Anti-her3 antibody and Anti-her3 antibody-drug conjugate and medical use thereof

Abstract

Provided are an anti-HER3 antibody and an anti-HER3 antibody-drug conjugate and a medical use thereof, specifically, the anti-HER3 antibody, and the anti-HER3 antibody-drug conjugate as represented by general formula (Pc-L-Y-D), wherein Pc is an anti-HER3 antibody, and L, Y and n are as defined in the description.

Description

Anti-HER3 antibodies and anti-HER3 antibody-drug conjugates and their medical uses Technical Field

[0001] The present disclosure relates to anti-HER3 antibodies, anti-HER3 antibody-exitecan analog conjugates, their preparation methods, pharmaceutical compositions containing them, and their use in preparing drugs for treating HER3-mediated diseases or conditions; in particular, their use in preparing anticancer drugs. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] 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 all contain three parts: an extracellular region, a transmembrane region, and an intracellular region. The extracellular region contains four domains, and the intracellular region contains an intracellular tyrosine kinase domain for signal transduction and a tail with tyrosine phosphorylation residues located in the cytoplasm. When ligands bind to extracellular domains I and III, cell signaling is initiated. Under normal circumstances, these receptors mediate cell division, migration, survival, and organ development. When EGFR family members mutate, the resulting abnormal signaling stimulates cell survival and is associated with cancer progression. The basic mechanism by which the HER3 receptor is activated and produces its physiological effects is similar to that of other HER3 family members. However, its ligands include neuregulin 1 (NRG-1) and neuregulin 2 (NRG-2). Furthermore, upon activation, HER3 cannot form homomers, but can only form heterodimers with EGFR or HER2. During heterodimerization, the intracellular domain of HER3 exhibits high tyrosine phosphatase activity. Structural analysis has revealed six P85 (PI-3K) binding sites within the HER3 intracellular domain. This specific structure enables HER3 to recruit up to six PI-3Ks to the P85 regulatory subunit upon interaction, thereby strongly activating the PI-3K signaling pathway. In fact, the HER3 / HER2 dimer is the most active of the HER3 dimers. EGFR is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, keratinocytes, and other cells. The EGFR signaling pathway plays a crucial role in physiological processes such as cell growth, proliferation, and differentiation.

[0004] HER3 is highly expressed in various common malignancies, 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 primarily due to increased mRNA transcription, which in turn increases protein translation. HER3 is often highly expressed together with HER2. High HER3 expression is closely linked to the occurrence and progression of various tumors, as well as the survival of subjects. Therefore, the research of anti-tumor drugs targeting HER3 is of great significance.

[0005] Summary of the Invention

[0006] The present disclosure relates to anti-HER3 antibodies, anti-HER3 antibody-exitecan analog conjugates and uses thereof.

[0007] The present 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 has an apparent affinity EC of less than 0.5 nM 50 Binding to HER3 protein, the apparent affinity EC 50 It was determined by ELISA method;

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

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

[0011] The present 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 has an apparent affinity EC of less than 0.5 nM 50 Binding to HER3 protein, the apparent affinity EC 50 It was determined by ELISA method;

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

[0014] c. The anti-HER3 antibody can be endocytosed by cells expressing human HER3. When the anti-HER3 antibody is determined by the method of Test Example 3, its IC 50 Less than 2nM;

[0015] d. The anti-HER3 antibody can be internalized by cells expressing human HER3. 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 of any of the above items 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 embodiments, the anti-HER3 antibody of any of the above 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 regions are determined according to the Chothia numbering convention.

[0020] In some embodiments, the anti-HER3 antibody of any of the above 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 regions are determined according to the IMGT numbering rules.

[0023] In some embodiments, the anti-HER3 antibody of any of the above 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 regions are determined according to the Kabat numbering convention. The present 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 regions are determined according to the Chothia numbering convention.

[0028] The present 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 regions are determined according to the IMGT numbering rules.

[0031] The present 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 regions described therein are determined according to the Kabat numbering convention.

[0034] In some embodiments, the anti-HER3 antibody of any of the above items is a human antibody or antigen-binding fragment.

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

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

[0037] In some embodiments, the anti-HER3 antibody of any of the above 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 above items 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 embodiments, the anti-HER3 antibody of any of the above comprises:

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

[0042] In some embodiments, the anti-HER3 antibody of any of the above comprises:

[0043] The heavy chain is shown in SEQ ID NO:27 and the light chain is shown in SEQ ID NO:28.

[0044] In some embodiments, the anti-HER3 antibody of any of the above items 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, and the apparent affinity EC50 is determined by ELISA;

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

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

[0048] In some embodiments, the anti-HER3 antibody of any of the above items 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, and the apparent affinity EC50 is determined by ELISA;

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

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

[0052] d. The anti-HER3 antibody can be internalized by cells expressing human HER3. 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, the present disclosure also provides an isolated anti-HER3 antibody, wherein the antibody competes for binding to human HER3 with the anti-HER3 antibody of any of the preceding items.

[0054] In some embodiments, the present disclosure also provides a nucleic acid molecule encoding the anti-HER3 antibody as described in any of the preceding items.

[0055] In some embodiments, the present disclosure also provides a host cell comprising the nucleic acid molecule as described in any of the preceding items.

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

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

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

[0059] In some embodiments, the present disclosure further provides an antibody-drug conjugate represented by 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] Ra and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group; or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0064] R 1 R is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

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

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

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

[0068] L is the joint unit;

[0069] Pc is an anti-HER3 antibody as described in any of the preceding items.

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

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

[0072] in:

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

[0074] R a and R b are the same or different and are each independently selected from hydrogen atoms, deuterium atoms, halogens and C 1-6alkyl;

[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 3-6 Cycloalkyl;

[0077] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0078] m is 0 or 1.

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

[0080]

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

[0082] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, 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)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl and straight-chain heteroalkyl of 1 to 8 chain atoms, said straight-chain heteroalkyl of 1 to 8 chain atoms containing 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl and straight chain heteroalkyl of 1 to 8 chain atoms are each independently optionally further selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6substituted by one or more substituents in the cycloalkyl group;

[0084] 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 is an integer from 1 to 20;

[0085] L 3 A peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, 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, wherein t is an integer from 1 to 6;

[0087] R 3 、R 4 and R 5 are the same or different and are each independently selected from hydrogen atom, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 hydroxyalkyl;

[0088] R 6 and R 7 are the same or different and are each independently selected from hydrogen atom, halogen, C 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 represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the linker unit -L- is -L 1 -L2 -L 3 -L 4 -,

[0090] L 1 for s 1 is an integer from 2 to 8;

[0091] L 2 is a chemical bond;

[0092] L 3 is a tetrapeptide residue;

[0093] L 4 -NR 5 (CR 6 R 7 )t-,R 5 、R 6 or R 7 The same or different, and each independently a hydrogen atom or a C 1-6 Alkyl, t is 1 or 2;

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

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

[0096] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein -L- is:

[0097]

[0098] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein -LY- is optionally selected from:

[0099]

[0100] In some embodiments, the antibody-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items is a general formula (Pc-L a -YD) shown in the antibody - drug conjugate or a pharmaceutically acceptable salt thereof:

[0101]

[0102] in,

[0103] Pc is the anti-HER3 antibody as described above;

[0104] m is an integer from 0 to 4; for example, m is selected from 0, 1, 2, 3 and 4;

[0105] n is 1 to 10, n is a decimal or integer; specifically, n is a decimal or integer between 2 and 8, including both endpoints; more specifically, n is a decimal or integer between 2 and 7, including both endpoints; alternatively, n is a decimal or integer between 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, or 7 and 8, including both endpoints;

[0106] R 1 is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; R 2 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic, aryl and heteroaryl; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0107] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl and straight-chain heteroalkyl of 1 to 8 chain atoms, said straight-chain heteroalkyl of 1 to 8 chain atoms containing 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl and straight chain heteroalkyl of 1 to 8 chain atoms are each independently optionally further selected from halogen, hydroxy, cyano, amino, C 1-8 Alkyl, chloro C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 substituted by one or more substituents of the cycloalkyl group;

[0108] 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 is an integer from 1 to 20;

[0109] L 3A peptide residue consisting of 2 to 7 amino acid residues, wherein the amino acid residues are selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and are optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

[0110] R 5 is selected from the group consisting of a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;

[0111] R 6 and R 7 are the same or different and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group and a hydroxyalkyl group.

[0112] In some embodiments, the antibody-drug conjugate of the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items is a general formula (Pc-L a -YD) shown in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof: wherein,

[0113] Pc is an anti-HER3 antibody as described in any of the preceding items;

[0114] m is an integer from 0 to 4; for example, m is selected from 0, 1, 2, 3 and 4;

[0115] n is 1 to 10, n is a decimal or integer; specifically, n is a decimal or integer between 2 and 8, including both endpoints; more specifically, n is a decimal or integer between 2 and 7, including both endpoints; alternatively, n is a decimal or integer between 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, or 7 and 8, including both endpoints;

[0116] R 1 Selected from halogen, 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, heterocyclyl, aryl and heteroaryl; R 2 Selected from hydrogen atom, halogen, 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 1-6alkyl, heterocyclyl, aryl and heteroaryl; or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 a cycloalkyl group or a heterocyclic group;

[0117] W is selected from C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl and straight-chain heteroalkyl of 1 to 8 chain atoms, said straight-chain heteroalkyl of 1 to 8 chain atoms containing 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 Alkyl, C 1-8 Alkyl-C 3-6 Cycloalkyl and straight chain heteroalkyl of 1 to 8 chain atoms are each independently optionally further selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl, chloro C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 substituted by one or more substituents of the cycloalkyl group;

[0118] 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 is an integer from 1 to 20;

[0119] L 3 The peptide residue is composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from the group consisting of phenylalanine (F), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and is optionally further selected from the group consisting of halogen, hydroxyl, cyano, amino, C 1-6 Alkyl, chloro C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 substituted by one or more substituents in the cycloalkyl group;

[0120] 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;

[0121] R 6 and R 7 are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 hydroxyalkyl;

[0122] The heterocyclyl group contains 3 to 6 ring atoms, 1 to 3 of which are heteroatoms selected from nitrogen, oxygen and sulfur.

[0123] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items, wherein the antibody-drug conjugate is:

[0124]

[0125] in:

[0126] n is 1 to 8, n is a decimal or an integer; specifically, n is a decimal or an integer between 2 and 8, including both endpoints; more specifically, n is a decimal or an integer between 2 and 7, including both endpoints; 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 both endpoints;

[0127] HER3-29 is an anti-HER3 antibody comprising a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO: 28.

[0128] In some embodiments, the antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any of the preceding items is preferably 3 to 8, and n is a decimal or an integer.

[0129] In some embodiments, the present disclosure also provides a method for preparing a compound of the general formula (Pc-L) as described in any of the preceding items. a -YD) or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0130]

[0131] Pc' and general formula (L a -YD) to obtain the compound represented by the general formula (Pc-L a -YD) shown in the compound;

[0132] in:

[0133] Pc' is obtained after reduction of Pc;

[0134] n、m、W、L 2 , L 3 、R 1 、R 2 、R 5 、R 6 and R 7 As defined in any of the preceding items.

[0135] In some embodiments, the present disclosure also provides a pharmaceutical composition comprising the anti-HER3 antibody as described in any of the preceding items, or the nucleic acid molecule as described in any of the preceding items, or the antibody-drug conjugate as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0136] In some embodiments, the present disclosure also provides the use of an anti-HER3 antibody as described in any of the preceding items, or a nucleic acid molecule as described in any of the preceding items, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, or a pharmaceutical composition as described in any of the preceding items in the preparation of a medicament for treating a HER3-mediated disease or condition.

[0137] In some embodiments, the present disclosure also provides use of an anti-HER3 antibody as described in any of the preceding items, or a nucleic acid molecule as described in any of the preceding items, or an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, or a pharmaceutical composition as described in any of the preceding items in the preparation of a medicament for treating and / or preventing tumors and cancers, wherein 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.

[0138] In some embodiments, the present disclosure further provides a kit comprising the anti-HER3 antibody as described in any of the preceding items, or the nucleic acid molecule as described in any of the preceding items, or the antibody-drug conjugate as described in any of the preceding items or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in any of the preceding items.

[0139] In some embodiments, the present disclosure further provides a method for preventing or treating a disease or condition, comprising administering to a subject a therapeutically effective amount of an anti-HER3 antibody as described in any of the preceding items, or a nucleic acid molecule as described in any of the preceding items, or an antibody-drug conjugate as described in any of the preceding items, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in any of the preceding items. In some embodiments, the disease or condition is preferably a tumor, an autoimmune disease, or an infectious disease; and in some embodiments, the disease or condition is a disease or condition associated with HER3.

[0140] In another aspect, the present disclosure provides a pharmaceutical composition comprising the anti-HER3 antibody, antibody-drug conjugate, or pharmaceutically acceptable salt thereof as described in any of the preceding items, 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 antibody-drug conjugate as described above.

[0141] In another aspect, the present disclosure provides use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, as a drug.

[0142] On the other hand, the present disclosure provides the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same as described in any of the preceding items in the preparation of a medicament for treating a HER3-mediated disease or condition. In some embodiments, the HER3-mediated disease or condition is a HER3-overexpressing cancer, a HER3-intermediate expressing cancer or a HER3-low expressing cancer.

[0143] On the other hand, the present disclosure provides the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, as described in any of the preceding items, in the preparation of a medicament for treating or preventing cancer. In some embodiments, the tumor and cancer 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.

[0144] On the other hand, the present disclosure further relates to a method for treating and / or preventing tumors, comprising administering to a subject in need thereof a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same as described in any of the preceding items; in some embodiments, the tumor is a cancer associated with high HER3 expression, a moderate expression cancer, or a low expression cancer.

[0145] On the other hand, the present disclosure further relates to a method for treating or preventing tumors or cancers, comprising administering to a subject in need thereof a therapeutically effective dose of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any of the preceding items, or a pharmaceutical composition comprising the same; wherein the tumors and cancers 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.

[0146] On the other hand, the present disclosure further provides an anti-HER3 antibody or antibody-drug conjugate thereof as described in any of the preceding items as a drug, in some embodiments, as a drug for treating cancer or tumors, more preferably as a drug for treating HER3-mediated cancer.

[0147] The active compound (e.g., a compound according to the present disclosure, or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate according to the present disclosure, or a pharmaceutically acceptable salt thereof) can be formulated for administration by any appropriate route, and the active compound can be in a unit dose form, or in a form that a subject can self-administer as a single dose. A unit dose of the active compound or composition described herein can be expressed in the form of a tablet, capsule, cachet, bottled solution, powder, granules, lozenge, suppository, reconstituted powder, or liquid formulation.

[0148] The dosage of the active compound or composition used in the methods of treatment of the present disclosure will generally vary with the severity of the disease, the weight of the subject and the relative efficacy of the active compound. However, as a general guide, a suitable unit dosage may be 0.1 mg to 1000 mg.

[0149] The pharmaceutical composition disclosed herein may contain, in addition to the active compound, one or more excipients selected from the following ingredients: fillers, diluents, binders, wetting agents, disintegrants or excipients, etc. Depending on the method of administration, the composition may contain 0.1 to 99% by weight of the active compound.

[0150] The HER3 antibodies and antibody-drug conjugates provided in the present disclosure have good affinity with cell surface antigens, good cellular endocytosis efficiency and strong tumor suppression efficiency, and have a wider drug application window, and are suitable for clinical drug applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0151] FIG1 : Binding activity of the antibodies and positive antibodies disclosed herein to HER3 protein.

[0152] FIG2 : Binding activity of the antibodies and positive antibodies of the present disclosure to MCF7 cells.

[0153] FIG3 : The endocytic activity of the antibodies and positive antibodies of the present disclosure was tested using DT3C.

[0154] FIG4 : The endocytic activity of the antibodies and positive antibodies of the present disclosure was tested using pHrodo.

[0155] FIG5 shows the efficacy of the disclosed ADC samples on SW620 xenograft tumors in nude mice. DETAILED DESCRIPTION

[0156] the term

[0157] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present disclosure, preferred methods and materials are described herein. In describing and claiming the present disclosure, the following terms will be used in accordance with the following definitions.

[0158] When a trade name is used in this disclosure, it is intended to include the formulation of the trade name product, the drug substance and the active drug portion of the trade name product.

[0159] The term "antibody-drug conjugate" (ADC) refers to an antibody linked to a biologically active drug, wherein the antibody can be coupled to the drug directly or via a linker.

[0160] The term "drug loading" refers to the average amount of drug loaded per antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of the amount of drug to the amount of antibody. Drug loading can range from 0-12 drugs attached per antibody, with illustrative examples being 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs, and this value can be a decimal or an integer. In certain embodiments, each antibody carries 1 to about 10 drugs; in certain 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, or about 8 drugs, and this value can be a decimal or an integer. Drug loading can be determined by conventional methods, such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0161] In one embodiment of the present disclosure, the cytotoxic drug is coupled to the sulfhydryl group of the antibody via a linker unit.

[0162] The loading capacity of the ligand cytotoxic drug conjugate can be controlled by the following non-limiting methods, including:

[0163] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0164] (2) Control reaction time and temperature,

[0165] (3) Select different reaction reagents.

[0166] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0167] The term "antibody" disclosed herein is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, or antigen-binding fragments thereof (also referred to as "antigen-binding portions"), as long as 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 interconnected by disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same class of Ig can be divided into different subclasses based on the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains based on the difference in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

[0168] The approximately 110 amino acids near the N-terminus of a full-length antibody heavy and light chain vary greatly in sequence and constitute the variable region (abbreviated as the Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL). The heavy and light chain variable regions include hypervariable regions (also called complementarity-determining regions, abbreviated as CDRs or HVRs) and relatively conserved framework regions (also called framework regions, abbreviated as FRs). Each VL and VH is composed of three CDRs and four FRs arranged from amino to carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0169] The "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region described in the present disclosure refer to variants of the heavy chain constant region or light chain constant region derived from humans that have been disclosed in the prior art and do not change the structure and function of the antibody variable region. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification and amino acid substitution of the heavy chain constant region, specific substitutions such as the YTE mutation known in the prior art, L234A and / or L235A mutation, S228P mutation, 265A (e.g., D265A) and / or 297A (e.g., N297A), and / or mutations to obtain a knob-into-hole structure (so that the antibody heavy chain has a knob-Fc and hole-Fc combination). These mutations have been shown to impart new properties to the antibody without changing the function of the antibody variable region.

[0170] In this disclosure, "human antibody" (HuMAb), "humanized antibody," "fully human antibody," and "completely human antibody" are used interchangeably to refer to an antibody whose amino acid sequence corresponds to the amino acid sequence of an antibody produced by a human or human cell, or is derived from a non-human source utilizing a human antibody repertoire or other human antibody encoding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0171] The term "antigen-binding fragment" or "functional fragment" or "antigen-binding portion" refers to one or more fragments of an intact antibody that retains the ability to specifically bind to an antigen. Fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Exemplary binding fragments encompassed by the term "antigen-binding fragment" include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a dsFv, a stable antigen-binding fragment formed by interchain disulfide bonds between VH and VL; and (vi) diabodies, bispecific antibodies, and multispecific antibodies comprising fragments such as scFv, dsFv, and Fab. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to join the two domains through an artificial peptide linker that enables them to form a single protein chain, wherein VL and VH pair to form a monovalent molecule, called a single-chain Fv (scFv) (see, e.g., 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 the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies can be of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.

[0172] The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including insertions, deletions or substitutions of 1, 2, 3 or more amino acids based on the original protein or polypeptide.

[0173] The term "antibody framework region" or "FR region" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0174] The term "complementarity determining region," "CDR," or "hypervariable region" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to 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 the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (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 convention, the "ABM" numbering convention, the "contact" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc MP, Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, for the classical format, following the Kabat convention, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); 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 the Chothia convention, the CDR amino acid residues 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 rule, the CDR amino acid residues in VH are numbered approximately as 27-38 (HCDR1), 56-65 (HCDR2), and 105-117 (HCDR3), and the CDR amino acid residues in VL are numbered approximately as 27-38 (LCDR1), 56-65 (LCDR2), and 105-117 (LCDR3).Following AbM convention, 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).

[0175] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound by an antibody (e.g., a specific site on a HER3 molecule). An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).

[0176] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody or antigen-binding fragment to a predetermined epitope on an antigen. Typically, the antibody or antigen-binding fragment binds to an antigen with a specific binding affinity of less than 10 -8 M, for example, less than approximately 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 Binds with an affinity (KD) of M or less.

[0177] When the term "compete" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it means competition between antigen binding proteins, which is determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., HER3 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1992).

[0013] The present invention relates to a method for the determination of the competitive inhibition of antigen binding proteins by direct solid-phase labeling of antigens, such as immunoprecipitants, antigen-binding proteins, and antigen-binding proteins. The method also includes methods ... Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to adjacent epitopes sufficiently close to the binding epitope of the reference antigen binding protein that the two epitopes sterically interfere with each other in binding. Additional details on methods for determining competitive binding are provided in the Examples herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more.In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0178] As used herein, the term "nucleic acid molecule" refers to a DNA molecule or an RNA molecule. A nucleic acid molecule can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0179] Amino acid sequence "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence, after aligning the amino acid sequences and, if necessary, introducing gaps to achieve maximum sequence identity, and not considering any conservative substitutions as part of the sequence identity. For the purpose of determining amino acid sequence identity percentage, alignment can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0180] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcRs (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. Primary cells that mediate ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. To assess the ADCC activity of a molecule of interest, in vitro and in vivo ADCC assays can be performed, such as those described in Clynes et al. (PNAS USA 95: 652-656 (1998)), U.S. Pat. Nos. US5500362 and US5821337.

[0181] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells or virions are eliminated by internalization by phagocytes (e.g., macrophages, neutrophils, and dendritic cells). The internalized antibody-coated target cells or virions are contained in a vesicle called a phagosome, which then fuses with one or more lysosomes to form a phagolysosome. ADCP can be evaluated by using in vitro cytotoxicity assays and videomicroscopy using macrophages as effector cells (e.g., van Bij et al., Journal of Hepatology, Vol. 53, No. 4, October 2010, pp. 677–685). "Complement-dependent cytotoxicity" or "CDC" refers to a cytotoxic effect in which complement is involved, i.e., the classical complement pathway is activated by binding of antibodies to corresponding antigens on cells or virions to form a complex, and the resulting membrane-attacking complex exerts a lytic effect on the target cell. CDC can be evaluated by in vitro assays (e.g., CDC assays using normal human serum as a complement source) or in a C1q concentration series. A decrease in CDC activity (e.g., a decrease in CDC activity due to the introduction of a second mutation into a polypeptide or antibody) can be measured by comparing the CDC activity of the polypeptide or antibody with the CDC activity of a parent polypeptide or antibody that does not have the second mutation within the same assay. To assess the ability of an antibody to induce CDC, the assay described by Romeuf et al. (Romeuf et al., Br J Haematol. 2008 Mar; 140(6): 635-43) can be used.

[0182] The antibodies or antibody fragments described herein can be coupled to effector molecules by any means. For example, the antibodies or antibody fragments can be attached to the cytotoxic drug by chemical or recombinant means. Chemical methods for preparing fusions or conjugates are known in the art and can be used to prepare immunoconjugates. The method for coupling the antibody or antibody fragment and the drug must be able to connect the antibody and the cytotoxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0183] In one embodiment, the antibody and the cytotoxic drug are both proteins and can be coupled using technology well known in the art. There are hundreds of cross-linking agents disclosed in the art that can couple two proteins. The cross-linking agent is generally selected based on the reactive functional groups available or inserted on the antibody or cytotoxic drug. In addition, if there is no reactive group, a photoactivatable cross-linking agent 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, dimethyl adipimidate and bis(diazobenzidine), and heterobifunctional agents: m-maleimidobenzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.

[0184] Cross-linking 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)mercapto-propionyl hydrazide). TPCH and TPMPH react with carbohydrate moieties of glycoproteins that have previously been oxidized by mild periodate treatment, thereby forming a hydrazone bond between the hydrazide portion of the cross-linker and the aldehyde generated by the periodate. The heterobifunctional cross-linkers GMBS (N-(γ-maleimidobutyryloxy)-succinimide) and SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane) react with primary amines to introduce a maleimido group onto the component. This maleimido group can then react with a sulfhydryl group on another component that can be introduced by the cross-linker, thereby forming a stable thioether bond between the components. If steric hindrance between the components interferes with the activity of any one component, a cross-linking agent can be used to introduce a long spacer arm between the components, such as succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Thus, there are many suitable cross-linking agents that can be used and each is selected based on its effect on the optimal immunoconjugate yield.

[0185] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which another DNA segment can be connected. In another embodiment, the vector is a viral vector, in which another DNA segment can be connected to a viral genome. The vector disclosed herein can be autonomously replicated in the host cell into which they have been introduced (e.g., bacterial vectors and additional mammalian vectors with a bacterial origin of replication) or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating (e.g., non-additional mammalian vectors) with the host genome.

[0186] Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as those described in Chapters 5-8 and 15 of the Cold Spring Harbor Laboratory Manual of Antibody Laboratory Techniques. The disclosed antibodies or antigen-binding fragments utilize genetic engineering methods to add one or more human FR regions to non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website (http: / / imgt.cines.fr) by comparing them with the IMGT Human Antibody Variable Region Germline Gene Database and MOE software, or from the Journal of Immunoglobulins, 2001, ISBN 012441351.

[0187] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacteria, microorganisms, plants, or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, 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 the present disclosure do not include cells from human embryos.

[0188] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred existing technology, mammalian expression systems result in 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 expanded in serum-free culture medium in a bioreactor to produce the antibody. The culture medium secreting the antibody can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0189] "Conservative modification" or "conservative substitution or replacement" 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, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity. Exemplary conservative replacements are as follows:

[0190] The original residues were conservatively substituted with Ala(A)Gly; SerArg(R)Lys; HisAsn(N)Gln; His; AspAsp(D)Glu; AsnCys(C)Ser; Ala; ValGln(Q)Asn; Glu

[0191] Glu(E)Asp;GlnGly(G)AlaHis(H)Asn;GlnIle(I)Leu;ValLeu(L)Ile;ValLys(K)Arg;HisMet(M)Leu;Ile ;TyrPhe(F)Tyr;Met;LeuPro(P)AlaSer(S)ThrThr(T)SerTrp(W)Tyr;PheTyr(Y)Trp;PheVal(V)Ile;Leu

[0192] “Exogenous” refers to substances produced outside the body of an organism, cell, or human body, depending on the circumstances. “Endogenous” refers to substances produced inside the body of a cell, organism, or human body, depending on the circumstances.

[0193] "Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage 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 × 100. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous; if 95 out of 100 positions in the two sequences match or are homologous, then the two sequences are 95% homologous. Typically, when aligning two sequences, the comparison is performed to give the maximum percentage homology. For example, the comparison can be performed using the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between each sequence over the entire length of each reference sequence. The following references relate to the BLAST algorithm commonly used for sequence analysis: 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) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al., (1997) Genome Res. 7: 649-656. Other conventional BLAST algorithms, such as those provided by NCBI BLAST, are also well known to those skilled in the art.

[0194] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It will also be understood that, due to deliberate or unintentional mutations, all progeny may not be precisely identical in DNA content. Mutant progeny that possess the same function or biological activity as screened for in the originally transformed cell are included. Where a different designation is intended, this is clear from the context.

[0195] As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a trace amount of a specific portion of nucleic acid, RNA and / or DNA is amplified as described, for example, in U.S. Patent No. 4,683,195. In general, sequence information from the ends of the target region or beyond is required so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5' terminal nucleotides of the two primers can be consistent with 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). As used herein, PCR is considered to be an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which includes using a known nucleic acid and a nucleic acid polymerase as a primer to amplify or generate a specific portion of a nucleic acid.

[0196] "Isolated" 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 medium. Generally, the term "isolated" is not intended to imply the complete absence of such materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with experimental or therapeutic uses of the compounds as described herein.

[0197] The term "drug" refers to a chemical substance that can alter or identify physiological functions and pathological states of the body and can be used to prevent, diagnose, and treat disease. Drugs include cytotoxic drugs. There is no strict distinction between drugs and poisons. Poisons are chemical substances that can have toxic effects on the body at relatively low doses, potentially damaging human health. Excessive doses of any drug can produce toxic reactions.

[0198] Cytotoxic drugs are substances that inhibit or prevent cell 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 cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188、Sm 153 、Bi 212 、P 32 and radioactive isotopes of Lu), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0199] In some embodiments, the toxin can be a small molecule toxin and its derivatives from bacteria, fungi, plants or animals, including camptothecin derivatives such as isatecan, maytansinoids and their derivatives (CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (WO 2016 / 127790 A1, compound 7), diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modeccin, α-sarcin, Aleurites fordii toxin, dianthin toxin, Phytolaca americana toxin (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, scutellaria baicalensis ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0200] The term "chemotherapeutic agent" is a chemical compound that can be used to treat tumors. This definition also includes anti-hormonal agents that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic treatment. They can themselves be hormones. Examples of chemotherapeutic agents include alkylating agents such as thiotepa, cyclosphamide (CYTOXAN), and thiophene. TM), alkyl sulfonates such as busulfan, improsulfan and piposulfan, aziridines such as benaodopa, carboquone, meturedopa and uredopa, aziridines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine, nitrogen mustard mustards such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, oxazolidinone hydrochloride; melphalan, novembichin, cholesteryl phenylacetic acid mustard, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclarubicin, dactinomycin, authramycin, azaserine, bleomycin, actinomycin C mycin, calicheamicin, carabicin, chromomycin, carzinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptozocin, streptozocin, tuberculin,Ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate, 5-fluorouracil (5-FU), folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate, the pterin analogs fludarabine, 6-mercaptopterin, thioimidazole, thioguanpterin, pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxitluridine, enocitabine, floxuridine, 5-FU, and androgens such as calusterone, dromostanol propionate propionate, epitiostanol, mepitiostane, testolactone, antiadrenal drugs such as aminoglutethimide, mitotane, trilostane, folic acid supplements such as frolinic acid, aceglucose lactone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, biasntrene, edatraxate, defofamine, colcemid, diaziquone, elfomithine, elliptinium acetate, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pintostatin, phenamet, pirarubicin, podophyllinic acid, 2-ethylhydrazide, procarbazine, Razoxane, sizofiran, spirogermanium, tricine, triazoline; 2,2',2"-trichlorrotriethylamine, urethan, vinblastine, dacarbazine, mannitol mustard, mitobronitol, dibromodulcitol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxanes such as paclitaxel ( Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel ( Rhone-Poulenc Rorer, Antony, France), chlorambucil, gemcitabine, 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfidine, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunorubicin, aminopterin; xeloda, ibandronate, CPT-11, topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), esperamicins, capecitabine, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. This definition also includes antihormonal agents that can modulate or inhibit the effects of hormones on tumors, such as antiestrogens including tamoxifen, raloxifene, the aromatase inhibitors 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene, and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

[0201] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain 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 various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which 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. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from one or more substituents selected from H atoms, D atoms, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0202] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0203] The term "alkylene" refers to a saturated straight or branched chain aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane, which is a straight or branched chain 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 an alkylene group containing 1 to 8 carbon atoms, and most preferably an alkylene group 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 may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably independently and optionally selected from one or more substituents of alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, cycloalkyloxy, heterocyclyloxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0204] The term "alkenyl" refers to an alkyl compound containing a carbon-carbon double bond in the molecule, wherein alkyl is as defined above. Alkenyl groups may be substituted or unsubstituted. When substituted, the substituents are preferably one or more of the following groups independently selected from hydrogen, alkyl, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0205] The term "alkynyl" refers to an alkyl compound containing a carbon-carbon triple bond in the molecule, wherein alkyl is as defined above. Alkyl groups may be substituted or unsubstituted. When substituted, the substituents are preferably one or more groups independently selected from hydrogen, alkyl, alkoxy, halogen, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0206] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 8 carbon atoms (e.g., 3, 4, 5, 6, 7, and 8) carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0207] The term "spiroalkyl" refers to a polycyclic group of 5 to 20 members, a carbon atom (called spiral atom) shared between the monocycles, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members (e.g., 7, 8, 9 or 10 members). According to the number of spiral atoms shared between the rings, the spiroalkyl group is divided into a single spiroalkyl group, a double spiroalkyl group or a multi-spiroalkyl group, preferably a single spiroalkyl group and a double spiroalkyl group. More preferably, it is 4 / 4 members, 4 / 5 members, 4 / 6 members, 5 / 5 members or 5 / 6 members of a single spiroalkyl group. Non-limiting examples of spiroalkyl groups include:

[0208]

[0209] The term "fused cycloalkyl" refers to a 5 to 20-membered, all-carbon polycyclic group in which each ring in the system shares a pair of adjacent carbon atoms with the other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members (e.g., 7, 8, 9 or 10 members). According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. Non-limiting examples of fused cycloalkyl include:

[0210]

[0211] The term "bridged cycloalkyl" refers to a 5 to 20-membered, all-carbon polycyclic group in which any two rings share two carbon atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system. Preferably, it is 6 to 14 members, more preferably 7 to 10 members (e.g., 7, 8, 9 or 10 members). Depending on the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

[0212]

[0213] The cycloalkyl ring includes a cycloalkyl group as described above (including a monocyclic, spirocyclic, condensed and bridged ring) fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl group, non-limiting examples of which include indanyl, tetrahydronaphthyl and benzocycloheptanyl, etc.; preferably phenylcyclopentyl or tetrahydronaphthyl.

[0214] The cycloalkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment, and the substituent is preferably independently selected from one or more substituents of hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0215] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl and cycloalkyl are as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy. Alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from H atoms, D atoms, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0216] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is a heteroatom selected from nitrogen, oxygen, sulfur, S(O) or S(O)2, 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, of which 1 to 4 (e.g., 1, 2, 3 and 4) are heteroatoms; more preferably, it contains 3 to 8 (e.g., 3, 4, 5, 6, 7 and 8) ring atoms, of which 1 to 3 are heteroatoms (e.g., 1, 2 and 3); more preferably, it contains 3 to 6 ring atoms, of which 1 to 3 are heteroatoms; and most preferably, it contains 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.

[0217] The term "spiroheterocyclyl" refers to a polycyclic heterocyclic group of 5 to 20 members, 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 no ring has a completely conjugated π electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members (e.g., 7, 8, 9 or 10 members). Spiroheterocyclyl is divided into monospiro heterocyclyl, dispiro heterocyclyl or polyspiro heterocyclyl according to the number of shared spiro atoms between rings, preferably monospiro heterocyclyl and dispiro heterocyclyl. It is more preferably 4 / 4 members, 4 / 5 members, 4 / 6 members, 5 / 5 members or 5 / 6 members monospiro heterocyclyl. Non-limiting examples of spiroheterocyclyl include:

[0218]

[0219] The term "fused heterocyclic group" refers to a polycyclic heterocyclic group of 5 to 20 members, each ring in the system sharing a pair of atoms adjacent to the other rings in the system, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, 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. Preferably, it is 6 to 14 members, more preferably 7 to 10 members (e.g., 7, 8, 9 or 10 members). According to the number of constituent rings, it can be divided into 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:

[0220]

[0221] The term "bridged heterocyclic group" refers to a polycyclic heterocyclic group of 5 to 14 members, wherein any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but no ring has a completely conjugated π electron system, 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. Preferably, it is 6 to 14 members, more preferably 7 to 10 members (e.g., 7, 8, 9 or 10 members). According to the number of constituent rings, it can be divided into 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:

[0222]

[0223] The heterocyclyl ring includes a heterocyclyl as described above (including monocyclic, spiro heterocyclic, fused heterocyclic and bridged heterocyclic rings) fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0224] wait.

[0225] The heterocyclic group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from one or more substituents of hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclic, aryl and heteroaryl.

[0226] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (fused polycyclic is a ring that shares adjacent pairs of carbon atoms) group having a conjugated π electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring includes an aryl ring as described above fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

[0227]

[0228] The aryl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably independently selected from one or more substituents of hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0229] The term "heteroaryl" refers to a heteroaromatic system containing 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-membered or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl and the like. The heteroaryl ring includes a heteroaryl group as described above fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0230]

[0231]

[0232] The heteroaryl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment, and the substituent is preferably independently selected from one or more substituents of hydrogen, halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0233] The term "amino-protecting group" is used to protect the amino group with a readily removable group in order to maintain the amino group intact during reactions elsewhere in the molecule. Non-limiting examples include (trimethylsilyl)ethoxymethyl, tetrahydropyranyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups may optionally be substituted with 1-3 substituents selected from halogen, alkoxy, or nitro.

[0234] The term "hydroxy protecting group" is a suitable group for protecting a hydroxy group known in the art, as described in the literature ("Protective Groups in Organic Synthesis", 5 Th Ed.TWGreene & P.GM Wuts) in the hydroxyl protecting group. As an example, preferably, the hydroxyl protecting group can be (C 1-10 Alkyl or aryl) 3 silyl, for example: 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, most preferably C 1-4 Alkoxy-substituted C 1-4 Alkyl, for example: methyl, tert-butyl, allyl, benzyl, methoxymethyl (MOM), ethoxyethyl, 2-tetrahydropyranyl (THP), etc.; can be (C 1-10 Alkyl or aromatic) acyl, for example: formyl, acetyl, benzoyl, p-nitrobenzoyl, etc.; can be (C 1-6 Alkyl or C 6-10 aryl)sulfonyl; can also be (C 1-6 Alkoxy or C 6-10 aryloxy)carbonyl.

[0235] The term "cycloalkyloxy" refers to a cycloalkyl-O- group in which cycloalkyl is as defined above.

[0236] The term "heterocyclyloxy" refers to a heterocyclyl-O- group in which heterocyclyl is as defined above.

[0237] The term "alkylthio" refers to an alkyl-S- group in which alkyl is as defined above.

[0238] The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.

[0239] The term "haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.

[0240] The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.

[0241] The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

[0242] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0243] The term "hydroxy" refers to -OH.

[0244] The term "mercapto" refers to -SH.

[0245] The term "amino" refers to -NH2.

[0246] The term "cyano" refers to -CN.

[0247] The term "nitro" refers to -NO2.

[0248] The term "oxo" refers to "=0".

[0249] The term "carbonyl" refers to C=O.

[0250] The term "carboxy" refers to -C(O)OH.

[0251] The term "carboxylate" refers to -C(O)O(alkyl), -C(O)O(cycloalkyl), (alkyl)C(O)O-, or (cycloalkyl)C(O)O-, wherein alkyl and cycloalkyl are as defined above.

[0252] The compounds disclosed herein include isotopic derivatives thereof. 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 a structure disclosed herein, with "deuterium" or "tritium" replacing hydrogen, or with 18 F-fluorine labeling ( 18 F isotope) instead of fluorine, or with 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 a carbon atom is replaced by a C-isotope) are within the scope of the present 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 pharmacodynamics, pharmacokinetics, or receptor studies. The various deuterated forms of the compounds disclosed herein refer to compounds in which each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of compounds with reference to relevant literature. Commercially available deuterated starting materials can be used when preparing deuterated forms of compounds, or they can be synthesized using deuterated reagents using conventional techniques, including but not limited to deuterated borane, trideuterated borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated iodoethane, and deuterated iodomethane. Deuterated compounds can generally retain activity comparable to undeuterated compounds, and when deuterated at certain specific sites, better metabolic stability can be achieved, thereby obtaining certain therapeutic advantages.

[0253] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and that the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0254] "Substituted" means that one or more hydrogen atoms, preferably 1 to 5, more preferably 1 to 3 hydrogen atoms, in a group are independently replaced by a corresponding number of substituents. Those skilled in the art will be able to determine (by experiment or theory) whether substitution is possible or not without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0255] A "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0256] The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio, and effective for the intended use.

[0257] As used herein, the singular form "a," "an," and "the" include plural references and vice versa unless the context clearly dictates otherwise.

[0258] When the term "about" is applied to a parameter such as pH, concentration, temperature, etc., it indicates that the parameter can vary by ±10%, and sometimes more preferably within ±5%. As will be understood by those skilled in the art, when a parameter is not critical, numbers are generally given for illustration purposes only and are not limiting.

[0259] The term "linker unit", "connecting unit" or "connecting fragment" refers to a chemical structure fragment or bond that is connected to a ligand (antibody in this disclosure) at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug.

[0260] The linker can include one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio) valerate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate ("SMCC", also referred to herein as "MCC") and N-succinimidyl (4-iodo-acetyl) aminobenzoate ("SIAB"). The linker can include an extender, a spacer, and an amino acid unit, and can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates release of the drug in the cell. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020) can be used.

[0261] Connector components include but are not limited to:

[0262] MC=6-maleimidocaproyl, the structure is as follows:

[0263]

[0264] Val-Cit or "vc" = valine-citrulline (an exemplary dipeptide in a protease-cleavable linker)

[0265] Citrulline = 2-amino-5-ureidopentanic acid

[0266] PAB = p-aminobenzyloxycarbonyl (an example of a "self-immolative" linker component)

[0267] Me-Val-Cit = N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to protect it from cleavage by cathepsin B)

[0268] MC(PEG)6-OH = Maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteines)

[0269] SPP = N-succinimidyl 4-(2-pyridylthio)pentanoate

[0270] SPDP = N-succinimidyl 3-(2-pyridyldithio) propionate

[0271] SMCC = succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate

[0272] IT = Iminothiolane

[0273] The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0274] The term "carrier" as used in the drugs disclosed herein refers to a system that can change the way a drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure. Preferred examples include micelles, microemulsions, gels, liquid crystals, vesicles, etc. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, making them excellent drug carriers.

[0275] The term "excipient" refers to any additive in a pharmaceutical preparation other than the main drug, also known as an adjuvant. Examples include binders, fillers, disintegrants, and lubricants in tablets; the base component of semisolid ointments and creams; and preservatives, antioxidants, flavorings, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0276] The term "diluent," also known as filler, primarily increases the weight and bulk of a tablet. The addition of a diluent not only maintains a certain volume but also reduces dosage variations of the main ingredient and improves the drug's compressibility. When the tablet contains an oily component, an absorbent is added to absorb the oil and maintain a "dry" state, facilitating tablet production. Examples include starch, lactose, inorganic calcium salts, and microcrystalline cellulose.

[0277] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0278] The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. The 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 added to a mixture of water and glycerol to form a microemulsion. The injection or microemulsion may be injected into the subject's bloodstream via local bolus injection. Alternatively, the solution or microemulsion may be administered in a manner that maintains a constant circulating concentration of the disclosed compound. To maintain such a constant concentration, a continuous intravenous drug delivery device may be used. An example of such a device is the Deltec CADD-PLUS™ 5400 intravenous pump.

[0279] The pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration. The suspension can be prepared according to known techniques using the above-mentioned suitable dispersants or wetting agents and suspending agents. Sterile injection preparations can also be sterile injection solutions or suspensions prepared in non-toxic parenteral acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. In addition, sterile fixed oils can be conveniently used as solvents or suspension media. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used. In addition, fatty acids such as oleic acid can also be used to prepare injections.

[0280] "Administer," "give," and "treat," as they apply 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 an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as it applies to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0281] The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugates of the present disclosure, or a salt of a compound described herein. Such salts are safe and effective when used in mammals and have the desired biological activity. The antibody-drug conjugates of the present disclosure contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 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.

[0282] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds disclosed herein, to a subject having one or more symptoms of a disease, and it is known that the therapeutic agent has a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated subject or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent that effectively alleviates any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0283] The details of one or more embodiments of the present disclosure are set forth in the above description. Although any methods and materials similar or identical to those described herein can be used to implement or test the present disclosure, preferred methods and materials are described below. Other features, objects, and advantages of the present disclosure will be apparent from the description and claims. In the description and claims, unless otherwise clearly indicated in the context, the singular includes the case of plural referents. Unless otherwise defined, all technical and scientific terms used herein have the common meaning understood by those of ordinary skill in the art to which the present disclosure belongs. All patents and publications cited in the description are incorporated by reference. The following examples are presented to more fully illustrate the preferred embodiments of the present disclosure. These examples should not be construed in any way to limit the scope of the present disclosure, which is defined by the claims.

[0284] The present invention is further described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0285] Experimental methods in the disclosed embodiments or test examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the raw material or commercial manufacturer. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Current Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0286] Example

[0287] Example 1: Construction of HER3 High-Expression Cell Line

[0288] The pCDH-Her3 lentiviral expression vector plasmid and the pVSV-G and pCMV-dR8.91 lentiviral system packaging vectors were transfected into 293T virus packaging cells using Lipofectamine 3000 transfection reagent. The virus-containing culture supernatant was collected, filtered, and subjected to ultrahigh-speed centrifugation. The concentrated virus was used to infect Chinese hamster ovary (CHO-K1) cells. After puromycin selection for two to three weeks, single-cell sorting was performed by FACS.

[0289] The expression of HER3 on the surface of lentivirus-infected CHO-K1 cells was detected by FACS, and monoclonal cell lines with high HER3 expression were selected.

[0290] The selected monoclonal cell lines are expanded and stored for subsequent experiments.

[0291] HuMan ErbB3 amino acid sequence (UniProtKB-P21860-1, AA Ser 20-Thr 643)

[0292]

[0293] HuMan ErbB3 nucleotide sequence

[0294]

[0295] Example 2: Production of anti-human HER3 monoclonal antibodies

[0296] 2.1 Preparation of positive control antibody

[0297] The positive control antibody U3 was prepared according to WO2007077028A2 (page 118, U1-59). The amino acid sequences of the heavy and light chains of U3 are as follows:

[0298] U3 heavy chain:

[0299]

[0300] U3 light chain:

[0301]

[0302] 2.2 Preparation of Antibodies Disclosed

[0303] Using a fully human natural phage antibody library and the antigen Biotinylated HuMan ErbB3 (purchased from Beijing Biopsies Biotechnology Co., Ltd., catalog number: ER3-H82E6), after panning, phage detection was performed using the ELISA method to obtain positive clones. The positive clones were sequenced, and after obtaining the sequence, the positive clone sequence was inserted into the protein expression vector Phr-IgG and expressed by 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. The constant region of the antibody of the fully human antibody molecule HER3-29:

[0304] Heavy chain constant region of human IgG1:

[0305]

[0306] Human kappa light chain constant region:

[0307]

[0308] The sequences of the heavy chain variable region and light chain variable region of the fully human antibody molecule HER3-29 are as follows:

[0309] HER3-29 heavy chain variable region:

[0310]

[0311] HER3-29 light chain variable region:

[0312]

[0313] The CDR sequences obtained using different numbering rules are as follows:

[0314] Table 1. CDR sequences obtained using the Chothia numbering rule

[0315] Antibody HER3-29 Sequence Number Heavy Chain CDR1GFTFDDY SEQ ID NO: 9 Heavy Chain CDR2SWNSGS SEQ ID NO: 10 Heavy Chain CDR3EGLPGLDY SEQ ID NO: 11 Light Chain CDR1RASQHVGTYLN SEQ ID NO: 12 Light Chain CDR2GAANLQS SEQ ID NO: 13 Light Chain CDR3QQSYNTPPFS SEQ ID NO: 14

[0316] Table 2. CDR sequences obtained by IMGT numbering rules

[0317] Antibody HER3-29 SEQ No. Heavy chain CDR1GFTFDDYASEQ ID NO: 15 Heavy chain CDR2ISWNSGSISEQ ID NO: 16 Heavy chain CDR3AKEGLPGLDYSEQ ID NO: 17 Light chain CDR1QHVGTYSEQ ID NO: 18 Light chain CDR2GAASEQ ID NO: 19 Light chain CDR3QQSYNTPPFSSEQ ID NO: 20

[0318] Table 3. CDR sequences obtained by Kabat numbering rules

[0319] Antibody HER3-29 sequence number

[0320] Heavy chain CDR1 DYAMH SEQ ID NO: 21 Heavy chain CDR2 GISWNSGSIGYADSVKG SEQ ID NO: 22 Heavy chain CDR3 EGLPGLDY SEQ ID NO: 23 Light chain CDR1 RASQHVGTYLN SEQ ID NO: 24 Light chain CDR2 GAANLQS SEQ ID NO: 25 Light chain CDR3 QQSYNTPPFS SEQ ID NO: 26

[0321] The heavy chain sequence and light chain sequence of the fully human antibody molecule HER3-29 are as follows:

[0322] HER3-29 heavy chain:

[0323]

[0324] HER3-29 light chain:

[0325]

[0326] Example 3: Preparation of ADC

[0327] Determination of DAR value of ADC

[0328] The DAR value calculation method of the ADC disclosed herein uses RP-HPLC (reverse phase high performance liquid chromatography), as follows:

[0329] 1. Determination method:

[0330] Naked antibody and test ADC sample (concentration 1 mg / mL) were reduced with 4 μL of DDT (Sigma), incubated in a 37°C water bath for 1 hour, and then removed to an inner tube. Detection was performed using an Agilent 1200 HPLC using an Agilent PLRP-S 1000A 8 μm 4.6 x 250 mm column, 80°C column temperature, DAD detector wavelength 280 nm, flow rate 1 mL / min, and injection volume 40 μL. The light and heavy chains were then identified by comparing the spectra of the sample with those of the naked antibody. The spectra of the test sample were then integrated to calculate the DAR value.

[0331] 2. Solution preparation

[0332] 1) 0.25M DTT solution:

[0333] Preparation example: Take 5.78 mg of DTT and add 150 μL of purified water to fully dissolve it to prepare 0.25 M DTT solution. Store at -20°C.

[0334] 2) Mobile phase A (0.1% TFA aqueous solution):

[0335] Preparation example: Measure 1000 mL of purified water into a graduated cylinder, add 1 mL of TFA (Sigma), mix thoroughly before use, and store at 2-8°C for 14 days.

[0336] 3) Mobile phase B (0.1% TFA acetonitrile solution):

[0337] Preparation example: Measure 1000 mL of acetonitrile into a measuring cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.

[0338] 3. Data Analysis

[0339] By comparing the spectra of the sample and the naked antibody, the positions of the light and heavy chains are distinguished, and then the spectrum of the test sample is integrated to calculate the DAR value.

[0340] The calculation formula is as follows:

[0341] Name connected drug number LC0LC+12HC0HC+12HC+24HC+36

[0342] Sum of LC peak areas = LC peak area + LC+1 peak area

[0343] Total HC peak area = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area

[0344] LC DAR = Σ(number of connected drugs * peak area percentage) / total LC peak area

[0345] HC DAR = Σ(number of connected drugs * peak area percentage) / total HC peak area

[0346] DAR=LC DAR+HC DAR

[0347] drug

[0348] The drug portion of the conjugates of the present disclosure can be any suitable drug. Particularly suitable drugs are described, for example, in PCT Publication No. WO2020063676A1 (incorporated herein by reference in its entirety). Compound 9A disclosed herein (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]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide, which has the following structure:

[0349]

[0350] The present disclosure uses the following method to prepare an antibody-drug conjugate as shown in the ADC formula (HER3-29-9A) by adjusting reaction parameters.

[0351]

[0352] Example 3-1 ADC-1

[0353] At 37°C, a solution of HER3-29 antibody in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 11.8 mL, 797 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 208.2 μL, 2.082 μMol). The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0354] Compound 9A (prepared with compound 9-A of Example 9 of WO2020063676, the entire contents of which are incorporated herein by reference) (8.6 mg, 8.006 μmol) was dissolved in 500 μL DMSO, added to the above reaction solution, placed in a water bath shaker, and oscillated at 25 ° C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain a PBS buffer solution (4.02 mg / mL, 27.9 mL) of the exemplary product ADC-1 of the conjugate HER3-29-9A, which was stored at 4 ° C. RP-HPLC calculated average value: DAR = 4.19.

[0355] Example 3-2 ADC-2

[0356] At 37°C, a solution of HER3-29 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 11.8 mL, 797 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 128 μL, 1.281 μmol). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0357] Compound 9A (6.88 mg, 6.405 μmol) was dissolved in 400 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the conjugate HER3-29-9A, ADC-2, in PBS buffer (4.24 mg / mL, 27.2 mL), which was stored at 4°C. RP-HPLC calculated average DAR = 2.91.

[0358] Example 3-3 ADC-3

[0359] At 37°C, a solution of HER3-29 antibody in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 3.1 mL, 209 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 111 μL, 1.111 μM). The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0360] Compound 9A (3.37 mg, 3.137 μmol) was dissolved in 120 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of the conjugate HER3-29-9A, ADC-3, in PBS buffer (1.48 mg / mL, 12.8 mL), which was stored at 4°C. RP-HPLC calculated average DAR = 7.27.

[0361] Example 3-4 ADC-4

[0362]

[0363] To a solution of antibody U3 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 3.1 mL, 209 nmol) at 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 111 μL, 1.111 μmol) was added. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0364] Compound 9A (3.37 mg, 3.137 μmol) was dissolved in 120 μL of DMSO and added to the above reaction solution. The mixture was shaken in a water bath at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of conjugate U3-9A, ADC-4, in PBS buffer (1.48 mg / mL, 12.8 mL), which was stored at 4°C. RP-HPLC calculated average DAR = 6.76.

[0365] Example 3-5 ADC-5

[0366]

[0367] Referring to Example 12 on page 156 of WO2015155998A1, U3-1402 was prepared as a positive control. At 37°C, a solution of antibody U3 in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 3.1 mL, 236 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 130 μL). The mixture was shaken in a water bath at 37°C for 3 hours, and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0368] Compound 1402 (3.67 mg, 3.54 μmol) was dissolved in 180 μL of DMSO and added to the above reaction solution. The mixture was placed in a water bath shaker 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 water at pH 6.5, containing 0.001 M EDTA) to obtain an exemplary product of conjugate U3-1402, ADC-5, in PBS buffer (1.53 mg / mL, 15.4 mL), which was stored at 4°C. RP-HPLC calculated average DAR = 6.97.

[0369] Test Case

[0370] Test Example 1: Binding experiment of antibody and free HER3 protein

[0371] HER3 protein was diluted to 1 μg / mL in PBS (B320, pH 7.4) and added to a 96-well microtiter plate at a volume of 100 μL / well. The plate was incubated overnight at 4°C. After discarding the liquid, 300 μL of 5% skim milk (BD, 232100) diluted in PBS was added to each well for blocking and incubation at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plate was washed three times with PBST (PBS, pH 7.4 containing 0.1% tween-20). 100 μL of serially diluted antibody solution was added to each well and 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 incubated at 37°C for 1 hour. After washing the plate three times with PBST, 100 μL of TMB colorimetric substrate (KPL, 5120-0077) was added to each well and incubated at room temperature for 10-15 minutes. 50 μL of 1 M H2SO4 was added to each well to terminate the reaction. The absorbance at 450 nm was read using a microplate reader. The binding curve of the antibody and antigen was fitted using software, as shown in Figure 1. EC was calculated. 50The results are shown in Table 4.

[0372] Table 4. Binding activity of antibodies to HER3 protein

[0373] Antibody HER3-29U3

[0374] EC 50 (nM)0.140.56

[0375] Conclusion: The disclosed antibody HER3-29 has better binding activity to HER3 protein than the control antibody U3.

[0376] Test Example 2: Binding experiment of antibody to cells expressing HER3

[0377] MCF7 cells (ATCC, HTB-22) were prepared with FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)) to a concentration of 1×10 6 Cells / mL of cell suspension were added to a 96-well round-bottom plate at 100 μL / well. After centrifugation to remove the supernatant, 50 μL / well of the antibody to be tested diluted with FACS buffer at different concentrations was added and incubated in a 4°C refrigerator in the dark for 1 hour. After washing three times with FACS buffer at 300g, Alexa Fluor 488 goat anti-human (Goat anti-HuMan) IgG (H+L) (invitrogen, A-11013) at a working concentration was added and incubated in a 4°C refrigerator in the dark for 40 minutes. After washing three times with FACS buffer at 300g, the geometric mean fluorescence intensity was detected on a BD FACSCantoII flow cytometer, as shown in Figure 2. EC 50 See Table 5 for values.

[0378] Table 5. Antibody binding activity at the cell level

[0379] Antibody HER3-29U3EC 50 (nM)0.0570.249

[0380] Conclusion: The disclosed antibody HER3-29 has better binding activity to cells expressing HER3 protein than the control antibody U3.

[0381] Test Example 3: DT3C Antibody Internalization Experiment

[0382] The purpose of this experiment is to indirectly reflect the internalization of HER3 antibodies based on the killing of cells by activated diphtheria toxin (DT) after DT3C protein enters cells. 50 The in vitro endocytic activity of the antibody was evaluated by measuring Imax.

[0383] DT3C is a recombinantly expressed fusion protein composed of Fragment A (toxin portion only) of diphtheria toxin and Fragment 3C (IgG-binding portion) of group G Streptococcus. This protein has a high affinity for the Fc structure of antibodies and enters cells together with the antibody during endocytosis. Under the action of intracellular furin, the toxic DT is released. DT inhibits EF2-ADP ribosylation, blocks protein translation, and ultimately leads to cell death. DT3C that does not enter cells has no cytotoxic activity. The endocytic activity of the antibody is evaluated based on the cell killing effect.

[0384] Fresh cell culture medium containing 20% ​​low IgG FBS was used to prepare a CHOK1 cell suspension expressing HER3 at a cell density of 2×10 4 cells / mL, 50 μL / well was added to the cell culture plate and cultured at 37°C in 5% carbon dioxide for 16 hours.

[0385] Prepare 4× concentration of DT3C in serum-free medium and filter it with a 0.22μm filter to form a sterile solution. Prepare 4× concentration of antibody in serum-free medium, mix 80μL DT3C (400nM) and 80μL antibody (66nM) in a 1:1 volume ratio, and incubate at room temperature for 30 minutes. Take 50μL of diluted antibody and add it to 50μL of cells and incubate in the incubator for three days. Add 50μL CTG (CellTiter-Glo) to each well. TM Reagent, G7573), incubated at room temperature in the dark for 10 minutes, and chemiluminescence was read on a Victor3. The results are shown in Figure 3 and Table 6.

[0386] Table 6. Endocytic activity of antibodies

[0387] Antibody HER3-29U3Imax48%19%IC 50 (nM)0.512.81

[0388] Conclusion: The endocytic activity of the disclosed antibody HER3-29 is better than that of the control antibody U3.

[0389] Test Example 4: pHrodo Antibody Internalization Experiment

[0390] The purpose of this experiment is to reflect the internalization of the HER3 antibody based on the change in fluorescence signal after the dye is internalized. The intensity of the fluorescence signal is used to evaluate the antibody's in vitro endocytic activity.

[0391] The pH-sensitive pHrodo iFL dye conjugated to the Fab fragment directly binds to the Fc region of the HER3 antibody without interfering with the antibody's antigen recognition. The pHrodo iFL dye is virtually non-fluorescent at neutral pH. During HER3 antibody endocytosis, the dye is internalized, and the fluorescence signal gradually increases as the pH decreases. The increase in fluorescence signal is used to evaluate the antibody's endocytic activity.

[0392] HER3 / CHOK1 cells were cultured in DMEM / F12 + 10% FBS + 10 μg / mL puromycin. On the first day of the experiment, a cell suspension was prepared in fresh cell culture medium at a density of 2 × 10 5 cells / mL, 100 μL / well was added to a 96-well cell culture plate and cultured at 37°C in 5% carbon dioxide for 24 hours.

[0393] Aspirate 50 μL of cell suspension from the plate and add 50 μL of the antibody and pHrodo dye mixture to each well. Set up two replicate wells for each antibody sample. Set up a dye-only group and an isotype IgG1 control group.

[0394] After 24 hours of incubation in the incubator, the culture medium was aspirated and 50 μL of trypsin was added to each well for 2 minutes. Digestion was terminated with 50 μL of fresh culture medium. Using a dispenser, cells from duplicate wells of the same sample were transferred to the same well of a round-bottom plate. Centrifuge at 1500 rpm for 2 minutes, discard the culture medium, and wash the cells once with FACS buffer (PBS + 2.5% FBS) and centrifuge at 1500 rpm for 2 minutes. Add 200 μL of FACS buffer (PBS + 2.5% FBS), resuspend the cells, and analyze the FITC signal using a flow cytometer. Data were analyzed using FlowJo 7.6; the results are shown in Figure 4 and Table 7.

[0395] Table 7. Antibody endocytic activity

[0396] Antibody HER3-29U3 FITC signal 373267

[0397] Conclusion: The endocytic activity of the disclosed antibody HER3-29 is better than that of the control antibody U3.

[0398] Test Example 5: ADC Molecular Cell Activity Experiment

[0399] The purpose of this experiment is to detect the cell killing effect of ADC samples. 50 and Imax were used to evaluate the in vitro activity of Her3-ADC.

[0400] MCF7 cells (human breast cancer cells), SW620 cells (human colon cancer cells, Nanjing Kebai, CBP60036), and WiDr cells (human colorectal cancer cells) were trypsinized, neutralized with fresh culture medium, centrifuged at 1000 rpm, and resuspended in culture medium. After counting, the cell suspension density was adjusted to 500 cells / well and added to a 96-well cell culture plate. No cells were plated in column 11, but only 135 μL of culture medium was added. The cells were cultured at 37°C in 5% carbon dioxide for 16 hours.

[0401] ADC samples were diluted to 15 μM (10× concentration) with PBS. This was the initial concentration, and five-fold dilutions were performed with PBS for a total of eight concentrations. 15 μL of the 10× concentration solution was added to each well. The cells were incubated at 37°C in a 5% CO2 atmosphere for 6 days.

[0402] 70 μL CTG was added to each well and incubated at room temperature in the dark for 10 minutes. A white base film was attached to the bottom of the cell culture plate and placed on a Victor3 to read the chemical reaction. The data of this experiment were processed using the data processing software GraphPad Prism 5.0, see Table 8.

[0403] Table 8. In vitro killing experiments of HER3-29-9A at different DAR values

[0404]

[0405] Conclusion: The ADC samples ADC-1, ADC-2, and ADC-3 disclosed herein have better cell killing activity than the positive controls ADC-4 and ADC-5.

[0406] In vivo biological evaluation

[0407] Test Example 6: In vivo efficacy evaluation in HER3-overexpressing CDX models

[0408] SW620 cells (5×10 6 The cells were inoculated subcutaneously in the right rib of Balb / c nude mice and divided into 9 groups of 8 mice each, for a total of 9 groups. The average group volume was 134.75 mm 3 ADC was injected intraperitoneally for a total of 3 times, once every 5 days. Each animal was injected with 0.1 mL / 10 g according to body weight. Tumor volume and body weight were measured twice a week and the data were recorded. Data were recorded using Excel statistical software: mean value was calculated as avg; SD value was calculated as STDEV; SEM value was calculated as STDEV / SQRT (number of animals in each group); GraphPad Prism software was used for graphing, and two-way ANOVA or one-way ANOVA was used for statistical analysis of data.

[0409] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L短 2

[0410] 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; T0 and C0 are the tumor volumes at the beginning of the experiment.

[0411] Tumor inhibition rate TGI (%) = 1-T / C (%). The results are shown in Figure 5 and Table 9.

[0412] Table 9. Efficacy of ADC on SW620 xenografts in nude mice

[0413]

[0414]

[0415] Conclusion: The therapeutic effects of ADC-1 and ADC-2 on SW620 xenograft tumors in nude mice are better than that of the positive control ADC-5.

Claims

1. An isolated anti-HER3 antibody, wherein the anti-HER3 antibody has one or more of the following characteristics: a. The anti-HER3 antibody binds to the HER3 protein with an apparent affinity EC of less than 0.5 nM, and the apparent affinity EC 50 is determined by the ELISA method; 50 ​ b. The anti-HER3 antibody binds to the HER3 protein expressed by MCF7 cells with an apparent affinity EC of less than 0.2 nM, and the apparent affinity EC 50 is determined by the FACS method; 50 ​ c. The anti-HER3 antibody can be internalized by cells expressing human HER3; preferably, when the anti-HER3 antibody is assayed by the DT3C antibody internalization experiment, its IC 50 is less than 2 nM; d. The anti-HER3 antibody can be internalized by cells expressing human HER3; preferably, when the anti-HER3 antibody is measured by the pHrodo antibody internalization assay, its FITC signal is greater than 300.

2. The isolated anti-HER3 antibody according to claim 1, wherein the anti-HER3 antibody 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; Preferably, 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 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 shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14 respectively; wherein the CDR regions are determined according to the Chothia numbering rule; or b. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 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 shown in SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20 respectively; wherein the CDR regions are determined according to the IMGT numbering rule; or c. The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 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 shown in SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26 respectively; wherein the CDR regions are determined according to the Kabat numbering rule.

3. An isolated anti-HER3 antibody, 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 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 shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14 respectively; The CDR regions as described above are determined according to the Chothia numbering rule; 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 regions as described above are 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 regions as described above are determined according to the Kabat numbering rules.

4. The isolated anti-HER3 antibody according to any one of claims 1 to 3, wherein the anti-HER3 antibody is a human antibody or an antigen-binding fragment.

5. The isolated anti-HER3 antibody according to any one of claims 1 to 4, which comprises 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 as shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:

8.

6. The isolated anti-HER3 antibody according to any one of claims 1 to 5, which comprises: 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: A heavy chain as shown in SEQ ID NO:27 and a light chain as shown in SEQ ID NO:

28.

7. The isolated anti-HER3 antibody according to any one of claims 1 to 6, wherein the anti-HER3 antibody has one or more of the following characteristics: a. The anti-HER3 antibody binds to the HER3 protein with an apparent affinity EC of less than 0.5 nM 50 and the apparent affinity EC 50 is determined by the ELISA method; b. The anti-HER3 antibody binds to the HER3 protein expressed by MCF7 cells with an apparent affinity EC of less than 0.2 nM 50 and the apparent affinity EC 50 is determined by the FACS method; c. The anti-HER3 antibody described above can be endocytosed by cells expressing human HER3; preferably, when the anti-HER3 antibody is measured by the DT3C antibody endocytosis assay, its IC 50 is less than 2 nM; d. The anti-HER3 antibody can be endocytosed by cells expressing human HER3; preferably, when the anti-HER3 antibody is measured by the pHrodo antibody endocytosis assay, its FITC signal is greater than 300.

8. An isolated anti-HER3 antibody, wherein the antibody competes with the anti-HER3 antibody according to any one of claims 1 to 7 for binding to human HER3.

9. A nucleic acid molecule encoding the isolated anti-HER3 antibody according to any one of claims 1 to 8.

10. A host cell comprising the nucleic acid molecule according to claim 9.

11. An immunoconjugate comprising: the isolated anti-HER3 antibody according to any one of claims 1 to 8 and an effector molecule, wherein, the effector molecule is conjugated to the anti-HER3 antibody; preferably, the effector molecule is selected from anti-tumor agents, immunomodulators, biologic response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

12. A method for immunodetecting or assaying HER3, the method comprising the step of contacting the isolated anti-HER3 antibody according to any one of claims 1 to 8 with a subject or a sample from a subject.

13. An antibody-drug conjugate represented by the general formula (Pc-L a -Y-D) or a pharmaceutically acceptable salt thereof: wherein, Pc is the isolated anti-HER3 antibody according to any one of claims 1 to 8; m is an integer from 0 to 4; n is from 1 to 10, n being a fraction or an integer; R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic group, aryl and heteroaryl; R 2 is selected from a hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic group, aryl and heteroaryl; or, R 1 and R 2 together with the carbon atom to which it is attached form a cycloalkyl or heterocyclic group; W is selected from C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl, and straight-chain heteroalkyls having from 1 to 8 chain atoms, said straight-chain heteroalkyls having from 1 to 8 chain atoms containing from 1 to 3 heteroatoms selected from N, O, and S, wherein said C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl, and straight-chain heteroalkyls having from 1 to 8 chain atoms are each independently optionally further substituted with one or more substituents selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl; L 2 selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 )p 1 C(O)- and chemical bonds, where p 1 is an integer from 1 to 20; L 3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residues are selected from amino acid residues formed by amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and are optionally further substituted by one or more substituents selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl; R 5 selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group; R 6 and R 7 are the same or different and each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.

14. The antibody-drug conjugate of the general formula (Pc-L a -Y-D) according to claim 13, or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate is: wherein: n is from 1 to 8, n being a fraction or an integer; preferably n is from 3 to 8, n being a fraction or an integer; HER3-29 is an anti-HER3 antibody comprising a heavy chain as shown in SEQ ID NO: 27 and a light chain as shown in SEQ ID NO:

28.

15. A method for preparing an antibody-drug conjugate of the general formula (Pc-L a -Y-D) as defined in claim 13 or a pharmaceutically acceptable salt thereof, wherein comprising the following steps: Pc’ is coupled with a compound represented by the general formula (L a -Y-D) to obtain a compound represented by the general formula (Pc-L a -Y-D); wherein: Pc’ is obtained by reducing Pc, and Pc, n, m, W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 and R 7 as defined in claim 13.

16. A pharmaceutical composition comprising the isolated anti-HER3 antibody according to any one of claims 1 to 8, or the nucleic acid molecule according to claim 9, or the antibody-drug conjugate according to claim 13 or 14 or a pharmaceutically acceptable salt thereof; and one or more pharmaceutically acceptable excipients, diluents or carriers.

17. Use of the isolated anti-HER3 antibody according to any one of claims 1 to 8, or the nucleic acid molecule according to claim 9, or the antibody-drug conjugate according to claim 13 or 14 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 16 in the manufacture of a medicament for the treatment of HER3-mediated diseases or disorders.

18. Use of the isolated anti-HER3 antibody according to any one of claims 1 to 8, or the nucleic acid molecule according to claim 9, or the antibody-drug conjugate according to claim 13 or 14 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 16 in the manufacture of a medicament for the treatment and / or prevention of tumors; preferably, the tumors 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.

19. A kit comprising the isolated anti-HER3 antibody according to any one of claims 1 to 8, or the nucleic acid molecule according to claim 9, or the antibody-drug conjugate according to claim 13 or 14 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 16.

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