Anti-her3 antibodies and anti-her3 antibody drug conjugates and medical uses thereof

Abstract

This document provides information on anti-HER3 antibodies and anti-HER3 antibody-drug conjugates, along with their pharmaceutical uses. Specifically, it describes anti-HER3 antibodies and anti-HER3 antibody-drug conjugates as shown in the general formula (Pc-L-Y-D), where Pc represents the anti-HER3 antibody, and L, Y, and n are as defined in the product information.

Description

[0001] This application is a divisional application of the invention patent application with application number 202180054438.6, application date October 14, 2021, entitled "Anti-HER3 antibody and anti-HER3 antibody drug conjugate and their pharmaceutical uses". Technical Field

[0002] This disclosure relates to anti-HER3 antibodies, anti-HER3 antibody-eczema analogue conjugates, methods of their preparation, pharmaceutical compositions comprising them, and their use in the preparation of medicaments for treating HER3-mediated diseases or conditions; particularly in the preparation of anticancer medicaments. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] HER3 (epidermal growth factor receptor 3, ErbB-3 or HER3) is a member of the epidermal growth factor receptor (EGFR) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). These receptors each consist of three parts: an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains four domains, while the intracellular domain contains one intracellular tyrosine kinase domain for signal transduction and a tail with tyrosine phosphorylated residues located in the cytoplasm. Cell signaling is initiated when the ligand binds to extracellular domains I and III. Under normal circumstances, these receptors mediate cell division, migration, survival, and organ development. Mutations in EGFR family members result in abnormal signal transduction that stimulates cell survival and is associated with cancer progression. The basic principle behind the activation and physiological effects of the HER3 receptor is similar to that of other family members, but the difference lies in that its ligands include neuroregulatory protein 1 (NRG-1) and neuroregulatory protein 2 (NRG-2), and after activation, HER3 cannot form homomers, but can only form heterodimers with EGFR or HER2. During the formation of heterodimers, the intracellular domain of HER3 exhibits high tyrosine phosphorylase activity. Structural analysis revealed that the intracellular domain of HER3 has six P85 (PI-3K subunit) binding sites. This specific structure determines that when HER3 interacts with the P85 regulatory subunit, it can recruit up to six PI-3K molecules to the regulatory subunit sites, thereby strongly activating the PI-3K signaling pathway. In fact, the HER3 / HER2 dimer is the most active HER dimer. EGFR is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, and other cells. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation, and differentiation.

[0005] HER3 is highly expressed in various common malignant tumors, such as breast cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, and melanoma. Unlike EGFR gene mutations, which lead to high-level expression or overactivation, HER3 has a low mutation rate. Its high expression is mainly due to increased mRNA transcription, resulting in increased protein translation, and it is often co-expressed with HER2. High HER3 expression is closely related to the occurrence, progression, and survival of various tumors, making research on anti-tumor drugs targeting HER3 of great significance. Summary of the Invention

[0006] This disclosure relates to anti-HER3 antibodies, anti-HER3 antibody-ecithecan analogue conjugates, and their uses.

[0007] This disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody has one or more of the following characteristics:

[0008] a. The anti-HER3 antibody described above has an apparent affinity EC of less than 0.5 nM. 50 The epigenetic affinity EC binds to the HER3 protein. 50 It was determined using the ELISA method;

[0009] b. The anti-HER3 antibody has an apparent affinity EC of less than 0.2 nM. 50 The epigenetic affinity EC binds to the HER3 protein expressed in MCF7 cells. 50 It was determined using the FACS method;

[0010] c. The anti-HER3 antibody described herein can be endocytosed by cells expressing human HER3.

[0011] This disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody has one or more of the following characteristics:

[0012] a. The anti-HER3 antibody described above has an apparent affinity EC of less than 0.5 nM. 50 The epigenetic affinity EC binds to the HER3 protein. 50 It was determined using the ELISA method;

[0013] b. The anti-HER3 antibody has an apparent affinity EC of less than 0.2 nM. 50 The epigenetic affinity EC binds to the HER3 protein expressed in MCF7 cells. 50 It was determined using the FACS method;

[0014] c. The anti-HER3 antibody described herein can be internalized by cells expressing human HER3. When the method of Test Example 3 is used to determine the anti-HER3 antibody, its IC50 value is [value missing]. 50 Less than 2 nM;

[0015] d. The anti-HER3 antibody can be endocytosed by cells expressing human HER3, and when the anti-HER3 antibody is measured using the method of Test Example 4, its FITC signal is greater than 300.

[0016] In some embodiments, the anti-HER3 antibody as described in any of the preceding embodiments comprises (1) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 7; and (2) LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 8.

[0017] In some implementations, the anti-HER3 antibody as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0018] a. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 respectively.

[0019] The CDR area mentioned above is determined according to the Chothia numbering rule.

[0020] In some implementations, the anti-HER3 antibody as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0021] b. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 respectively.

[0022] The CDR area mentioned above is determined according to the IMGT numbering rules.

[0023] In some implementations, the anti-HER3 antibody as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0024] c. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 respectively.

[0025] The CDR region is determined according to the Kabat numbering rule. This disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0026] a. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 respectively.

[0027] The CDR area mentioned above is determined according to the Chothia numbering rule.

[0028] This disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0029] b. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 respectively.

[0030] The CDR area mentioned above is determined according to the IMGT numbering rules.

[0031] This disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0032] c. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 respectively.

[0033] The CDR area mentioned above is determined according to the Kabat numbering rules.

[0034] In some implementations, the anti-HER3 antibody, as described in any of the preceding embodiments, is a human antibody or an antigen-binding fragment.

[0035] In some implementations, the anti-HER3 antibody as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0036] The amino acid sequence of the heavy chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 7, and / or the amino acid sequence of the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 8;

[0037] In some implementations, the anti-HER3 antibody as described in any of the preceding embodiments comprises a heavy chain variable region and a light chain variable region, wherein:

[0038] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8; or

[0039] In some embodiments, the anti-HER3 antibody as described in any of the preceding embodiments further comprises an antibody heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from human IgG1, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, and the light chain constant region is selected from human antibody κ and λ chain constant regions and conventional variants thereof; more preferably, the antibody comprises a heavy chain constant region as shown in SEQ ID NO: 5 and a light chain constant region as shown in SEQ ID NO: 6.

[0040] In some implementations, the anti-HER3 antibody, as described in any of the preceding embodiments, comprises:

[0041] Heavy chains having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 27, and / or light chains having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 28;

[0042] In some implementations, the anti-HER3 antibody, as described in any of the preceding embodiments, comprises:

[0043] Heavy chains as shown in SEQ ID NO: 27 and light chains as shown in SEQ ID NO: 28.

[0044] In some implementations, the anti-HER3 antibody, as described in any of the preceding embodiments, has one or more of the following characteristics:

[0045] a. The anti-HER3 antibody binds to the HER3 protein with an apparent affinity EC50 of less than 0.5 nM, wherein the apparent affinity EC50 is determined by an ELISA method;

[0046] b. The anti-HER3 antibody binds to the HER3 protein expressed on MCF7 cells with an epigenetic affinity EC50 of less than 0.2 nM, wherein the epigenetic affinity EC50 is determined by FACS method;

[0047] c. The anti-HER3 antibody described herein can be endocytosed by cells expressing human HER3.

[0048] In some implementations, the anti-HER3 antibody, as described in any of the preceding embodiments, has one or more of the following characteristics:

[0049] a. The anti-HER3 antibody binds to the HER3 protein with an apparent affinity EC50 of less than 0.5 nM, wherein the apparent affinity EC50 is determined by an ELISA method;

[0050] b. The anti-HER3 antibody binds to the HER3 protein expressed on MCF7 cells with an epigenetic affinity EC50 of less than 0.2 nM, wherein the epigenetic affinity EC50 is determined by FACS method;

[0051] c. The anti-HER3 antibody can be internalized by cells expressing human HER3, and when the anti-HER3 antibody is measured using the method in Test Example 3, its IC50 is less than 2 nM;

[0052] d. The anti-HER3 antibody can be endocytosed by cells expressing human HER3, and when the anti-HER3 antibody is measured using the method of Test Example 4, its FITC signal is greater than 300.

[0053] In some embodiments, this disclosure also provides an isolated anti-HER3 antibody, wherein said antibody competitively binds to human HER3 with an anti-HER3 antibody as described in any of the preceding embodiments.

[0054] In some implementations, this disclosure also provides a nucleic acid molecule that encodes an anti-HER3 antibody as described in the previous one.

[0055] In some implementations, this disclosure also provides a host cell containing nucleic acid molecules as described in the previous one.

[0056] In some embodiments, this disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-HER3 antibody as described in any of the preceding claims, or a nucleic acid molecule as described above, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0057] In some embodiments, this disclosure also provides an immunoconjugate comprising an anti-HER3 antibody as described in any of the preceding claims and an effector molecule, wherein the effector molecule is conjugated to the anti-HER3 antibody; preferably, the effector molecule is selected from radioisotopes, antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0058] In some embodiments, this disclosure also provides an in vivo or in vitro method for the immune detection or determination of HER3, the method comprising the step of contacting a subject or a sample from a subject with an anti-HER3 antibody as described in any of the preceding claims.

[0059] In some embodiments, this disclosure also provides an antibody-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof:

[0060]

[0061] in:

[0062] Y is selected from -O-(CR) a R b ) m -CR 1 R 2 -C(O)-、-O-CR 1 R 2 -(CR a R b ) m -、-O-CR 1 R 2 -、-NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0063] R a and R b They may be the same or different, and each is independently selected from hydrogen, deuterium, halogen, alkyl, haloalkyl, deuteralkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, and heterocyclic groups; or, R a and R b Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0064] R 1 Selected from halogens, haloalkyls, deuteralkyls, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclics, aryl groups, and heteroaryl groups; R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups; or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0065] Or, R a and R 2 Together with the carbon atom attached to it, it forms a cycloalkyl or heterocyclic group;

[0066] m is an integer from 0 to 4;

[0067] n is between 1 and 10, and n is a decimal or an integer;

[0068] L represents the connector unit;

[0069] Pc is the anti-HER3 antibody as described in the previous one.

[0070] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the general formula (Pc-LYD) described in any of the preceding embodiments, is a number from 1 to 8, where n is a decimal or an integer. In some embodiments, n is a number from 3 to 8, where n is a decimal or an integer.

[0071] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, as shown in the general formula (Pc-LYD) described in the preceding one,

[0072] in:

[0073] Y is -O-(CR) a R b ) m -CR 1 R 2 -C(O)-;

[0074] R a and R b They may be the same or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, and carbon atoms. 1-6 alkyl;

[0075] R 1 Halogenated C 1-6 Alkyl or C 3-6 cycloalkyl;

[0076] R 2 Selected from hydrogen atoms, halogenated C 1-6 Alkyl and C 3-6cycloalkyl;

[0077] Or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 cycloalkyl;

[0078] m is 0 or 1.

[0079] In some embodiments, the antibody-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, wherein Y is selected from:

[0080]

[0081] The O end of Y is connected to the connector unit L.

[0082] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the general formula (Pc-LYD) described in the preceding claim, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0083] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- and -C(O)-WC(O)-, where W is selected from C. 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 chain atoms, wherein the straight-chain heteroalkyl groups of 1 to 8 chain atoms contain 1 to 3 heteroatoms selected from N, O, and S, wherein the C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 chain atoms are each independently and optionally further selected from halogens, hydroxyl groups, cyano groups, amino groups, C6 groups. 1-6 Alkyl, Halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 One or more substituents in the cycloalkyl group are substituted;

[0084] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20;

[0085] L 3 It is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0086] L 4 Selected from -NR 5 (CR 6 R 7 ) t -、-C(O)NR 5 -C(O)NR 5 (CH2) t - and chemical bonds, where t is an integer from 1 to 6;

[0087] R 3 R 4 and R 5 Whether the atoms are the same or different, and each is independently selected from hydrogen atoms, C atoms 1-6 Alkyl, Halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 Hydroxyalkyl;

[0088] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, Halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 Hydroxyalkyl.

[0089] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the general formula (Pc-LYD) described in the preceding claim, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0090] L 1 for s 1 Integers between 2 and 8;

[0091] L 2 It is a chemical bond;

[0092] L 3 It is a tetrapeptide residue;

[0093] L 4 For -NR 5 (CR 6 R 7 )t-,R 5 R 6 Or R 7 They may be the same or different, and each is independently a hydrogen atom or a carbon atom. 1-6 Alkyl group, where t is 1 or 2;

[0094] The L mentioned therein 1 The terminal is connected to the PC, L 4 The end is connected to Y.

[0095] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the general formula (Pc-LYD) described in the preceding one, L 3 It is a tetrapeptide residue of GGFG.

[0096] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the general formula (Pc-LYD) described in the preceding claim, wherein -L- is:

[0097] .

[0098] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, represented by the general formula (Pc-LYD) as described in any of the preceding embodiments, is of the general formula (Pc-L). a The antibody-drug conjugate or its pharmaceutically acceptable salt as shown in (-YD):

[0099]

[0100] in,

[0101] Pc is the anti-HER3 antibody as described above;

[0102] m is an integer from 0 to 4; for example, m is selected from 0, 1, 2, 3, and 4.

[0103] n is 1 to 10, and n is a decimal or an integer; specifically, n is a decimal or an integer between 2 and 8, including the two endpoint values; more specifically, n is a decimal or an integer between 2 and 7, including the endpoint values; alternatively, n is a decimal or an integer between 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7 or 7 and 8, including the endpoint values;

[0104] R 1Selected from halogens, haloalkyls, deuteralkyls, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclics, aryl groups, and heteroaryl groups; R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups; or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups;

[0105] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 chain atoms, wherein the straight-chain heteroalkyl groups of 1 to 8 chain atoms contain 1 to 3 heteroatoms selected from N, O, and S, wherein the C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 chain atoms are each independently and optionally further selected from halogens, hydroxyl groups, cyano groups, amino groups, C6 groups. 1-8 Alkyl, Chlorinated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 The cycloalkyl group is substituted by one or more substituents;

[0106] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20;

[0107] L 3 It is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl;

[0108] R 5 Selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups;

[0109] R 6 and R 7They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

[0110] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, represented by the general formula (Pc-LYD) as described in any of the preceding embodiments, is of the general formula (Pc-L). a The antibody-drug conjugate or its pharmaceutically acceptable salt as shown in (-YD) is: wherein,

[0111] Pc is the anti-HER3 antibody as described in the previous item;

[0112] m is an integer from 0 to 4; for example, m is selected from 0, 1, 2, 3, and 4.

[0113] n is 1 to 10, and n is a decimal or an integer; specifically, n is a decimal or an integer between 2 and 8, including the two endpoint values; more specifically, n is a decimal or an integer between 2 and 7, including the endpoint values; alternatively, n is a decimal or an integer between 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7 or 7 and 8, including the endpoint values;

[0114] R 1 Selected from halogens, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 3-6 cycloalkyl, C 3-6 cycloalkyl C 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, heterocyclic, aryl, and heteroaryl; R 2 Selected from hydrogen atoms, halogens, and halogenated C atoms 1-6 Alkyl, deuterated C 1-6 Alkyl, C 3-6 cycloalkyl, C 3-6 cycloalkyl C 1-6 Alkyl, C 1-6 Alkoxy C 1-6 Alkyl, heterocyclic, aryl, and heteroaryl; or, R 1 and R 2 Together with the carbon atoms it is attached to, they form C 3-6 cycloalkyl or heterocyclic groups;

[0115] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 chain atoms, wherein the straight-chain heteroalkyl groups of 1 to 8 chain atoms contain 1 to 3 heteroatoms selected from N, O, and S, wherein the C 1-8 Alkyl, C 1-8 Alkyl-C 3-6Cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 chain atoms are each independently and optionally further selected from halogens, hydroxyl groups, cyano groups, amino groups, C6 groups. 1-6 Alkyl, Chlorinated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 The cycloalkyl group is substituted by one or more substituents;

[0116] L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20;

[0117] L 3 A peptide consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q), and aspartic acid (D), and optionally further selected from halogens, hydroxyl groups, cyano groups, amino groups, and C groups. 1-6 Alkyl, Chlorinated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 One or more substituents in the cycloalkyl group are substituted;

[0118] R 5 Selected from hydrogen atoms, C 1-6 Alkyl, Halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 Hydroxyalkyl;

[0119] R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, and carbon atoms. 1-6 Alkyl, Halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 Hydroxyalkyl;

[0120] The heterocyclic group contains 3 to 6 ring atoms, of which 1 to 3 are heteroatoms selected from nitrogen, oxygen and sulfur.

[0121] In some embodiments, the antibody-drug conjugate of the general formula (Pc-LYD) as described in any of the preceding embodiments, or a pharmaceutically acceptable salt thereof, is:

[0122]

[0123] in:

[0124] n is 1 to 8, where n is a decimal or an integer; specifically, n is a decimal or an integer between 2 and 8, including the two endpoint values; more specifically, n is a decimal or an integer between 2 and 7, including the endpoint values; alternatively, n is a decimal or an integer between 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, or 7 and 8, including the endpoint values.

[0125] HER3-29 is an anti-HER3 antibody that contains a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 28.

[0126] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt, as shown in the general formula (Pc-LYD) described in any of the preceding embodiments, is preferably 3 to 8, where n is a decimal or an integer.

[0127] In some embodiments, this disclosure also provides a pharmaceutical composition comprising an anti-HER3 antibody as described in any of the preceding claims, or a nucleic acid molecule as described in any of the preceding claims, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, and one or more pharmaceutically acceptable excipients, diluents or carriers.

[0128] In some embodiments, this disclosure also provides the use of an anti-HER3 antibody as described in any of the preceding claims, or a nucleic acid molecule as described in any of the preceding claims, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims, in the preparation of a medicament for treating HER3-mediated diseases or conditions.

[0129] In some embodiments, this disclosure also provides the use of an anti-HER3 antibody as described in any of the preceding claims, or a nucleic acid molecule as described in any of the preceding claims, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims, in the preparation of a medicament for treating and / or preventing tumors and cancers, wherein said tumors and cancers are selected from breast cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, and melanoma.

[0130] In some embodiments, this disclosure also provides a kit comprising an anti-HER3 antibody as described in any of the preceding claims, or a nucleic acid molecule as described in any of the preceding claims, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, or a pharmaceutical composition as described in any of the preceding claims.

[0131] In some embodiments, this disclosure also provides a method for preventing or treating a disease or condition, the method comprising administering to a subject a therapeutically effective amount of an anti-HER3 antibody as described in any of the preceding embodiments, or a nucleic acid molecule as described in any of the preceding embodiments, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding embodiments, or a pharmaceutical composition as described in any of the preceding embodiments. In some embodiments, the disease or condition is preferably a tumor, an autoimmune disease, or an infectious disease; in some embodiments, the disease or condition is a HER3-related disease or condition.

[0132] On the other hand, this disclosure provides a pharmaceutical composition comprising an anti-HER3 antibody, an antibody-drug conjugate, or a pharmaceutically acceptable salt thereof as described in any of the preceding claims, and one or more pharmaceutically acceptable excipients, diluents, or carriers. In some embodiments, a unit dose of the pharmaceutical composition contains 0.1-3000 mg or 1-1000 mg of the anti-HER3 antibody or the antibody-drug conjugate as described above.

[0133] On the other hand, this disclosure provides for the use of antibody-drug conjugates or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising them as described in any of the preceding claims as pharmaceuticals.

[0134] On the other hand, this disclosure provides the use of an antibody-drug conjugate as described in any of the preceding claims or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising therethe in the preparation of a medicament for treating HER3-mediated diseases or conditions, in some embodiments of which the HER3-mediated diseases or conditions are HER3-high, medium, or low expression cancers.

[0135] On the other hand, this disclosure provides the use of antibody-drug conjugates as described in any of the preceding claims or pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising such conjugates in the preparation of medicaments for the treatment or prevention of cancer, wherein in some embodiments the tumors and cancers are selected from breast cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, and melanoma.

[0136] On the other hand, this disclosure further relates to a method for treating and / or preventing tumors, the method comprising administering to a subject in need of the thereof a therapeutically effective dose of an antibody-drug conjugate as described in any of the preceding claims or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the thereof; in some embodiments, wherein the tumor is a cancer associated with high HER3 expression, a moderately expressed cancer, or a low expressed cancer.

[0137] On the other hand, this disclosure further relates to a method for treating or preventing tumors or cancer, the method comprising administering to a subject in need of the thereof a therapeutically effective dose of an antibody-drug conjugate as described in any of the preceding claims or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the thereof; wherein the tumor and cancer are, in some embodiments, selected from breast cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, and melanoma.

[0138] On the other hand, this disclosure further provides anti-HER3 antibodies or antibody-drug conjugates thereof as described in the preceding one as medicines, in some embodiments as medicines for treating cancer or tumors, more preferably as medicines for treating HER3-mediated cancers.

[0139] The active compound (e.g., a compound according to this disclosure or a pharmaceutically acceptable salt thereof, a ligand-drug conjugate according to this disclosure or a pharmaceutically acceptable salt thereof) can be formulated in a form suitable for administration via any appropriate route, either in a unit dose or in a manner that allows the subject to self-administer a single dose. The unit dose of the active compound or composition disclosed herein can be expressed as a tablet, capsule, sachet, bottled liquid, powder, granules, lozenge, suppository, regenerated powder, or liquid formulation.

[0140] The dosage of the active compound or composition used in the treatment methods disclosed herein will generally vary depending on the severity of the disease, the subject's weight, and the relative efficacy of the active compound. However, as a general guideline, a suitable unit dose may be 0.1 mg to 1000 mg.

[0141] In addition to the active compound, the pharmaceutical compositions disclosed herein may contain one or more excipients selected from the following: fillers, diluents, binders, wetting agents, disintegrants, or excipients. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0142] The HER3 antibody and antibody-drug conjugate disclosed herein have good affinity for cell surface antigens, good endocytosis efficiency and strong tumor inhibition efficiency, and have a wider drug application window, making them suitable for clinical drug application. Attached Figure Description

[0143] Figure 1 The binding activity of the antibodies and positive antibodies to the HER3 protein disclosed herein.

[0144] Figure 2 The binding activity of the antibodies and positive antibodies disclosed herein with MCF7 cells.

[0145] Figure 3 The endocytic activity of the disclosed antibodies and positive antibodies was tested using DT3C.

[0146] Figure 4 The endocytic activity of the antibodies and positive antibodies disclosed herein was tested using pHrodo.

[0147] Figure 5 This disclosure describes the efficacy of ADC samples against SW620 xenograft tumors in tumor-bearing nude mice. Detailed Implementation

[0148] the term

[0149] Unless otherwise specified, all technical and scientific terms used herein are consistent with the common understanding of one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used to perform or test this disclosure, preferred methods and materials are described herein. In describing and claiming protection for this disclosure, the following terms are used in accordance with the definitions below.

[0150] When a trade name is used in this disclosure, it is intended to include the formulation of the product under that trade name, the pharmaceutical part of the product under that trade name, and the active pharmaceutical part.

[0151] The term "antibody-drug conjugate" (ADC) refers to the linking of an antibody to a biologically active drug. The antibody can be directly conjugated to the drug or via a linker unit.

[0152] The term "drug loading" refers to the average number of drugs carried by each antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of drug amount to antibody amount. Drug loading can range from 0 to 12 drugs per antibody, exemplarily 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 drugs. This value can be a decimal or an integer. In some embodiments, each antibody carries 1 to about 10 drugs; in other embodiments, each antibody carries about 1 to about 9, 1 to about 8, about 3 to about 7, about 3 to about 6, about 3 to about 5, about 2, about 3, about 4, about 5, about 6, about 7, and about 8 drugs. This value can be a decimal or an integer. Drug loading can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0153] In one embodiment of this disclosure, the cytotoxic drug is coupled to the thiol group of the antibody via a linker unit.

[0154] The loading of ligand-cytotoxic drug conjugates can be controlled using the following non-limiting methods, including:

[0155] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0156] (2) Control the reaction time and temperature.

[0157] (3) Choose different reaction reagents.

[0158] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0159] The term "antibody" is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, or their antigen-binding fragments (also called "antigen-binding portions"), provided they exhibit the desired antigen-binding activity. A full-length antibody is an immunoglobulin (Ig) comprising at least two heavy chains and two light chains linked together by disulfide bonds. The amino acid composition and sequence of the constant regions of the heavy chains of immunoglobulins differ, thus their antigenicity varies. Accordingly, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains of µ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge regions and the number and position of disulfide bonds in the heavy chains can further subdivide into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into κ chains or λ chains based on differences in their constant regions. Each of the five types of Ig can have either a κ chain or a λ chain.

[0160] The sequence of approximately 110 amino acids near the N-terminus of both the full-length antibody heavy and light chains varies considerably and is termed the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are termed the constant region. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The heavy and light chain variable regions include hypervariable regions (also known as complementarity-determining regions, abbreviated as CDR or HVR) and relatively conserved framework regions (also known as frame regions, abbreviated as FR). Each VL and VH consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0161] The “conventional variants” of the human antibody heavy chain constant region and human antibody light chain constant region described in this disclosure refer to variants of the heavy chain constant region or light chain constant region derived from humans that do not alter the structure and function of the antibody variable region, as disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that involve site-specific modifications and amino acid substitutions in the heavy chain constant region. Specific substitutions include known prior art mutations such as YTE mutations, L234A and / or L235A mutations, S228P mutations, 265A (e.g., D265A) and / or 297A (e.g., N297A), and / or mutations that obtain a knock-in-hole structure (resulting in the antibody heavy chain having a knock-Fc and hole-Fc combination). These mutations have been shown to give antibodies new properties without altering the function of the antibody variable region.

[0162] In this disclosure, the terms "human antibody" (HuMAb), "human-derived antibody," "fully human antibody," and "completely human antibody" are used interchangeably. Their amino acid sequences correspond to the amino acid sequences of antibodies produced by humans or human cells, or to non-human amino acid sequences derived from human antibody libraries or other human antibody coding sequences. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues.

[0163] The terms “antigen-binding fragment” or “functional fragment” or “antigen-binding portion” refer to one or more fragments of a complete antibody that retain the ability to specifically bind to an antigen. Fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments covered in the term “antigen-binding fragment” include: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by disulfide bridges on the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of the antibody; (v) dsFv, a stable antigen-binding fragment formed by interchain disulfide bonds between VH and VL; and (vi) bispecific, bispecific, and multispecific antibodies comprising fragments such as scFv, dsFv, and Fab. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be joined using recombinant methods via artificial peptide linkers that enable them to form single protein chains, where VL and VH pair to form a monovalent molecule called a single-chain Fv (scFv) (see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0164] The term "amino acid difference" or "amino acid mutation" refers to an amino acid change or mutation in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one, two, three, or more amino acids.

[0165] The term "antibody framework region" or "FR region" refers to a portion of the variable domain VL or VH, which serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0166] The terms "complementarity-determining region," "CDR," or "hypervariant region" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of a CDR can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule, the “ABM” numbering rule, the “contact” numbering rule (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains[J]. 2001), and the ImMunoGenTics (IMGT) numbering rule (see Lefranc MP, Dev. Comp. Immunol., 27, 55-77(2003)), etc. For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 27-38 (HCDR1), 56-65 (HCDR2), and 105-117 (HCDR3), while the CDR amino acid residues in VL are approximately numbered 27-38 (LCDR1), 56-65 (LCDR2), and 105-117 (LCDR3).Following the AbM rule, the CDR amino acids in VH are numbered 26-35 (HCDR1), 50-58 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).

[0167] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound to an antibody (e.g., a specific site on the HER3 molecule). Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0168] The terms "specific binding," "selective binding," "selectively binding," and "specific binding" refer to the binding of an antibody or antigen-binding fragment to a pre-defined epitope on an antigen. Typically, the antibody or antigen-binding fragment binds at a frequency of approximately less than 10-1. -8 M, for example, approximately less than 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or lower affinity (KD) binding.

[0169] As used herein, the term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0170] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, after aligning the amino acid sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0171] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcRs (such as natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to target cells and subsequently cause lysis of the target cells. Primary cells and NK cells that regulate ADCC express only FcyRIII, while monocytes express FcyRI, FcyRII, and FCYRIII. To assess the ADCC activity of a target molecule, in vitro and in vivo ADCC assays can be performed, as described by Clynes et al. (PNASUSA 95:652-656(1998)), US Patent Nos. US5500362 and US5821337, etc.

[0172] Antibody-dependent cytotoxicity (ADCP) refers to the mechanism by which antibody-coated target cells or viruses are eliminated through internalization by phagocytes (e.g., macrophages, neutrophils, and dendritic cells). The internalized antibody-coated target cells or viruses are contained in vesicles called phagosomes, which then fuse with one or more lysosomes to form phagolysosomes. ADCP can be evaluated using in vitro cytotoxicity assays with macrophages as effector cells and videomicroscopy (e.g., van Bij et al., Journal of Hepatology, Vol. 53, No. 4, October 2010, pp. 677–685). Complement-dependent cytotoxicity (CDC) refers to complement-mediated cytotoxicity, where antibodies bind to the corresponding antigens on cells or viruses, activating the classical complement pathway and forming a complex that lyses the target cells. CDC can be evaluated in vitro (e.g., CDC assays using normal human serum as the complement source) or in C1q concentration series. A decrease in CDC activity (e.g., due to the introduction of a second mutation into the peptide or antibody) can be measured by comparing the CDC activity of the peptide or antibody with that of its parent peptide or antibody without the second mutation, using the same assay. To assess the antibody's ability to induce CDC, the assay can be performed as described by Romeuf et al. (Romeuf et al., Br J Haematol. 2008 Mar; 140(6):635-43).

[0173] The antibodies or antibody fragments described in this disclosure can be conjugated to effector molecules in any manner. For example, antibodies or antibody fragments can be attached to cytotoxic drugs by chemical or recombinant methods. Chemical methods for preparing fusions or conjugates are known in the art and can be used to prepare immunoconjugates. Methods for conjugating antibodies or antibody fragments to drugs must be able to link the antibody to the cytotoxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0174] In one embodiment, both the antibody and the cytotoxic drug are proteins and can be conjugated using techniques well known in the art. Hundreds of cross-linking agents disclosed in the art exist that can conjugate two proteins. Cross-linking agents are generally selected based on reactive functional groups available or inserted on the antibody or cytotoxic drug. Alternatively, if no reactive groups are available, photoactivated cross-linking agents can be used. In some cases, it may be necessary to include a spacer between the antibody and the cytotoxic drug. Cross-linking agents known in the art include homobifunctional agents: glutaraldehyde, dimethylhexamethyleneimide, and bis(diazobenzidine), and heterobifunctional agents: m-maleimide-benzoyl-N-hydroxysuccinimide and sulfonyl-m-maleimide-benzoyl-N-hydroxysuccinimide.

[0175] Crosslinking agents that can be used to couple effector molecules to antibody fragments include, for example, TPCH (S-(2-thiopyridyl)-L-cysteine ​​hydrazide) and TPMPH (S-(2-thiopyridyl)mercaptopropionylhydrazide). TPCH and TPMPH react with the carbohydrate moiety of glycoproteins that have been previously oxidized by mild periodate treatment, thereby forming a hydrazone bond between the hydrazide moiety of the crosslinking agent and the aldehyde generated by periodate. Heterobifunctional crosslinking agents GMBS (N-(γ-maleimide-butyryloxy)-succinimide) and SMCC (succinimide-4-(N-maleimide-methyl)cyclohexane) react with primary amines, thereby introducing a maleimide group onto the component. This maleimide group can then react with a thiohydrogen group on another component that can be introduced by the crosslinking agent, thereby forming a stable thioether bond between the components. If steric hindrance between components interferes with the activity of any one component, a cross-linking agent can be used to introduce long spacer arms between the components, such as 3-(2-pyridyldithio)propionic acid n-succinimide ester (SPDP). Therefore, many suitable cross-linking agents exist, which can be used and are selected individually based on their effect on optimal immunoconjugate yield.

[0176] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0177] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. The disclosed antibodies or antigen-binding fragments utilize genetic engineering methods to add one or more human-derived FR regions to a non-human CDR region. Human FR germline sequences can be obtained by comparing against the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from the journal Immunoglobulins, 2001 ISBN012441351.

[0178] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line), 293 cells, and NSO cells. In some embodiments, the host cells in this disclosure do not contain cells derived from human embryos.

[0179] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human HER3. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, e.g., at -70°C, or lyophilized.

[0180] "Conservative modification" or "conservative substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0181] "Exogenous" refers to substances that are produced outside of an organism, cell, or human body, depending on the circumstances. "Endogenous" refers to substances that are produced inside a cell, organism, or human body, depending on the circumstances.

[0182] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. Two compared sequences are homologous at positions occupied by the same bases or amino acid monomer subunits; for example, if every position in two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared multiplied by 100. For example, in optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made when aligning two sequences to give the maximum percentage of homology. For example, comparisons can be performed using the BLAST algorithm, where the algorithm's parameters are chosen to give the maximum match between the sequences over the entire length of each reference sequence. The following references relate to BLAST algorithms frequently used in sequence analysis: BLAST (BLAST ALGORITHMS): Altschul, SF et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993) Nature Genet. 3:266-272; Madden, TL et al., (1996) Meth. Enzymol. 266:131-141; Altschul, SF et al., (1997) NucleicAcids Res. 25:3389-3402; Zhang, J. et al., (1997) Genome Res. 7:649-656. Other common BLAST algorithms, such as those provided by NCBIBLAST, are also well-known to those skilled in the art.

[0183] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, the terms “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of the number of transfections. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0184] As used herein, “polymerase chain reaction” or “PCR” refers to a procedure or technique in which trace amounts of specific portions of nucleic acids, RNA, and / or DNA are amplified as described, for example, in U.S. Patent No. 4,683,195. Generally, sequence information from the ends or beyond of the target region is required to allow the design of oligonucleotide primers; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified. The 5' nucleotides of both primers may be congruent to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA, etc. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, NY). The PCR used herein is considered an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying nucleic acid test samples, which involves using known nucleic acids as primers and a nucleic acid polymerase to amplify or produce specific portions of nucleic acids.

[0185] "Separated" refers to a purified state, and in this context means that the specified molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "separated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compound as described herein.

[0186] The term "drug" refers to a chemical substance that can alter or reveal the physiological functions and pathological states of an organism, and can be used to prevent, diagnose, and treat diseases. Drugs include cytotoxic drugs. There is no strict boundary between drugs and poisons; poisons are chemical substances that can cause harm to the body even in small doses, damaging human health. Excessive dosage of any drug can produce toxic reactions.

[0187] Cytotoxic drugs are substances that inhibit or prevent cellular function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations; however, due to their lack of specificity, they can also induce apoptosis in normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., Atmospheric iodine). 211 I 131 I 125 Y 90 Re 186 Re188 、Sm 153 Bi 212 P 32 (and radioactive isotopes of Lu), chemotherapy drugs, antibiotics, and nucleolysins.

[0188] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are included, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, and 2,3-dimethylbutyl. Alkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any usable connection point. The substituents are preferably independently selected independently from one or more substituents chosen from H atoms, D atoms, halogens, alkyl groups, alkoxy groups, haloalkyl groups, haloalkoxy groups, cycloalkyloxy groups, heterocyclic oxy groups, hydroxyl groups, hydroxyalkyl groups, cyano groups, amino groups, nitro groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups.

[0189] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein the alkyl group is as defined above.

[0190] The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably alkylene containing 1 to 8 carbon atoms, and most preferably alkylene containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), etc. Alkylene groups can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable linking point, preferably independently selected independently from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, cycloalkyloxy, heterocyclic alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, heterocyclic alkylthio, and oxo.

[0191] The term "alkenyl" refers to an alkyl compound containing a carbon-carbon double bond in its molecule, wherein the definition of alkyl is as described above. Alkenyl groups can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from hydrogen, alkyl, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl substituents.

[0192] The term "alkynyl" refers to an alkyl compound containing a carbon-carbon triple bond in its molecule, where alkyl is defined as described above. The alkynyl group can be substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from hydrogen, alkyl, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl substituents.

[0193] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 carbon atoms (e.g., 3, 4, 5, 6, 7, and 8), and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, and cyclooctyl; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0194] The term "spirocycloalkyl" refers to a 5- to 20-membered polycyclic group that shares a single carbon atom (called a spiro atom) between its rings. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Spirocycloalkyl groups are classified as monospirocycloalkyl, bispirocycloalkyl, or polyspirocycloalkyl groups based on the number of shared spiro atoms between the rings, with monospirocycloalkyl and bispirocycloalkyl groups being preferred. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocycloalkyl group. Non-limiting examples of spirocycloalkyl groups include:

[0195]

[0196] The term "fused-ring alkyl" refers to a 5- to 20-membered polycyclic aromatic hydrocarbon group in which each ring shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused-ring alkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. Non-limiting examples of fused-ring alkyl groups include:

[0197]

[0198] The term "bridged cycloalkyl" refers to a 5- to 20-membered polycyclic carbon group in which any two rings share two non-directly bonded carbon atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

[0199]

[0200] The cycloalkyl ring includes cycloalkyl groups (including monocyclic, spirocyclic, fused, and bridged rings) fused to an aryl, heteroaryl, or heterocyclic alkyl ring as described above, wherein the ring connected to the parent structure is a cycloalkyl group. Non-limiting examples include indanyl, tetrahydronaphthyl, and benzocycloheptyl, etc.; preferably phenylcyclopentyl or tetrahydronaphthyl.

[0201] The cycloalkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably selected independently from one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0202] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl and cycloalkyl are defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, independently selected from H atoms, D atoms, halogens, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0203] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O) or S(O)2, but excluding the ring portion of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) ring atoms, wherein 1 to 4 (e.g., 1, 2, 3 and 4) are heteroatoms; more preferably, it contains 3 to 8 ring atoms (e.g., 3, 4, 5, 6, 7 and 8), wherein 1 to 3 are heteroatoms (e.g., 1, 2 and 3); even more preferably, it contains 3 to 6 ring atoms, wherein 1 to 3 are heteroatoms; most preferably, it contains 5 or 6 ring atoms, wherein 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups.

[0204] The term "spiroheterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which one or more ring atoms share a single atom (called a spiro atom) between the rings, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O) or S(O)2, and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6 to 14-membered, more preferably 7 to 10-membered (e.g., 7, 8, 9, or 10-membered). Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or polyspirocyclic groups according to the number of shared spiro atoms between the rings, with monospirocyclic and bispirocyclic groups being preferred. More preferably, it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocyclic group. Non-limiting examples of spirocyclic groups include:

[0205]

[0206] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O), or S(O)₂, and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0207]

[0208] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O), or S(O)₂, and the remaining ring atoms are carbon. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered (e.g., 7, 8, 9, or 10-membered). Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic groups, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0209]

[0210] The heterocyclic ring comprises a heterocyclic group (including monocyclic, spirocyclic, fused heterocyclic, and bridged heterocyclic rings) fused to an aryl, heteroaryl, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0211]

[0212] The heterocyclic group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclicoxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0213] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (fused polycyclic is a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring comprises an aryl ring fused to a heteroaryl, heterocyclic, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples include:

[0214]

[0215] The aryl group can be substituted or unsubstituted. When substituted, the substituent can be replaced at any usable connection point. The substituent is preferably independently selected independently from one or more substituents chosen from hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl, and heteroaryl. The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered (e.g., 5, 6, 7, 8, 9, or 10-membered), more preferably 5- or 6-membered, such as furanyl, thiophene, pyridinyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, etc. The heteroaryl ring comprises a heteroaryl group fused to an aryl, heterocyclic, or cycloalkyl ring as described above, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:

[0216]

[0217] The heteroaryl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably independently selected independently from one or more substituents selected from hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclic oxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0218] The term "amino protecting group" is used to protect the amino group by means of an easily removable group, so that the amino group remains unchanged when other parts of the molecule react. Non-limiting examples include (trimethylsilyl)ethoxymethyl, tetrahydropyranyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl, etc. These groups may optionally be replaced by 1-3 substituents selected from halogens, alkoxy groups, or nitro groups.

[0219] The term "hydroxyl protecting group" is a suitable group known in the art for the protection of hydroxyl groups; see reference ("Protective Groups in Organic Synthesis", 5). Th The hydroxyl protecting group in Ed.TWGreene & P.GMWuts. As an example, preferably, the hydroxyl protecting group can be (C 1-10 Alkyl or aryl) 3-silyl, such as: triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, etc.; can be C 1-10 Alkyl or substituted alkyl, preferably alkoxy or aryl-substituted alkyl, more preferably C 1-6 alkoxy-substituted C 1-6 alkyl or phenyl substituted C 1-6 Alkyl group, C is the most preferred. 1-4 alkoxy-substituted C 1-4 Alkyl groups, such as methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), etc.; can be (C 1-10 Alkyl or aromatic acyl group, such as formyl, acetyl, benzoyl, p-nitrobenzoyl, etc.; can be (C 1-6 Alkyl or C 6-10 aryl)sulfonyl; or (C 1-6 Alkoxy or C 6-10 Aryloxy)carbonyl.

[0220] The term "cycloalkyloxy" refers to cycloalkyl-O-, where the cycloalkyl group is as defined above.

[0221] The term “heterocyclic oxy group” refers to the heterocyclic group -O-, where the heterocyclic group is as defined above.

[0222] The term "alkylthio" refers to alkyl-S-, where the alkyl group is as defined above.

[0223] The term "halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0224] The term "haloalkoxy" refers to an alkoxy group that is substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0225] The term “deuterated alkyl” refers to an alkyl group that is replaced by one or more deuterium atoms, wherein the alkyl group is as defined above.

[0226] The term "hydroxyalkyl" refers to an alkyl group that has been substituted with a hydroxyl group, wherein the alkyl group is as defined above.

[0227] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0228] The term "hydroxyl group" refers to -OH.

[0229] The term "thiol" refers to -SH.

[0230] The term "amino" refers to -NH2.

[0231] The term "cyano" refers to -CN.

[0232] The term "nitro" refers to -NO2.

[0233] The term "oxo" refers to "=O".

[0234] The term "carbonyl" refers to C=O.

[0235] The term "carboxyl group" refers to -C(O)OH.

[0236] The term "carboxylic acid ester group" refers to -C(O)O (alkyl), -C(O)O (cycloalkyl), (alkyl)C(O)O- or (cycloalkyl)C(O)O-, where alkyl and cycloalkyl are as defined above.

[0237] The compounds disclosed herein contain their isotopic derivatives. The term "isotopic derivative" refers to a compound whose structure differs only in the presence of one or more isotopically enriched atoms. For example, a compound having the structure disclosed herein, using "deuterium" or "tritium" instead of hydrogen, or using... 18 F-fluorine labeling ( 18 F isotopes) can be used instead of fluorine, or... 11 C-, 13 C-, or 14 C-enriched carbon ( 11 C-, 13 C-, or 14 C-carbon labeling; 11 C-, 13 C-, or 14Compounds in which carbon atoms are replaced by C-isotopes are within the scope of this disclosure. Such compounds can be used as analytical tools or probes in, for example, biological assays, or as in vivo diagnostic imaging tracers for diseases, or as tracers for pharmacodynamic, pharmacokinetic, or receptor studies. The various deuterated forms of compounds disclosed herein refer to compounds in which each available hydrogen atom bonded to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds by referring to relevant literature. Commercially available deuteration starting materials can be used in the preparation of deuterated forms of compounds, or they can be synthesized using conventional techniques with deuteration reagents, including but not limited to deuterated boranes, trideuterated borane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane. Deuterated compounds generally retain activity comparable to undeuterated compounds, and better metabolic stability can be achieved when deuterated at certain specific sites, thus providing certain therapeutic advantages.

[0238] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of the event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0239] "Substituted" refers to one or more hydrogen atoms in a group, preferably 1 to 5, more preferably 1 to 3 hydrogen atoms, which are independently substituted by the corresponding number of substituents. Those skilled in the art can determine possible or impossible substitutions without much effort (through experimentation or theory). For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0240] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0241] As used herein, the term "pharmaceutically acceptable" means that these compounds, materials, compositions, and / or dosage forms are suitable for contact with a subject's tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, within reasonable medical judgment, have a reasonable benefit / risk ratio, and are effective for their intended use.

[0242] As used herein, the singular forms of “a,” “an,” and “the” include plural references, and vice versa, unless the context clearly indicates otherwise.

[0243] When the term "about" is applied to parameters such as pH, concentration, temperature, etc., it indicates that the parameter can vary by ±10%, and sometimes more preferably within ±5%. As those skilled in the art will understand, when a parameter is not critical, figures are usually given for illustrative purposes only and not as limitations.

[0244] The terms “connector unit,” “linking unit,” or “linking fragment” refer to a chemical structural fragment or bond that is linked at one end to a ligand (antibody in this disclosure) and at the other end to a drug. It may also be linked to other connectors before being linked to a drug.

[0245] For the preparation of conventional pharmaceutical compositions, please refer to the Chinese Pharmacopoeia.

[0246] The term "carrier" used in the context of this disclosure refers to a system that can alter the way a drug enters the body and its distribution within the body, control the rate of drug release, and deliver the drug to the target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, high-molecular-weight surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferred examples being micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

[0247] The term "excipient" refers to any additive in a pharmaceutical preparation other than the active pharmaceutical ingredient (API). Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in liquid preparations.

[0248] The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. The addition of diluents not only ensures a specific volume but also reduces dosage deviations of the main components and improves the compressibility of the drug. When the tablet contains oily components, absorbents are added to absorb the oil and maintain a "dry" state, facilitating tablet formation. Examples of absorbents include starch, lactose, inorganic salts of calcium, and microcrystalline cellulose.

[0249] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0250] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Water, Ringer's solution, and isotonic sodium chloride solution may be used as acceptable solvents and media. A sterile injectable formulation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated with a mixture of water and glycerol to form a microemulsion. The injection solution or microemulsion can be injected into the bloodstream of the subject by local large-volume injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the disclosed compound. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous pump.

[0251] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oil suspension for intramuscular and subcutaneous administration. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. The sterile injectable formulation may also be a sterile injectable solution or suspension prepared in a non-toxic, parenteral-acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol. Furthermore, a sterile fixative oil may be conveniently used as a solvent or suspension medium. For this purpose, any blended fixative oil, including synthetic mono- or diglycerides of glycerol, may be used. Additionally, fatty acids such as oleic acid may also be used to prepare the injectable formulation.

[0252] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0253] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to a salt of the antibody-drug conjugate disclosed herein, or a salt of the compound described herein, which is safe and effective in mammalian use and has the intended biological activity. The antibody-drug conjugate disclosed herein contains at least one amino group and can therefore form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0254] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any of the compounds disclosed herein, to a subject who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases to induce the regression of such symptoms or inhibit their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (such as treatments or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0255] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or the same as those described herein may be used to implement or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the description and claims, the singular form includes plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0256] The following embodiments are used to further describe the present invention, but these embodiments are not intended to limit the scope of the present invention.

[0257] Experimental methods not specifically described in the examples or test cases disclosed herein are generally performed under standard conditions or as recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Manual, Cold Spring Harbor Laboratory; Methods in Modern Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents not specifically named are commercially available, standard reagents.

[0258] Example

[0259] Example 1: Construction of HER3-overexpressing cell lines

[0260] The pCDH-Her3 lentiviral expression vector plasmid and the pVSV-G, pCMV-dR8.91 lentiviral system packaging vector were transfected into 293T virus packaging cells using Lipofectamine 3000 transfection reagent. The culture supernatant containing the virus was collected, filtered, and ultracentrifuged. The concentrated virus was used to infect Chinese hamster ovary cells CHO-K1. After two to three weeks of selection with puromycin, single-cell sorting was performed using FACS.

[0261] HER3 expression on the surface of lentivirally infected CHO-K1 cells was detected by FACS, and monoclonal cell lines with high HER3 expression were selected.

[0262] The selected monoclonal cell lines were expanded and stored for subsequent experiments.

[0263] HuMan ErbB3 Amino Acid Sequence (UniProtKB - P21860-1, AA Ser 20 - Thr 643)

[0264] SEVGNSQAVCPGTLNGLSVTGDAENQYQTLYKLYERCEVVMGNLEIVLTGHNADLSFLQWIREVTGYVLVAMNEFSTLPLPNLRVVRGTQVYDGKFAIFVMLNYNTNSSHALRQLRLTQLTEILSGGVYIEKNDKLCHMDTIDWRDIVRDRDAEIVVKDNGRSCPPCHEVCKGRCWGPGSEDCQTLTKTICAPQCNGHCFGPNPNQCCHDECAGGCSGPQDTDCFACRHFNDSGACVPRCPQPLVYNKLTFQLEPNPHTKYQYGGVCVASCPHNFVVDQTSCVRACPPDKMEVDKNGLKMCEPCGGLCPKACEGTGSGSRFQTVDSSNIDGFVNCTKILGNLDFLITGLNGDPWHKIPALDPEKLNVFRTVREITGYLNIQSWPPHMHNFSVFSNLTTIGGRSLYNRGFSLLIMKNLNVTSLGFRSLKEISAGRIYISANRQLCYHHSLNWTKVLRGPTEERLDIKHNRPRRDCVAEGKVCDPLCSSGGCWGPGPGQCLSCRNYSRGGVCVTHCNFLNGEPREFAHEAECFSCHPECQPMEGTATCNGSGSDTCAQCAHFRDGPHCVSSCPHGVLGAKGPIYKYPDVQNECRPCHENCTQGCKGPELQDCLGQTLVLIGKTHLT

[0265] SEQ ID NO: 1

[0266] HuMan ErbB3 Nucleotide Sequence

[0267]

[0268] SEQ ID NO: 2

[0269] Example 2: Production of anti-human HER3 monoclonal antibody

[0270] 2.1 Preparation of positive control antibodies

[0271] Positive control antibody U3 was prepared according to WO2007077028A2 (page 118, U1-59). The heavy and light chain amino acid sequences of U3 are as follows:

[0272] U3 heavy chain:

[0273] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSLKLSSVTAADTAVYYCARDKWTWYFDLWGRGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0274] SEQ ID NO: 3

[0275] U3 Light Chain:

[0276] DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLAWYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPRTFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0277] SEQ ID NO: 4

[0278] 2.2 Antibody Preparation Disclosed

[0279] Using a fully human natural phage antibody library and the antigen Biotinylated HuMan ErbB3 (purchased from Beijing Biosys Biotechnology Co., Ltd., catalog number: ER3-H82E6), positive clones were obtained after panning and phage detection using ELISA. The positive clones were sequenced, and the sequences were inserted into the protein expression vector Phr-IgG, which was then expressed in HEK293 and Expi-CHO-S cells. After purification, FACS and endocytosis activity verification experiments were performed to obtain the fully human antibody molecule HER3-29.

[0280] The constant region of the fully human antibody molecule HER3-29:

[0281] The heavy chain constant region of human IgG1:

[0282] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0283] SEQ ID NO: 5

[0284] Human κ light chain constant region:

[0285] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0286] SEQ ID NO: 6

[0287] The heavy chain variable region and light chain variable region sequences of the fully human antibody molecule HER3-29 are as follows:

[0288] HER3-29 heavy chain variable region:

[0289] QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKEGLPGLDYWGQGTLVTVSS

[0290] SEQ ID NO: 7

[0291] HER3-29 light chain variable region:

[0292] DIQMTQSPSSLSASIGDRATITCRASQHVGTYLNWYQQKPGKTPKLLISGAANLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPPFSFGQGTKVEIK

[0293] SEQ ID NO: 8

[0294] The CDR sequences obtained under different numbering rules are as follows:

[0295] Table 1. CDR sequences obtained by Chothia numbering rules

[0296]

[0297] Table 2. CDR Sequences Obtained by IMGT Numbering Rules

[0298]

[0299] Table 3. CDR sequences obtained by the Kabat numbering rules

[0300]

[0301] The heavy chain sequence and light chain sequence of the fully human antibody molecule HER3-29 are as follows:

[0302] HER3-29 heavy chain:

[0303] QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKEGLPGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0304] SEQ ID NO: 27

[0305] HER3-29 light chain:

[0306] DIQMTQSPSSLSASIGDRATITCRASQHVGTYLNWYQQKPGKTPKLLISGAANLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPPFSFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0307] SEQ ID NO: 28

[0308] Example 3: Preparation of ADC

[0309] DAR value determination of ADC

[0310] The method for calculating the DAR value of the ADC disclosed herein uses RP-HPLC (reversed-phase high-performance liquid chromatography), as detailed below:

[0311] 1. Measurement method:

[0312] The naked antibody and the ADC sample (concentration 1 mg / mL) were reduced with 4 μL of DDT (sigma) and incubated in a 37℃ water bath for 1 hour. After the reaction, the sample was transferred to an inner tube. Detection was performed using an Agilent 1200 high-performance liquid chromatograph (HPLC). The column used was an Agilent PLRP-S 1000A 8μm 4.6*250 mm column, with a column temperature of 80℃, a DAD detector wavelength of 280 nm, a flow rate of 1 mL / min, and an injection volume of 40 μL. The positions of the light and heavy chains were then distinguished by comparing the spectra of the sample and the naked antibody. The DAR value was calculated by integrating the spectra of the detected sample.

[0313] 2. Solution preparation

[0314] 1) 0.25M DTT solution:

[0315] Preparation example: Take 5.78 mg of DTT, add 150 μL of purified water to dissolve it completely, and prepare a 0.25 M DTT solution. Store at -20℃.

[0316] 2) Mobile phase A (0.1% TFA aqueous solution):

[0317] Preparation example: Measure 1000 mL of purified water with a graduated cylinder, add 1 mL of TFA (sigma), mix thoroughly before use, and store at 2-8℃ for 14 days.

[0318] 3) Mobile phase B (0.1% TFA acetonitrile solution):

[0319] Preparation example: Measure 1000 mL of acetonitrile with a graduated cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8℃ for 14 days.

[0320] 3. Data Analysis

[0321] By comparing the spectra of the sample with those of the bare antibody, the positions of the light and heavy chains are distinguished. Then, the DAR value is calculated by integrating the spectrum of the tested sample.

[0322] The calculation formula is as follows:

[0323]

[0324] Total LC peak area = LC peak area + LC+1 peak area

[0325] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area

[0326] LC DAR = Σ(Number of drugs connected * Percentage of peak area) / Total LC peak area

[0327] HC DAR = Σ(Number of drugs linked * Percentage of peak area) / Total peak area of ​​HC

[0328] DAR = LC DAR + HC DAR

[0329] drug

[0330] The pharmaceutical portion of this disclosure of conjugates may be any suitable pharmaceutical product. Particularly suitable pharmaceutical products are described, for example, in PCT Publication No. WO2020063676A1 (included herein by reference in its entirety). The disclosed compound 9A (i.e., compound 9-A of Example 9 of WO2020063676A1) is N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexahexa-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexanoamide, which has the following structure:

[0331]

[0332] This disclosure describes the preparation of antibody-drug conjugates, as shown in the general formula ADC (HER3-29-9A), by adjusting reaction parameters using the following method.

[0333]

[0334] Example 3-1 ADC-1

[0335] At 37 °C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 208.2 µL, 2.082 µMol) was added to the PBS buffered aqueous solution of antibody HER3-29 (pH=6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 11.8 mL, 797 nmol). The solution was placed in a water bath and shaken at 37 °C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25 °C in a water bath.

[0336] Compound 9A (prepared from compound 9-A of Example 9, WO2020063676, the entire contents of which are incorporated herein by reference) (8.6 mg, 8.006 µmol) was dissolved in 500 µL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffered product ADC-1 of the conjugate HER3-29-9A (4.02 mg / mL, 27.9 mL), which was stored at 4 °C. RP-HPLC average: DAR = 4.19.

[0337] Example 3-2 ADC-2

[0338] At 37 °C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 128 µL, 1.281 µmol) was added to the PBS buffered aqueous solution of antibody HER3-29 (pH=6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 11.8 mL, 797 nmol). The solution was placed in a water bath and shaken for 3 hours at 37 °C, after which the reaction was stopped. The reaction solution was then cooled to 25 °C in a water bath.

[0339] Compound 9A (6.88 mg, 6.405 µmol) was dissolved in 400 µL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain PBS buffered product ADC-2 of the conjugate HER3-29-9A (4.24 mg / mL, 27.2 mL), which was stored at 4 °C. RP-HPLC calculated average: DAR = 2.91.

[0340] Example 3-3 ADC-3

[0341] At 37 °C, a prepared tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (10 mM, 111 µL, 1.111 µMol) was added to the PBS buffered aqueous solution of antibody HER3-29 (pH=6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 3.1 mL, 209 nmol). The solution was placed in a water bath and shaken for 3 hours at 37 °C, after which the reaction was stopped. The reaction solution was then cooled to 25 °C in a water bath.

[0342] Compound 9A (3.37 mg, 3.137 µmol) was dissolved in 120 µL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain 12.8 mL of PBS buffered product ADC-3, an exemplary product of conjugate HER3-29-9A, and stored at 4 °C. The average value calculated by RP-HPLC was DAR = 7.27.

[0343] Example 3-4 ADC-4

[0344]

[0345] At 37 °C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 111 µL, 1.111 µmol) was added to a PBS buffered aqueous solution of antibody U3 (pH=6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 3.1 mL, 209 nmol). The solution was placed in a water bath and shaken for 3 hours at 37 °C, after which the reaction was stopped. The reaction solution was then cooled to 25 °C in a water bath.

[0346] Compound 9A (3.37 mg, 3.137 µmol) was dissolved in 120 µL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product ADC-4 of conjugate U3-9A in PBS buffer (1.48 mg / mL, 12.8 mL), which was stored at 4 °C. RP-HPLC calculated average: DAR = 6.76.

[0347] Example 3-5 ADC-5

[0348]

[0349] U3-1402 was prepared as a positive control, referring to Example 12 on page 156 of the WO2015155998A1 specification. At 37 °C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 130 µL) was added to the PBS buffered aqueous solution of antibody U3 (pH=6.5, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 3.1 mL, 236 nmol). The solution was placed in a water bath and shaken at 37 °C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25 °C in a water bath.

[0350] Compound 1402 (3.67 mg, 3.54 µmol) was dissolved in 180 µL DMSO and added to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.5 containing 0.001 M EDTA) to obtain the exemplary product ADC-5 of conjugate U3-1402 in PBS buffer (1.53 mg / mL, 15.4 mL), which was stored at 4 °C. RP-HPLC calculated average: DAR = 6.97.

[0351] Test case

[0352] Test Example 1: Antibody Binding to Free HER3 Protein Experiment

[0353] HER3 protein was diluted to 1 μg / mL with PBS (Yuanpei Biotechnology, B320) buffer at pH 7.4 and added to 100 μL / well of a 96-well microplate. The plate was incubated overnight at 4°C. After discarding the buffer, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added to each well for blocking, and the plate was incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20). Then, 100 μL of serially diluted antibody solution was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, the plate was washed three times with PBST, and 100 μL of mouse anti-human IgG (H+L) (Jackson ImmunoResearch, 209-035-088, 1:8000 dilution) was added to each well, and the plate was incubated at 37°C for 1 hour. After washing the plate three times with PBST, add 100 μL TMB chromogenic substrate (KPL, 5120-0077) to each well and incubate at room temperature for 10-15 minutes. Stop the reaction by adding 50 µL of 1 M H₂SO₄ to each well. Read the absorbance at 450 nm using a microplate reader. Use software to fit the antibody-antigen binding curve, see [link to table]. Figure 1 Calculate EC 50 The values ​​are shown in Table 4.

[0354] Table 4. Antibody binding activity to HER3 protein

[0355]

[0356] Conclusion: The antibody HER3-29 disclosed in this study exhibits superior binding activity to the HER3 protein compared to the control antibody U3.

[0357] Test Example 2: Antibody Binding Experiment with Cells Expressing HER3

[0358] MCF7 cells (ATCC, HTB-22) were prepared into 1×10⁶ cells using FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)). 6Cell suspension at 100 µL / well was added to 96-well round-bottom plates. After centrifugation to remove the supernatant, 50 µL / well of different concentrations of the test antibody diluted with FACS buffer was added, and the plates were incubated at 4°C in the dark for 1 hour. After washing three times with FACS buffer at 300 g, working concentration of Alexa Fluor 488 Goat anti-Human IgG (H+L) (invitrogen, A-11013) was added, and the plates were incubated at 4°C in the dark for 40 minutes. After washing three times with FACS buffer at 300 g, the geometric mean fluorescence intensity was detected on a BD FACSCantoII flow cytometer. Figure 2 EC 50 The values ​​are shown in Table 5.

[0359] Table 5. Antibody-cell binding activity

[0360]

[0361] Conclusion: The antibody HER3-29 disclosed in this study exhibits superior binding activity to cells expressing HER3 protein compared to the control antibody U3.

[0362] Test Example 3: DT3C Antibody Internalization Assay

[0363] The purpose of this experiment is to indirectly reflect the endocytosis of HER3 antibodies by observing the cell-killing effect of activated diphtheria toxin (DT) after the DT3C protein enters the cell. According to IC... 50 Imax was used to evaluate the in vitro endocytic activity of the antibody.

[0364] DT3C is a recombinant fusion protein composed of fragment A (toxin only) of diphtheria toxin and fragment 3C (IgG binding portion) of group G streptococcus. This protein exhibits high affinity for the Fc structure of antibodies and enters the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, it releases the toxic DT. DT inhibits EF2-ADP ribosylation activity, blocking protein translation and ultimately leading to cell death. DT3C that does not enter the cell does not possess cytotoxic activity. The endocytic activity of the antibody is evaluated based on its cytotoxic effect.

[0365] A suspension of recombinant CHOK1 cells expressing HER3 was prepared using fresh cell culture medium containing 20% ​​low IgG FBS, with a cell density of 2 × 10⁶ cells / year. 4 Cells / mL, added to cell culture plates at 50µL / well, and cultured at 37°C for 16 hours with 5% CO2.

[0366] DT3C was prepared at a concentration of 4× using serum-free medium and filtered through a 0.22µm filter to obtain a sterile solution. Antibody was prepared at a concentration of 4× using serum-free medium. 80µL of DT3C (400nM) and 80µL of antibody (66nM) were mixed at a 1:1 volume ratio and incubated at room temperature for 30 minutes. 50µL of the diluted antibody was added to 50µL of cells and incubated for three days. 50µL of CTG (CellTiter-Glo™ reagent, G7573) was added to each well and incubated at room temperature in the dark for 10 minutes. Chemiluminescence was read on a Victor3 microarray. Results are shown in the figure below. Figure 3 See Table 6.

[0367] Table 6. Antibody endocytic activity

[0368]

[0369] Conclusion: The endocytic activity of the antibody HER3-29 disclosed in this study is superior to that of the control antibody U3.

[0370] Test Example 4: pHrodo Antibody Internalization Assay

[0371] The purpose of this experiment is to reflect the endocytosis of HER3 antibodies based on changes in fluorescence signal after dye internalization. The in vitro endocytic activity of the antibody is evaluated based on the intensity of the fluorescence signal.

[0372] The pH-sensitive pHrodo iFL dye, coupled with the Fab fragment, can directly bind to the Fc region of the HER3 antibody without affecting antigen recognition. The pHrodo iFL dye exhibits almost no fluorescence at neutral pH. During HER3 antibody endocytosis, the dye is simultaneously internalized, and the fluorescence signal gradually increases as the pH decreases. The enhancement of the fluorescence signal is used to evaluate the antibody's endocytic activity.

[0373] HER3 / CHOK1 cells were cultured in DMEM / F12 + 10% FBS + 10µg / mL puromycin. On the first day of the experiment, a cell suspension was prepared using culture medium containing fresh cells at a density of 2×10⁻⁶ cells / mL. 5 Cells / mL, add 100µL / well to a 96-well cell culture plate and incubate at 37°C for 24 hours with 5% CO2.

[0374] Aspirate 50 µL of cell culture from the plate and add 50 µL of a mixture of antibody and pHrodo dye to each well. Two replicates are prepared for each antibody sample. A dye-only group and an isotype IgG1 control group are also included.

[0375] After 24 hours of incubation, the culture medium was aspirated, and 50 µL of trypsin was added to each well for digestion for 2 minutes. Digestion was then stopped with 50 µL of fresh culture medium. Cells from replicate wells of the same sample were transferred to the same well of a round-bottom plate using a pipette. The cells were centrifuged at 1500 rpm for 2 minutes, the culture medium was discarded, and the cells were washed once with FACS Buffer (PBS + 2.5% FBS) and centrifuged at 1500 rpm for 2 minutes. The cells were resuspended in 200 µL of FACS Buffer (PBS + 2.5% FBS), and the FITC signal was detected by flow cytometry. Data were analyzed using Flowjo 7.6, and the results are shown below. Figure 4 See Table 7.

[0376] Table 7. Antibody endocytosis activity

[0377]

[0378] Conclusion: The endocytic activity of the antibody HER3-29 disclosed in this study is superior to that of the control antibody U3.

[0379] Test Example 5: ADC Molecular Cellular Activity Assay

[0380] The purpose of this experiment was to detect the cell-killing effect of ADC samples, based on IC50. 50 We used Imax to evaluate the in vitro activity of Her3-ADC.

[0381] MCF7 cells (human breast cancer cells), SW620 cells (human colon cancer cells, Nanjing Kebai, CBP60036), and WiDr cells (human colorectal cancer cells) were digested with trypsin, neutralized with fresh culture medium, centrifuged at 1000 rpm, resuspended in culture medium, counted, and the cell suspension density was adjusted to 500 cells / well. The cells were then added to 96-well cell culture plates. In column 11, no cells were seeded, only 135 µL of culture medium was added. The cells were cultured at 37°C for 16 hours with 5% CO2.

[0382] The ADC sample was diluted to 15 µM (10× concentration) with PBS. Using this as the initial concentration, each sample was diluted five-fold with PBS, resulting in a total of eight concentrations. 15 µL of the 10× concentration solution was added to each well. The sample was incubated at 37°C with 5% CO2 for 6 days.

[0383] Add 70 µL of CTG to each well and incubate at room temperature in the dark for 10 minutes. Place the white backing membrane on the bottom of the cell culture plate and read the chemiluminescence on a Victor3 plate. Data from this experiment were processed using GraphPad Prism 5.0 software; see Table 8.

[0384] Table 8. In vitro killing experiments of HER3-29-9A with different DAR values

[0385]

[0386] Conclusion: The ADC samples ADC-1, ADC-2, and ADC-3 disclosed in this study exhibited superior cell-killing activity compared to the positive controls ADC-4 and ADC-5.

[0387] In vivo bioactivity evaluation

[0388] Test Example 6: In vivo efficacy evaluation of a HER3-overexpressing CDX model

[0389] SW620 cells (5 × 10⁻⁶) 6 (8 mice per group) were inoculated subcutaneously into the right rib area of ​​Balb / c nude mice. After 7 days, the mice were divided into 9 groups of 8 mice each. The average group volume was 134.75 mm². 3 The ADC was administered via intraperitoneal injection three times, once every five days. Each animal received 0.1 mL / 10 g of the ADC based on its body weight. Tumor volume and body weight were measured twice weekly, and data were recorded. Data were recorded using Excel statistical software: mean values ​​were calculated as averages (avg); SD values ​​were calculated as STDEV; SEM values ​​were calculated as STDEV / SQRT (number of animals per group). GraphPadPrism software was used for plotting, and two-way ANOVA or one-way ANOVA was used for statistical analysis.

[0390] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0391] The relative tumor proliferation rate T / C (%) = (T-T0) / (C-C0) × 100%, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; and T0 and C0 are the tumor volumes at the beginning of the experiment.

[0392] Tumor inhibition rate (TGI) (%) = 1 - T / C (%). Results are shown below. Figure 5 And Table 9.

[0393]

[0394]

[0395] Conclusion: The efficacy of ADC-1 and ADC-2 in treating SW620 xenograft tumors in nude mice disclosed in this study is superior to that of the positive control ADC-5.

Claims

1. A pharmaceutical composition comprising a therapeutically effective amount of isolated anti-HER3 antibody, and a pharmaceutically acceptable carrier, diluent, or excipient, wherein a suitable unit dose may be 0.1 to 1000 mg, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region, wherein: a. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 respectively. The CDR area as described above is determined according to the Chothia numbering rules; or b. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 respectively. The CDR area as described above is determined according to the IMGT numbering rules; or c. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23 respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26 respectively. The CDR area, as described above, is determined according to the Kabat numbering rules.

2. The pharmaceutical composition according to claim 1, wherein the anti-HER3 antibody is a human antibody or an antigen-binding fragment.

3. The pharmaceutical composition according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein: The amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 7, and / or the amino acid sequence of the light chain variable region has at least 90% sequence identity with SEQ ID NO: 8; Preferably, the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

4. The pharmaceutical composition according to any one of claims 1 to 3, comprising: A heavy chain having at least 85% sequence identity with SEQ ID NO: 27, and / or a light chain having at least 85% sequence identity with SEQ ID NO: 28; Preferably, the anti-HER3 antibody comprises: Heavy chains as shown in SEQ ID NO: 27 and light chains as shown in SEQ ID NO:

28.

5. A pharmaceutical composition comprising a therapeutically effective amount of a general formula (Pc-L) a The antibody-drug conjugate shown in (-YD), and pharmaceutically acceptable carriers, diluents, or excipients, wherein a suitable unit dose may be 0.1 to 1000 mg, wherein the general formula (Pc-L) is used. a -YD) is shown below: in, Pc is the isolated anti-HER3 antibody as described in any one of claims 1 to 4; m is an integer from 0 to 4; n is between 1 and 10, and n is a decimal or an integer; R 1 Selected from halogens, haloalkyls, deuteralkyls, cycloalkyls, cycloalkylalkyls, alkoxyalkyls, heterocyclics, aryl groups, and heteroaryl groups; R 2 Selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups; or, R 1 and R 2 Together with the carbon atoms attached to it, they form cycloalkyl or heterocyclic groups; W is selected from C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl groups and straight-chain heteroalkyl groups of 1 to 8 chain atoms, wherein the straight-chain heteroalkyl groups of 1 to 8 chain atoms contain 1 to 3 heteroatoms selected from N, O, and S, wherein the C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 The cycloalkyl group and the straight-chain heteroalkyl group with 1 to 8 chain atoms are each independently and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl; L 2 Selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- and chemical bonds, where p 1 Integers from 1 to 20; L 3 It is a peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acids formed from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuteralkyl, alkoxy and cycloalkyl. R 5 Selected from hydrogen atoms, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups; R 6 and R 7 They may be the same or different, and each is independently selected from hydrogen atoms, halogens, alkyl groups, haloalkyl groups, deuteralkyl groups, and hydroxyalkyl groups.

6. The pharmaceutical composition according to claim 5, wherein the antibody-drug conjugate is: in: n can be 1 to 8, where n is a decimal or an integer; preferably, n can be 3 to 8, where n is a decimal or an integer. HER3-29 is an anti-HER3 antibody that contains a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO:

28.

7. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a medicament for treating HER3-mediated diseases or conditions.

8. Use of the pharmaceutical composition according to any one of claims 1 to 6 in the preparation of a medicament for treating and / or preventing tumors; Preferably, the tumor is selected from breast cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, and melanoma.

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