-adsorbed fetal bovine serum + 1% penicillin / streptomycin + 200 μg / ml hygromycin B + 2 μg / ml puromycin) and cultured at 37°C and 5% CO2 for 24 hours. On the second day, when the cells reached approximately 80% confluence, they were analyzed using Lipofectamine. TM The 3000 transfection kit (purchased from Thermofisher, catalog number L3000008) was used to add 5 μg of plasmid (pLVX-humanTL1A(FL)-IRES-puro, the nucleotide sequence encoding the full-length amino acid sequence of human TL1A (NCBI sequence number: NP_005109.2) was cloned into the pLVX-IRES-Puro lentiviral vector, and viral particles were prepared in HEK293T cells) to 250 μl of Opti-MEM. Simultaneously, 10 μl of lipo3000 was added and gently mixed with a pipette, labeled tube A. 10 μl of lipo3000 was then added to 250 μl of... In Opti-MEM, tube B was used and allowed to stand for 5 minutes. The solution in tube B was then added dropwise to tube A using a pipette and allowed to stand for 15 minutes. The DNA-lipo3000 mixture was then added dropwise to the cells and shaken well. The cells were then placed in an incubator at 37°C and 5% CO2 and cultured for 48 hours to obtain HEK293-GR-humanTL1A cells.

[0398] Full-length hTL1A (NCBI: NP_005109.2): Used to construct the human TL1A overexpressing cell line HEK293-GR-humanTL1A

[0399] HEK293-GR-humanTL1A cells were collected and resuspended in assay medium (DMEM + 10% carbon-adsorbed fetal bovine serum). Cells were then cultured at 5 × 10⁶ cells / mL.4 The cells were seeded at 50,000 cells / well (50 μl) in 96-well plates (purchased from Corning, item number 210102961). The anti-TL1A antibody-drug conjugates to be tested and the control ADC were diluted in 6 points with 4-fold gradient starting from 3.12 nM in the above-mentioned assay medium. The diluted drugs were added to the 96-well cell plates at 50 μl per well. After mixing, the plates were incubated in a 37 °C, 5% (v / v) CO2incubator for 24 hours. After the incubation, the 96-well plates were equilibrated at room temperature for 5 min, and 50 μl / well of Bright-lite luciferase detection reagent (purchased from Vazyme, item number 7E562J1) was added to the plates. After shaking at room temperature for 10 min, the plates were detected for the fluorescence signal value using a PE Envision instrument-US Lumi module. The activation curve of the drug was plotted with the drug concentration as the abscissa and the fluorescence signal value RLU as the ordinate, and the EC50and maximum luminescence intensity Emaxof the activation curve were calculated by four-parameter fitting (GraphPad Prism 10). 50 max The results are shown in Table 14 and Figure 10.

[0400] Table 14 Activity of anti-TL1A antibody-drug conjugates in GRE reporter gene assay

[0401] Test Example 13 K562-luc-GRE reporter gene detection of bystander effect of anti-IL-4R antibody-drug conjugates

[0402] ​The 293T-IL4R cells obtained in Test Example 1 were collected, resuspended with assay medium (1640 + 1% carbon-absorbed fetal bovine serum + 1% sodium pyruvate + 1% non-essential amino acids + 1% penicillin / streptomycin double-antibiotic), and plated in a 96-well plate (Costar: 3917) at 2500 cells / 100 μL / well, and allowed to adhere overnight. The antibody-drug working solution was prepared with assay medium (2x final concentration) so that the final concentration of the drug was 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.0128 nM. The 293-IL4R overnight culture supernatant was discarded, 100 μL / well of fresh assay medium and 100 μL / well of drug working solution were added, mixed gently, and the final volume was 200 μL / well. The plate was incubated in a 37°C, 5% CO2 incubator for 5 days. The culture supernatant on the 5th day was collected and centrifuged at 400g for 5 minutes. 50 μL / well of K562-GRE cells and 50 μL / well of the 5th day culture supernatant were added, mixed, and incubated at 37°C for 24 hours. The substrate (Meilunbio, MA0521) and fluorescence detection buffer were mixed at 1:50 to obtain a substrate reaction solution. After 100 μL / well of the substrate reaction solution was added to the 96-well plate, it was mixed on a shaker (400 rpm, 10 minutes). The fluorescence signal value of the plate was detected using a PE Envision microplate reader-US Lumi module. The activation curve of the drug was plotted with the drug concentration as the abscissa and the fluorescence signal value RLU as the ordinate, and the four-parameter fitting (GraphPad Prism 10) was performed to calculate the EC50 and maximum luminescence intensity Emax of the activation curve. 50 and the maximum luminescence intensity E max The results are shown in Table 15 and Figure 11.

[0403] Table 15 Anti-IL-4R antibody-drug conjugate bystander effect

[0404] Test Example 14, K562-luc-GRE reporter gene detection of bystander effect of anti-TNFα antibody-drug conjugate

[0405] The nucleotide sequence encoding the amino acid sequence of human TNFα (NCBI sequence number: NP_000585.2, lacking amino acids 77-88) was cloned into the pCDNA5 / FRT (purchased from Invitrogen, catalog number V6010-20) vector, and the Flpin CHO cell line (purchased from Thermo) was transfected with the plasmid Flipin CHO-TNF-α cells were obtained through screening using the 3000 Transfection Kit (Invitrogen, catalog number: L3000-015). Flipin CHO-TNF-α cells were resuspended in assay medium (1640 + 1% carbon-adsorbed fetal bovine serum + 1% sodium pyruvate + 1% non-essential amino acids + 1% penicillin / streptomycin) and seeded at 2500 cells / 100 μL / well in 96-well plates (Costar: 3917) and incubated overnight. Antibody working solutions (2× final concentration) were prepared using assay medium to achieve final drug concentrations of 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.0128 nM. Discard the overnight supernatant of Flipin CHO-TNF-α culture, add 100 μL / well of fresh assay medium and 100 μL / well of drug working solution, mix gently to a final volume of 200 μL / well, and incubate the plate at 37°C in a 5% CO2 incubator for 5 days. Collect the culture supernatant on day 5 and centrifuge at 400g for 5 minutes. Add 50 μL / well of K562-GRE cells and 50 μL / well of day 5 culture supernatant, mix well, and incubate at 37°C for 24 hours. Mix the substrate (Meilunbio, MA0521) and fluorescence detection buffer at a 1:50 ratio to obtain the substrate reaction solution. Add 100 μL / well of the substrate reaction solution to a 96-well plate and mix on a shaker (400 rpm, 10 minutes). Detect the fluorescence signal value in the well plate using a PE Envision microplate reader with a US Lumi module. Plot the drug activation curve with drug concentration on the x-axis and RLU fluorescence signal value on the y-axis. Perform four-parameter fitting (GraphPad Prism10) and calculate the EC50 of the activation curve. 50 and maximum luminous intensity E max The results are shown in Table 16 and Figure 12.

[0406] Table 16 Bystander Effect of Anti-TNFα Antibody-Drug Conjugates

[0407] Test Example 15: Bystander Effect of K562-luc-GRE Reporter Gene Detection of Anti-TL1A Antibody-Drug Conjugate

[0408] The full-length amino acid sequence encoding human TL1A (NCBI: NP_005109.2) was cloned into the pcDNA3.1 vector (purchased from Clontech) and a plasmid was prepared. The plasmid was then transfected into the CHO-K1 cell line (purchased from ATCC). The CHOK1-TL1A cell line with good growth, high fluorescence intensity and single clone was obtained by screening after transfection (3000 Transfection Kit, purchased from Invitrogen, item number: L3000-015) and was continuously expanded and cryopreserved in liquid nitrogen. CHOK1-TL1A cells were resuspended with assay medium (1640 + 1% carbon adsorbed fetal bovine serum + 1% sodium pyruvate + 1% non-essential amino acids + 1% penicillin / streptomycin double antibody), and 2500 cells / 100 μL / well were plated in a 96-well plate (Costar: 3917) for overnight adhesion. Antibody drug working solution (2x final concentration) was prepared with assay medium, and the final drug concentration was 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, and 0.0128 nM. The overnight culture supernatant of CHOK1-TL1A was discarded, and 100 μL / well of fresh assay medium and 100 μL / well of drug working solution were added, mixed gently, and the final volume was 200 μL / well. The plate was incubated at 37°C in a 5% CO2 incubator for 5 days. The culture supernatant on the 5th day was collected and centrifuged at 400g for 5 minutes. 50 μL / well of K562-GRE cells and 50 μL / well of 5th day culture supernatant were added and mixed, and incubated at 37°C for 24 hours. The substrate (Meilunbio, MA0521) and fluorescence detection buffer were mixed at 1:50 to obtain the substrate reaction solution. After adding 100 μL / well of substrate reaction solution to the 96-well plate, it was mixed on a shaker (400 rpm, 10 minutes). The PE Envision microplate reader-US Lumi module was used to detect the fluorescence signal value in the plate. The activation curve of the drug was plotted with the drug concentration as the abscissa and the fluorescence signal value RLU as the ordinate, and the four-parameter fitting (GraphPad Prism10) was used to calculate the EC 50 and maximum luminescence intensity E max . The results are shown in Table 17 and Figure 13.

[0409] Table 17 Anti-TL1A antibody-drug conjugate bystander effect

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structure of Ab-(L-D) n , wherein, the Ab is an antibody or an antigen-binding fragment thereof; L is a linker unit; D is a drug unit of the formula n is 1-16.

2. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1, wherein, Connecting subunit L is selected from Its a-end is covalently connected to Ab, and its b-end is covalently connected to drug unit D, where L 1 The peptide residues are composed of 1 to 8 amino acids, and the peptide residues are further optionally coated with halogens, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4-7 membered heterocyclic groups, and (C1-C6 alkyl)-(C=O)-(NCH2CH2). j1 -(OCH2CH2) j2 One or more substituents are selected from (OC1-C6 alkyl), wherein j1 and j2 are each independently selected from integers 0 to 20; Preferably, said L 1 is a peptide residue consisting of 2 or 3 amino acids selected from glycine, alanine or glutamic acid, said peptide residue being further optionally substituted by (Ci-C6alkyl)-(C=0)-(NCH2CH2) j1 -(OCH2CH2) j2 -(OCi-C6alkyl), said j1 and j2 are each independently selected from integers 1 to 12; preferably, said peptide residue is further optionally substituted by substituted.

3. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein, The linker unit L is selected from which is covalently attached at the a end to Ab and at the b end to the drug unit D.

4. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein, The antibody-drug conjugate or pharmaceutically acceptable salt thereof is selected from the following compounds of Formula (I) or a pharmaceutically acceptable salt thereof:

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein, The Ab specifically binds one or more antigens selected from the group consisting of: IL-4R, TNFa, AXL, BAFFR, BCMA, BCR, BDCA2, BDCA4, BTLA, BTNL2 BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD163, CD168, CD 177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR’s, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNAR, IFNAR1, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL6R, IL5R, IL7R, IL9R, Integrins, LAG3, LIFR, MAG / Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, OX40 (CD134), PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4, IL2R-y, TL1A, and VTCN1; or. the Ab is selected from an antibody or an antigen-binding fragment that specifically binds to IL-4R, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and / or a light chain variable region (VL); the heavy chain variable region comprising CDR1, CDR2 and CDR3 selected from the VH as set forth in any one of SEQ ID NO. 1, 3, 5 and 7; the light chain variable region comprising CDR1, CDR2 and CDR3 selected from the VL as set forth in any one of SEQ ID NO. 2, 4, 6 and 8; Preferably, the heavy chain variable region comprises CDR1, CDR2 and CDR3 as set forth in: (1) SEQ ID NO. 11, 12 and 13, respectively; (2) SEQ ID NO. 17, 18 and 19, respectively; (3) SEQ ID NO. 23, 24 and 25, respectively; (4) SEQ ID NO. 29, 30 and 31, respectively; or (5) a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or having at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid insertion, deletion and / or substitution, preferably a substitution to a conservative amino acid, to the sequence as set forth in any one of (1) to (4) above, respectively; Preferably, the light chain variable region comprises CDR1, CDR2 and CDR3 as set forth in: (1) SEQ ID NO. 14, 15 and 16, respectively; (2) SEQ ID NO. 20, 21 and 22, respectively; (3) SEQ ID NO. 26, 27 and 28, respectively; (4) SEQ ID NO. 32, 33 and 34, respectively; or (5) a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or having at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid insertion, deletion and / or substitution, preferably a substitution to a conservative amino acid, to the sequence as set forth in any one of (1) to (4) above, respectively.

6. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of claim 5, wherein, the antibody or antigen-binding fragment comprising: a heavy chain variable region comprising an amino acid sequence having at least 80% identity to the sequence as set forth in any one of SEQ ID NO. 1, 3, 5 and 7; or / and a light chain variable region comprising an amino acid sequence having at least 80% identity to the sequence as set forth in any one of SEQ ID NO. 2, 4, 6 and 8; Preferably, (1) the heavy chain variable region comprises the sequence as set forth in SEQ ID NO. 1, and the light chain variable region comprises the sequence as set forth in SEQ ID NO. 2; (2) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO. 3, and the light chain variable region comprises a sequence as set forth in SEQ ID NO. 4; (3) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO. 5, and the light chain variable region comprises a sequence as set forth in SEQ ID NO. 6; (4) the heavy chain variable region comprises a sequence as set forth in SEQ ID NO. 7, and the light chain variable region comprises a sequence as set forth in SEQ ID NO. 8; (5) the heavy chain variable region and / or the light chain variable region comprises a sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in any one of (1)-(4), respectively; or, a sequence which has at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the sequence set forth in any one of (1)-(4), respectively; the mutations can be selected from insertions, deletions and / or substitutions, preferably conservative amino acid substitutions.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 1 to 4, wherein the Ab is an antibody or an antigen-binding fragment thereof that specifically binds TNFα, the antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and / or a light chain variable region (VL); the heavy chain variable region comprises CDR1, CDR2 and CDR3 of a VH as set forth in SEQ ID NO. 40; alternatively, the heavy chain variable region comprises CDRs which are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1, CDR2 and CDR3 of a VH as set forth in SEQ ID NO. 40, respectively, or have at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid insertions, deletions and / or substitutions, preferably conservative amino acid substitutions; the light chain variable region comprises CDR1, CDR2 and CDR3 of a VL as set forth in SEQ ID NO. 41; alternatively, the light chain variable region comprises CDRs which are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to CDR1, CDR2 and CDR3 of a VL as set forth in SEQ ID NO. 41, respectively, or have at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid insertions, deletions and / or substitutions, preferably conservative amino acid substitutions.

8. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 7, wherein, the antibody or antigen binding fragment comprises a heavy chain variable region comprising a sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO. 40; or, a sequence which differs from the sequence set forth in SEQ ID NO. 40 by up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, which can be selected from insertions, deletions and / or substitutions, which substitutions are preferably conservative amino acid substitutions; and / or a light chain variable region comprising a sequence which is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence set forth in SEQ ID NO. 41 ; or, a sequence which differs from the sequence set forth in SEQ ID NO. 41 by up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations, which can be selected from insertions, deletions and / or substitutions, which substitutions are preferably conservative amino acid substitutions; Preferably, the heavy chain variable region comprises the sequence set forth in SEQ ID NO. 40 and the light chain variable region comprises the sequence set forth in SEQ ID NO.

41.

9. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 1 to 4, wherein the Ab is an antibody or antigen binding fragment thereof that specifically binds TL1A, the antibody or antigen binding fragment thereof comprising a heavy chain variable region (VH) and / or a light chain variable region (VL); the heavy chain variable region comprises CDR1, CDR2 and CDR3 of the VH set forth in SEQ ID NO. 44; or, the heavy chain variable region comprises CDRs which are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to, or have at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid insertions, deletions and / or substitutions, preferably conservative amino acid substitutions, to CDR1, CDR2 and CDR3 of the VH set forth in SEQ ID NO. 44, respectively; the light chain variable region comprises CDR1, CDR2 and CDR3 of the VL set forth in SEQ ID NO. 45; Alternatively, the light chain variable region comprises CDRs that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, or have at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions, in CDR1, CDR2, and CDR3 of the VL of SEQ ID NO.

45.

10. The antibody-drug conjugate or pharmaceutically acceptable salt thereof according to claim 9, wherein, The antibody or antigen binding fragment comprises: a heavy chain variable region comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO. 44; or, a sequence that differs from the sequence of SEQ ID NO. 44 by at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations; the mutations can be selected from insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions; and / or a light chain variable region comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO. 45; or, a sequence that differs from the sequence of SEQ ID NO. 45 by at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations; the mutations can be selected from insertions, deletions, and / or substitutions, preferably conservative amino acid substitutions. Preferably, the heavy chain variable region comprises the sequence of SEQ ID NO. 44, and the light chain variable region comprises the sequence of SEQ ID NO.

45.

11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof of any one of claims 5 to 10, wherein, The antibody or antigen binding fragment further comprises a heavy chain constant region and / or a light chain constant region; preferably, the heavy chain constant region is selected from IgG, e.g., IgG1, IgG2, IgG3, or IgG4, which can be selected from human IgG, e.g., human IgG4; alternatively, the heavy chain constant region can be selected from an Fc region, a CH3 region, or a complete heavy chain constant region, which can be a human Fc region; alternatively, the heavy chain constant region has the sequence of SEQ ID NO. 9, 39, 42, or 46; the light chain constant region is selected from a kappa chain or a lambda chain, preferably a kappa chain; alternatively, the light chain constant region has the sequence of SEQ ID NO.

10.

12. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 5 to 11, wherein, The antibody or antigen binding fragment is: (1) a chimeric antibody or fragment thereof; (2) a humanized antibody or fragment thereof; or (3) a fully human antibody or fragment thereof.

13. The antibody-drug conjugate or pharmaceutically acceptable salt thereof of any one of claims 5 to 12, wherein, the antibody or antigen-binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific molecule (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, an intact antibody, a fragment of an intact antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, a Fab, a Fab', a Fab'-SH, a F(ab')2, a Fd, a Fv, a scFv, or a diabody; Preferably, the multispecific molecule further comprises an antibody or antigen-binding fragment that specifically binds to an antigen other than IL4R or binds to a different epitope of IL4R than the antibody or antigen-binding fragment of any of the above; Preferably, the antigen other than IL4R is selected from the group consisting of (1) a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA); (2) an immune checkpoint; (3) a target that recruits and / or activates immune cells.

14. A drug-linker compound or a pharmaceutically acceptable salt thereof, having the general structure of X-L-D, wherein, X is selected from halogen, OS(O)2CH3, or OS(O)2CF3; The linker unit L is selected from covalently attached to X at the a terminus and covalently attached to a drug unit D at the b terminus, wherein L 1 is a peptide residue consisting of 1 to 8 amino acids, which is further optionally substituted with one or more substituents selected from the group consisting of halogen, CN, =0, C1-C6 alkyl, OH, 0(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, 4- to 7-membered heterocyclyl, and (C1-C6 alkyl)-(C=0)-(NCH2CH2) j1 -(OCH2CH2) j2 -(OC1-C6 alkyl), j1and j2are each independently selected from an integer from 0 to 20; D is a drug unit of the formula 15. The drug-linker compound of claim 14, or a pharmaceutically acceptable salt thereof, wherein, L is selected from wherein the a-terminus is covalently attached to X and the b-terminus is covalently attached to the drug unit D.

16. The drug-linker compound of claim 14 or 15, or a pharmaceutically acceptable salt thereof, wherein, X is selected from Br or I.

17. The drug-linker compound of any one of claims 14 to 16, or a pharmaceutically acceptable salt thereof, wherein, the drug-linker compound or pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

18. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

19. Use of the antibody-drug conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 18, in the manufacture of a medicament for preventing or treating an inflammatory disease or an autoimmune disease; or for use in preventing or treating an inflammatory disease or an autoimmune disease. Preferably, the inflammatory disease or autoimmune disease is selected from atopic dermatitis.

20. A method of preparing the antibody-drug conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, comprising the step of conjugating the antibody to the drug-linker compound of any one of claims 14 to 17.

21. A method of treating an inflammatory disease or an autoimmune disease in a mammal, comprising administering to a mammal in need of treatment a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 18.

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